Sample records for difficile spore germination
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Francis, Michael B; Sorg, Joseph A
2016-01-01
Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis , 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile , spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC , spoVAD , and spoVAE . Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis ), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.
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Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores
Kochan, Travis J.; Kaiser, Alyssa M.; Hastie, Jessica L.; Giordano, Nicole P.; Smith, Ashley D.
2017-01-01
Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI. PMID:28704538
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Nerandzic, Michelle M.; Donskey, Curtis J.
2017-01-01
Background Clostridium difficile is a leading cause of healthcare-associated infections worldwide. Prevention of C. difficile transmission is challenging because spores are not killed by alcohol-based hand sanitizers or many commonly used disinfectants. One strategy to control spores is to induce germination, thereby rendering the spores more susceptible to benign disinfection measures and ambient stressors. Methods/Results C. difficile spores germinated on skin after a single application of cholic acid-class bile salts and co-germinants; for 4 C. difficile strains, recovery of viable spores from skin was reduced by ~0.3 log10CFU to 2 log10CFU after 2 hours and ~1 log10CFU to > 2.5 log10CFU after 24 hours. The addition of taurocholic acid to 70% and 30% ethanol significantly enhanced reduction of viable spores on skin and on surfaces. Desiccation, and to a lesser extent the presence of oxygen, were identified as the stressors responsible for reductions of germinated spores on skin and surfaces. Additionally, germinated spores became susceptible to killing by pH 1.5 hydrochloric acid, suggesting that germinated spores that remain viable on skin and surfaces might be killed by gastric acid after ingestion. Antibiotic-treated mice did not become colonized after exposure to germinated spores, whereas 100% of mice became colonized after exposure to the same quantity of dormant spores. Conclusions Germination could provide a new approach to reduce C. difficile spores on skin and in the environment and to render surviving spores less capable of causing infection. Our findings suggest that it may be feasible to develop alcohol-based hand sanitizers containing germinants that reduce spores on hands. PMID:29167835
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Donnelly, M. Lauren; Fimlaid, Kelly A.
2016-01-01
ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms. PMID:27044622
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Shaban, Lamyaa; Chen, Ying; Fasciano, Alyssa C; Lin, Yinan; Kaplan, David L; Kumamoto, Carol A; Mecsas, Joan
2018-04-01
Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O 2 conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291.
Girinathan, Brintha P; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N; Sorg, Joseph A; Dupuy, Bruno; Govind, Revathi
2017-01-01
Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile -associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB , are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR . A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 a re linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile . In this study, we showed that a mutation in tcdR , the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291.
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Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291
Girinathan, Brintha P.; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N.; Dupuy, Bruno
2017-01-01
ABSTRACT Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291. PMID:28217744
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Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.
Koenigsknecht, Mark J; Theriot, Casey M; Bergin, Ingrid L; Schumacher, Cassie A; Schloss, Patrick D; Young, Vincent B
2015-03-01
Clostridium difficile infection (CDI) following antibiotic therapy is a major public health threat. While antibiotic disruption of the indigenous microbiota underlies the majority of cases of CDI, the early dynamics of infection in the disturbed intestinal ecosystem are poorly characterized. This study defines the dynamics of infection with C. difficile strain VPI 10463 throughout the gastrointestinal (GI) tract using a murine model of infection. After inducing susceptibility to C. difficile colonization via antibiotic administration, we followed the dynamics of spore germination, colonization, sporulation, toxin activity, and disease progression throughout the GI tract. C. difficile spores were able to germinate within 6 h postchallenge, resulting in the establishment of vegetative bacteria in the distal GI tract. Spores and cytotoxin activity were detected by 24 h postchallenge, and histopathologic colitis developed by 30 h. Within 36 h, all infected mice succumbed to infection. We correlated the establishment of infection with changes in the microbiota and bile acid profile of the small and large intestines. Antibiotic administration resulted in significant changes to the microbiota in the small and large intestines, as well as a significant shift in the abundance of primary and secondary bile acids. Ex vivo analysis suggested the small intestine as the site of spore germination. This study provides an integrated understanding of the timing and location of the events surrounding C. difficile colonization and identifies potential targets for the development of new therapeutic strategies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.
De Sordi, Luisa; Butt, M Adil; Pye, Hayley; Kohoutova, Darina; Mosse, Charles A; Yahioglu, Gokhan; Stamati, Ioanna; Deonarain, Mahendra; Battah, Sinan; Ready, Derren; Allan, Elaine; Mullany, Peter; Lovat, Laurence B
2015-01-01
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.
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Effects of natural products on several stages of the spore cycle of Clostridium difficile in vitro.
Roshan, Niloufar; Riley, Thomas V; Hammer, Katherine A
2018-04-19
To investigate the effect of natural products on the spore cycle of C. difficile in vitro. Twenty-two natural products were investigated using four C. difficile strains. Effects on sporulation, determined using microscopy and a conventional spore recovery assay, showed that fresh onion bulb extract (6.3% v v -1 ) and coconut oil (8% v v -1 ) inhibited sporulation in all four isolates by 66-86% and 51-88%, respectively, compared to untreated controls. Fresh ginger rhizome extract (25% v v -1 ) was also inhibitory, although to a lesser extent. Using a standard spore germination and outgrowth assay, germination was unaffected by the 22 products, whereas outgrowth was significantly reduced by artichoke extract (18.8 mg ml -1 ), fresh onion bulb extract (25% v v -1 ), Leptospermum honeys (8% w v -1 ) and allicin (75 mg ml -1 ) (P < 0.01). Sporicidal activity, investigated using a standard plate recovery assay, was minimal. Three of the 22 natural products (13%) showed inhibitory effects on sporulation of C. difficile and six products (27%) reduced vegetative outgrowth of C. difficile. This study shows the potential of natural products to inhibit different stages of C. difficile sporulation and encourages further investigation in this field. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Rätsep, M; Kõljalg, S; Sepp, E; Smidt, I; Truusalu, K; Songisepp, E; Stsepetova, J; Naaber, P; Mikelsaar, R H; Mikelsaar, M
2017-10-01
Clostridium difficile infection (CDI) is one of the most prevalent healthcare associated infections in hospitals and nursing homes. Different approaches are used for prevention of CDI. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients. Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic Lactobacillus plantarum Inducia (DSM 21379) to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum Inducia, prebiotic xylitol (Xyl) and their combination as a synbiotic (Syn) product; c) the suppression of germination of C. difficile spores in vitro and in vivo in animal model of C. difficile infection with Inducia, Xyl and Syn treatment. The VPI strain 10463 (ATCC 43255), epidemic strain (M 13042) and clinical isolates (n = 12) of C. difficile from Norway and Estonia were susceptible and contrarily L. plantarum Inducia resistant to vancomycin, metronidazole and ciprofloxacin. The intact cells of Inducia, natural and neutralized cell free supernatant inhibited in vitro the growth of tested C. difficile reference strain VPI and Estonian and Norwegian clinical isolates of C. difficile after co-cultivation. This effect against C. difficile sustained in liquid media under ampicillin (0.75 μg/ml) and Xyl (5%) application. Further, incubation of Inducia in the media with 5% Xyl fully stopped germination of spores of C. difficile VPI strain after 48 h. In infection model the 48 hamsters were administered ampicillin (30 mg/kg) and 10-30 spores of C. difficile VPI strain. They also received five days before and after the challenge a pretreatment with a synbiotic (single daily dose of L. plantarum Inducia 1 ml of 10 10 CFU/ml and 20% xylitol in 1 ml by orogastric gavage). The survival rate of hamsters was increased to 78% compared to 13% (p = 0.003) survival rate of hamsters who received no treatment. When administered Xyl the survival rate of hamsters reached 56% vs.13% (p = 0.06). In both Syn (6/9, p = 0.003) and Xyl (3/9, p = 0.042) groups the number of animals not colonized with C. difficile significantly increased. In conclusion, the combination of xylitol with L. plantarum Inducia suppresses the germination of spores and outgrowth into vegetative toxin producing cells of C. difficile and reduces the colonization of gut with the pathogen. Putative therapeutical approach includes usage of the synbiotic during antimicrobial therapy for prevention of CDI and its potential to reduce recurrences of CDI. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee
2017-01-01
The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis , indicating that this protein would not be a good target for inhibiting spore formation.
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Ribis, John W.; Ravichandran, Priyanka; Putnam, Emily E.; Pishdadian, Keyan
2017-01-01
ABSTRACT The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation. PMID:28959733
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Inactivation of Clostridium difficile in sewage sludge by anaerobic thermophilic digestion.
Xu, Changyun; Salsali, Hamidreza; Weese, Scott; Warriner, Keith
2016-01-01
There has been an increase in community-associated Clostridium difficile infections with biosolids derived from wastewater treatment being identified as one potential source. The current study evaluated the efficacy of thermophilic digestion in decreasing levels of C. difficile ribotype 078 associated with sewage sludge. Five isolates of C. difficile 078 were introduced (final density of 5 log CFU/g) into digested sludge and subjected to anaerobic digestion at mesophilic (36 or 42 °C) or thermophilic (55 °C) temperatures for up to 60 days. It was found that mesophilic digestion at 36 °C did not result in a significant reduction in C. difficile spore levels. In contrast, thermophilic sludge digestion reduced endospore levels at a rate of 0.19-2.68 log CFU/day, depending on the strain tested. The mechanism of lethality was indirect - by stimulating germination then inactivating the resultant vegetative cells. Acidification of sludge by adding acetic acid (6 g/L) inhibited the germination of spores regardless of the sludge digestion temperature. In conclusion, thermophilic digestion can be applied to reduce C. difficile in biosolids, thereby reducing the environmental burden of the enteric pathogen.
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Memory of Germinant Stimuli in Bacterial Spores
Wang, Shiwei; Faeder, James R.; Setlow, Peter
2015-01-01
ABSTRACT Bacterial spores, despite being metabolically dormant, possess the remarkable capacity to detect nutrients and other molecules in their environment through a biochemical sensory apparatus that can trigger spore germination, allowing the return to vegetative growth within minutes of exposure of germinants. We demonstrate here that bacterial spores of multiple species retain memory of transient exposures to germinant stimuli that can result in altered responses to subsequent exposure. The magnitude and decay of these memory effects depend on the pulse duration as well as on the separation time, incubation temperature, and pH values between the pulses. Spores of Bacillus species germinate in response to nutrients that interact with germinant receptors (GRs) in the spore’s inner membrane, with different nutrient types acting on different receptors. In our experiments, B. subtilis spores display memory when the first and second germinant pulses target different receptors, suggesting that some components of spore memory are downstream of GRs. Furthermore, nonnutrient germinants, which do not require GRs, exhibit memory either alone or in combination with nutrient germinants, and memory of nonnutrient stimulation is found to be more persistent than that induced by GR-dependent stimuli. Spores of B. cereus and Clostridium difficile also exhibit germination memory, suggesting that memory may be a general property of bacterial spores. These observations along with experiments involving strains with mutations in various germination proteins suggest a model in which memory is stored primarily in the metastable states of SpoVA proteins, which comprise a channel for release of dipicolinic acid, a major early event in spore germination. PMID:26604257
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Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells
Edwards, Adrianne N.; Karim, Samiha T.; Pascual, Ricardo A.; Jowhar, Lina M.; Anderson, Sarah E.; McBride, Shonna M.
2016-01-01
Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands. PMID:27833595
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Moist-Heat Resistance, Spore Aging, and Superdormancy in Clostridium difficile▿†
Rodriguez-Palacios, Alexander; LeJeune, Jeffrey T.
2011-01-01
Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log10 within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log10 within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log10, 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores. PMID:21398481
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Contribution of Spores to the Ability of Clostridium difficile To Adhere to Surfaces
Joshi, Lovleen Tina; Phillips, Daniel S.; Williams, Catrin F.; Alyousef, Abdullah
2012-01-01
Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species. PMID:22923404
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Bertolo, Lisa; Boncheff, Alexander G; Ma, Zuchao; Chen, Yu-Han; Wakeford, Terra; Friendship, Robert M; Rosseau, Joyce; Weese, J Scott; Chu, Michele; Mallozzi, Michael; Vedantam, Gayatri; Monteiro, Mario A
2012-06-01
Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a D-glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester-phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Crowther, Grace S; Baines, Simon D; Todhunter, Sharie L; Freeman, Jane; Chilton, Caroline H; Wilcox, Mark H
2013-01-01
First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.
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The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile
Hong, Huynh A; Ferreira, William T; Hosseini, Siamand; Anwar, Saba; Hitri, Krisztina; Wilkinson, Anthony J; Vahjen, Wilfried; Zentek, Jürgen; Soloviev, Mikhail; Cutting, Simon M
2017-01-01
Abstract Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease. PMID:28968845
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Weingarden, Alexa R; Chen, Chi; Zhang, Ningning; Graiziger, Carolyn T; Dosa, Peter I; Steer, Clifford J; Shaughnessy, Megan K; Johnson, James R; Sadowsky, Michael J; Khoruts, Alexander
2016-09-01
To test whether ursodeoxycholic acid (UDCA) is inhibitory to Clostridium difficile and can be used in the treatment of C. difficile-associated ileal pouchitis. The restoration of secondary bile metabolism may be the key mechanism for fecal microbiota transplantation (FMT) in treating recurrent C. difficile infections (RCDI). Therefore, it is possible that exogenous administration of inhibitory bile acids may be used directly as nonantibiotic therapeutics for this indication. The need for such a treatment alternative is especially significant in patients with refractory C. difficile-associated pouchitis, where the efficacy of FMT may be limited. We measured the ability of UDCA to suppress the germination and the vegetative growth of 11 clinical isolate strains of C. difficile from patients treated with FMT for RCDI. In addition, we used oral UDCA to treat a patient with RCDI pouchitis that proved refractory to multiple antibiotic treatments and FMT. UDCA was found to be inhibitory to the germination and the vegetative growth of all C. difficile strains tested. Fecal concentrations of UDCA from the patient with RCDI pouchitis exceeded levels necessary to inhibit the germination and the growth of C. difficile in vitro. The patient has remained infection free for over 10 months after the initiation of UDCA. UDCA can be considered as a therapeutic option in patients with C. difficile-associated pouchitis. Further studies need to be conducted to define the optimal dose and duration of such a treatment. In addition, bile acid derivatives inhibitory to C. difficile that are able to achieve high intracolonic concentrations may be developed as therapeutics for RCDI colitis.
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Microbiologic factors affecting Clostridium difficile recurrence.
Chilton, C H; Pickering, D S; Freeman, J
2018-05-01
Recurrent Clostridium difficile infection (rCDI) places a huge economic and practical burden on healthcare facilities. Furthermore, rCDI may affect quality of life, leaving patients in an rCDI cycle and dependant on antibiotic therapy. To discuss the importance of microbiologic factors in the development of rCDI. Literature was drawn from a search of PubMed from 2000 onwards with the search term 'recurrent Clostridium difficile infection' and further references cited within these articles. Meta-analyses and systematic reviews have shown that CDI and rCDI risk factors are similar. Development of rCDI is attendant on many factors, including immune status or function, comorbidities and concomitant treatments. Studies suggest that poor bacterial diversity is correlated with clinical rCDI. Narrow-spectrum gut microflora-sparing antimicrobials (e.g. surotomycin, cadazolid, ridinilazole) are in development for CDI treatment, while microbiota therapeutics (faecal microbiota transplantation, nontoxigenic C. difficile, stool substitutes) are increasingly being explored. rCDI can only occur when viable C. difficile spores are present, either within the gut lumen after infection or when reacquired from the environment. C. difficile spore germination can be influenced by gut environmental factors resulting from dysbiosis, and spore outgrowth may be affected stage by some antimicrobials (e.g. fidaxomicin, ramoplanin, oritavancin). rCDI is a significant challenge for healthcare professionals, requiring a multifaceted approach; optimized infection control to minimize reinfection; C. difficile-targeted antibiotics to minimize dysbiosis; and gut microflora restoration to promote colonization resistance. These elements should be informed by our understanding of the microbiologic factors involved in both C. difficile itself and the gut microbiome. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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Crowther, Grace S; Chilton, Caroline H; Todhunter, Sharie L; Nicholson, Scott; Freeman, Jane; Baines, Simon D; Wilcox, Mark H
2014-08-01
Biofilms are characteristic of some chronic or recurrent infections and this mode of growth tends to reduce treatment efficacy. Clostridium difficile infection (CDI) is associated with a high rate of recurrent symptomatic disease. The presence and behaviour of C. difficile within intestinal biofilms remains largely unexplored, but may factor in recurrent infection. A triple-stage chemostat gut model designed to facilitate the formation of intestinal biofilm was inoculated with a pooled human faecal emulsion. Bacterial populations were allowed to equilibrate before simulated CDI was induced by clindamycin (33.9 mg/L, four times daily, 7 days) and subsequently treated with vancomycin (125 mg/L, four times daily, 7 days). Indigenous gut microbiota, C. difficile total viable counts, spores, cytotoxin and antimicrobial activity in planktonic and biofilm communities were monitored during the 10 week experimental period. Vancomycin successfully treated the initial episode of simulated CDI, but ∼18 days after therapy cessation, recurrent infection occurred. Germination, proliferation and toxin production were evident within planktonic communities in both initial and recurrent CDI. In contrast, sessile C. difficile remained in dormant spore form for the duration of the experiment. The effects of and recovery from clindamycin and vancomycin exposure for sessile populations was delayed compared with responses for planktonic bacteria. Intestinal biofilms provide a potential reservoir for C. difficile spore persistence, possibly facilitating their dispersal into the gut lumen after therapeutic intervention, leading to recurrent infection. Therapeutic options for CDI could have increased efficacy if they are more effective against sessile C. difficile. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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An in silico evaluation of treatment regimens for recurrent Clostridium difficile infection
Blanco, Natalia; Foxman, Betsy; Malani, Anurag N.; Zhang, Min; Walk, Seth; Rickard, Alexander H.
2017-01-01
Background Clostridium difficile infection (CDI) is a significant nosocomial infection worldwide, that recurs in as many as 35% of infections. Risk of CDI recurrence varies by ribotype, which also vary in sporulation and germination rates. Whether sporulation/germination mediate risk of recurrence and effectiveness of treatment of recurring CDI remains unclear. We aim to assess the role of sporulation/germination patterns on risk of recurrence, and the relative effectiveness of the recommended tapered/pulsing regimens using an in silico model. Methods We created a compartmental in-host mathematical model of CDI, composed of vegetative cells, toxins, and spores, to explore whether sporulation and germination have an impact on recurrence rates. We also simulated the effectiveness of three tapered/pulsed vancomycin regimens by ribotype. Results Simulations underscored the importance of sporulation/germination patterns in determining pathogenicity and transmission. All recommended regimens for recurring CDI tested were effective in reducing risk of an additional recurrence. Most modified regimens were still effective even after reducing the duration or dosage of vancomycin. However, the effectiveness of treatment varied by ribotype. Conclusion Current CDI vancomycin regimen for treating recurrent cases should be studied further to better balance associated risks and benefits. PMID:28800598
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Spore formation and toxin production in Clostridium difficile biofilms.
Semenyuk, Ekaterina G; Laning, Michelle L; Foley, Jennifer; Johnston, Pehga F; Knight, Katherine L; Gerding, Dale N; Driks, Adam
2014-01-01
The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.
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Spore Formation and Toxin Production in Clostridium difficile Biofilms
Semenyuk, Ekaterina G.; Laning, Michelle L.; Foley, Jennifer; Johnston, Pehga F.; Knight, Katherine L.; Gerding, Dale N.; Driks, Adam
2014-01-01
The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection. PMID:24498186
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Clostridium difficile shows no trade-off between toxin and spore production within the human host.
Blanco, Natalia; Walk, Seth; Malani, Anurag N; Rickard, Alexander; Benn, Michele; Eisenberg, Marisa; Zhang, Min; Foxman, Betsy
2018-05-01
This study aimed to describe the correlation between Clostridium difficile spore and toxin levels within the human host. In addition, we assessed whether overgrowth of Candida albicans modified this association. We measured toxin, spore and Candida albicans levels among 200 successively collected stool samples that tested positive for C. difficile, and PCR ribotyped these C. difficile isolates. Analysis of variance and linear regression were used to test the association between spore and toxin levels. Kruskal-Wallis tests and t-tests were used to compare the association between spore or toxin levels and host, specimen, or pathogen characteristics. C. difficile toxin and spore levels were positively associated (P<0.001); this association did not vary significantly with C. albicans overgrowth [≥5 logs of C. albicans colony-forming units (c.f.u.) g -1 ]. However, ribotypes 027 and 078-126 were significantly associated with higher levels of toxin and spores, and C. albicans overgrowth. The strong positive association observed between in vivo levels of C. difficile toxin and spores suggests that patients with more severe C. difficile infections may have increased spore production, enhancing C. difficile transmission. Although, on average, spore levels were higher in toxin-positive samples than in toxin-negative/PCR-positive samples, spores were found in almost all toxin-negative samples. The ubiquity of spore production among toxin-negative and formed stool samples emphasizes the importance of following infection prevention and control measures for all C. difficile-positive patients during their entire hospital stay.
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Inactivation of Clostridium difficile spores by microwave irradiation.
Ojha, Suvash Chandra; Chankhamhaengdecha, Surang; Singhakaew, Sombat; Ounjai, Puey; Janvilisri, Tavan
2016-04-01
Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 10(7) CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time-temperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Warriner, K; Xu, C; Habash, M; Sultan, S; Weese, S J
2017-03-01
Clostridium difficile is a significant pathogen with over 300 000 cases reported in North America annually. Previously, it was thought that C. difficile was primarily a clinically associated infection. However, through the use of whole genome sequencing it has been revealed that the majority of cases are community acquired. The source of community-acquired C. difficile infections (CDI) is open to debate with foodborne being one route considered. Clostridium difficile fits the criteria of a foodborne pathogen with respect to being commonly encountered in a diverse range of foods that includes meat, seafood and fresh produce. However, no foodborne illness outbreaks have been directly linked to C. difficile there is also no conclusive evidence that its spores can germinate in food matrices. This does not exclude food as a potential vehicle but it is likely that the pathogen is also acquired through zoonosis and the environment. The most significant factor that defines susceptibility to CDI is the host microbiome and functioning immune system. In this respect, effective control can be exercised by reducing the environmental burden of C. difficile along with boosting the host defences against the virulent enteric pathogen. © 2016 The Society for Applied Microbiology.
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Evolutionary clade affects resistance of Clostridium difficile spores to Cold Atmospheric Plasma
NASA Astrophysics Data System (ADS)
Connor, Mairéad; Flynn, Padrig B.; Fairley, Derek J.; Marks, Nikki; Manesiotis, Panagiotis; Graham, William G.; Gilmore, Brendan F.; McGrath, John W.
2017-02-01
Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Spores produced by C. difficile are robust and can remain viable for months, leading to prolonged healthcare-associated outbreaks with high mortality. Exposure of C. difficile spores to a novel, non-thermal atmospheric pressure gas plasma was assessed. Factors affecting sporicidal efficacy, including percentage of oxygen in the helium carrier gas admixture, and the effect on spores from different strains representing the five evolutionary C. difficile clades was investigated. Strains from different clades displayed varying resistance to cold plasma. Strain R20291, representing the globally epidemic ribotype 027 type, was the most resistant. However all tested strains displayed a ~3 log reduction in viable spore counts after plasma treatment for 5 minutes. Inactivation of a ribotype 078 strain, the most prevalent clinical type seen in Northern Ireland, was further assessed with respect to surface decontamination, pH, and hydrogen peroxide concentration. Environmental factors affected plasma activity, with dry spores without the presence of organic matter being most susceptible. This study demonstrates that cold atmospheric plasma can effectively inactivate C. difficile spores, and highlights factors that can affect sporicidal activity.
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Nerandzic, Michelle M.; Rackaityte, Elze; Jury, Lucy A.; Eckart, Kevin; Donskey, Curtis J.
2013-01-01
Background Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe®) would reduce the burden of spores on skin. Methods Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths. Results Spore removal from hands was enhanced with Vashe soak (>2.5 log10 reduction) versus soap and water wash or soak (~2.0 log10 reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log10 spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin. Conclusions Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin. PMID:23844234
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Hasan, Jafrul A; Japal, Knoxley M; Christensen, Erick R; Samalot-Freire, Luisa C
2011-01-01
Clostridium difficile is a strict anaerobic spore-forming bacterium, and an increasingly common nosocomial pathogen. The U.S. Environmental Protection Agency (EPA) is responsible for the registration of disinfectants, including products designed to treat environmental surfaces contaminated with spores of C. difficile. Product efficacy data are required for registration; however, there is a lack of methodology for generating high-quality spore suspensions for evaluating product performance. As such, a study was carried out to select a suitable C. difficile strain and to develop a stand-alone method to prepare a spore suspension that meets specific criteria necessary for quantitative testing of disinfectants. The criteria are: (1) a spore titer of > 8 log10/mL, (2) > or = 90% spores to vegetative cells, and (3) resistance of spores (determined by viability) to 2.5 M hydrochloric acid (HCl). Several strains of C. difficile (toxigenic and nontoxigenic) were grown on various media (solid and liquid) for varying lengths of time to determine the best combination of incubation conditions and media to optimize spore production and quality. Once the spore production procedure was optimized, a toxigenic strain of C. difficile [American Type Culture Collection (ATCC) 43598] was selected for use in trials to verify repeatability from one production run to the next. The spore suspension was initiated by spreading vegetative cells of C. difficile (ATCC 43598) on CDC anaerobic 5% sheep blood agar plates and incubating for 7-10 days at 36 +/- 1 degrees C under anaerobic conditions. Spores were harvested when > or = 90% of the cells converted to spores as determined by observation using phase-contrast microscopy. The spores were washed three times with saline-Tween-80, resuspended in cold deionized water, heated to 70 degrees C for 10 min, evaluated microscopically for quality, and enumerated on cycloserine-cefoxitin-fructose agar containing horse blood and taurocholate. The spore suspension was used to inoculate brushed stainless steel carriers (1 cm in diameter) with and without a soil load in accordance with the Standard Quantitative Carrier Disk Test Method (ASTM E-2197-02) to determine carrier load. Once it was determined that > 6 log10 spores/carrier could be recovered, spores were evaluated for resistance to HCI. The sporulation method presented in this report is simple and repeatable and results in spore suspension of high titer (> 8 log10/mL) and quality (> or = 90% spores to vegetative cells) that met acid resistance criteria (spores were resistant to 2.5 M HCI for 10 min). In addition, recovery from brushed stainless steel carriers with and without soil load was > 6 log10 spores/carrier. A 6 log10 performance standard was set forth in the EPA's interim guidance for generating data to support a label claim for effectiveness against C. difficile spores on hard, nonporous surfaces. This precollaborative investigation successfully demonstrated the use of a methodology for in vitro production of C. difficile spores (ATCC 43598) necessary for conducting efficacy tests. A proposal will be submitted to the AOAC INTERNATIONAL Methods Committee on Antimicrobial Efficacy Testing for a collaborative study; see Appendix.
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Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.
Tseng, Shawn; Abramzon, Nina; Jackson, James O; Lin, Wei-Jen
2012-03-01
Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.
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Zhang, Pengfei; Liang, Jintao; Yi, Xuan; Setlow, Peter
2014-01-01
Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (Trelease) greater than the time of germinant removal (Tb) increased, while the time Tlag − Tb, where Tlag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔTrelease = Trelease − Tlag) were increased; Factors affecting average Trelease values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants. PMID:24769693
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Improving culture media for the isolation of Clostridium difficile from compost.
Dharmasena, Muthu; Jiang, Xiuping
2018-06-01
This study was to optimize the detection methods for Clostridium difficile from the animal manure-based composts. Both autoclaved and unautoclaved dairy composts were inoculated with a 12-h old suspension of a non-toxigenic C. difficile strain (ATCC 43593) and then plated on selected agar for vegetative cells and endospores. Six types of enrichment broths supplemented with taurocholate and l-cysteine were assessed for detecting a low level of artificially inoculated C. difficile (ca. 5 spores/g) from dairy composts. The efficacy of selected enrichment broths was further evaluated by isolating C. difficile from 29 commercial compost samples. Our results revealed that using heat-shock was more effective than using ethanol-shock for inducing endospore germination, and the highest endospore count (p < 0.05) was yielded at 60 °C for 25 min. C. difficile agar base, supplemented with 0.1% l-cysteine, 7% defibrinated horse blood, and cycloserine-cefoxitin (CDA-CYS-H-CC agar) was the best medium (p < 0.05) for recovering vegetative cells from compost. C. difficile endospore populations from both types of composts enumerated on both CDA-CYS-H-CC agar supplemented with 0.1% sodium taurocholate (CDA-CYS-H-CC-T agar) and brain heart infusion agar supplemented with 0.5% yeast extract, 0.1% l-cysteine, cycloserine-cefoxitin, and 0.1% sodium taurocholate (BHIA-YE-CYS-CC-T agar) media were not significantly different from each other (p > 0.05). Overall, enrichment of inoculated compost samples in broths containing moxalactam-norfloxacin (MN) produced significantly higher (p < 0.05) spore counts than in non-selective broths or broths supplemented with CC. Enrichment in BHIB-YE-CYS-MN-T broth followed by culturing on an agar containing 7% horse blood and 0.1% taurocholate provided a more sensitive and selective combination of media for detecting a low population of C. difficile from environmental samples with high background microflora. Copyright © 2018 Elsevier Ltd. All rights reserved.
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New amino acid germinants for spores of the enterotoxigenic Clostridium perfringens type A isolates.
Udompijitkul, Pathima; Alnoman, Maryam; Banawas, Saeed; Paredes-Sabja, Daniel; Sarker, Mahfuzur R
2014-12-01
Clostridium perfringens spore germination plays a critical role in the pathogenesis of C. perfringens-associated food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Germination is initiated when bacterial spores sense specific nutrient germinants (such as amino acids) through germinant receptors (GRs). In this study, we aimed to identify and characterize amino acid germinants for spores of enterotoxigenic C. perfringens type A. The polar, uncharged amino acids at pH 6.0 efficiently induced germination of C. perfringens spores; L-asparagine, L-cysteine, L-serine, and L-threonine triggered germination of spores of most FP and NFB isolates; whereas, L-glutamine was a unique germinant for FP spores. For cysteine- or glutamine-induced germination, gerKC spores (spores of a gerKC mutant derivative of FP strain SM101) germinated to a significantly lower extent and released less DPA than wild type spores; however, a less defective germination phenotype was observed in gerAA or gerKB spores. The germination defects in gerKC spores were partially restored by complementing the gerKC mutant with a recombinant plasmid carrying wild-type gerKA-KC, indicating that GerKC is an essential GR protein. The gerKA, gerKC, and gerKB spores germinated significantly slower with L-serine and L-threonine than their parental strain, suggesting the requirement for these GR proteins for normal germination of C. perfringens spores. In summary, these results indicate that the polar, uncharged amino acids at pH 6.0 are effective germinants for spores of C. perfringens type A and that GerKC is the main GR protein for germination of spores of FP strain SM101 with L-cysteine, L-glutamine, and L-asparagine. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile
Hong, Huynh A.; Hitri, Krisztina; Hosseini, Siamand; Kotowicz, Natalia; Bryan, Donna; Mawas, Fatme; Wilkinson, Anthony J.; van Broekhoven, Annie; Kearsey, Jonathan
2017-01-01
ABSTRACT Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26–39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26–39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26–39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile. These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26–39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI. PMID:28167669
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Hospital Clostridium difficile outbreak linked to laundry machine malfunction.
Sooklal, Shelini; Khan, Ayesha; Kannangara, Saman
2014-06-01
Clostridium difficile is a gram-positive, spore-forming anaerobic bacillus that is associated with diarrheal disease. C difficile is shed in the feces of affected individuals and its spores can survive on surfaces for prolonged periods of time. These spores can contaminate a hospital environment by spread through health care workers and suboptimal environmental cleaning practices. We report an outbreak of health care facility-onset C difficile infection that was eventually linked to contaminated mop pads after a laundry machine malfunction. Copyright © 2014 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.
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The housefly Musca domestica as a mechanical vector of Clostridium difficile.
Davies, M P; Anderson, M; Hilton, A C
2016-11-01
Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/- 66.9 for vegetative cell suspension and 288.2 +/- 83.2 for spore suspension). After 1h, this had reduced (21.2 +/- 11.4 for vegetative cell suspension and 19.9 +/- 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/- 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/- 0.58. C. difficile could be recovered from fly excreta for up to 96h. This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.
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Krawczyk, Antonina O; de Jong, Anne; Omony, Jimmy; Holsappel, Siger; Wells-Bennik, Marjon H J; Kuipers, Oscar P; Eijlander, Robyn T
2017-04-01
Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn 1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K + (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis , including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores. Copyright © 2017 American Society for Microbiology.
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Krawczyk, Antonina O.; de Jong, Anne; Omony, Jimmy; Holsappel, Siger; Wells-Bennik, Marjon H. J.; Eijlander, Robyn T.
2017-01-01
ABSTRACT Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA2mob required higher HA temperatures for efficient germination than spores lacking spoVA2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores. PMID:28130296
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REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES1
Lee, W. H.; Ordal, Z. John
1963-01-01
Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207–217. 1963.—It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates. Images PMID:16561987
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Warn, Peter; Thommes, Pia; Sattar, Abdul; Corbett, David; Flattery, Amy; Zhang, Zuo; Black, Todd; Hernandez, Lorraine D; Therien, Alex G
2016-11-01
Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Janezic, Sandra; Mlakar, Sabina; Rupnik, Maja
2018-04-23
Clostridium difficile is an anaerobic, spore-forming bacterium that causes intestinal infections. Although C. difficile is still predominantly considered as a nosocomial pathogen, there has been an increase in the number of community-associated infections. Since C. difficile is ubiquitous and can be isolated from nearly any environment, one of the possibilities for community acquisition could be exposure to spores in the domestic environment. The aim of this study was to evaluate the presence of C. difficile spores on shoes, slippers and on dog paws and to explore the importance of these surfaces as vectors for the dissemination of C. difficile in a domestic environment. Overall, C. difficile was present in 14 (70%) of 20 households and in 31 of 90 (34%) collected samples. Shoes and slippers had the highest positivity rates, 19 of 44 (43%) and 6 of 21 (28%), respectively, followed by dog paws 6 of 25 (24%). Thirteen C. difficilePCR ribotypes were identified with half of the isolates belonging to ribotype 014/020, which is the predominant type circulating in human population and is also commonly found in the environment (e.g. soil and water) in Slovenia. In three households, identical PCR ribotypes were found on dog paws, shoes and slippers. To understand the fine-scale genetic relatedness of these isolates, we sequenced the genomes. Low level of single nucleotide variant (SNV) differences between isolates from the same households, consistent with a recent transmission from a common source, were seen for isolates of PCR ribotype 014/020 but not for PCR ribotype 010. Our results suggest that shoe soles and dog paws could serve for the dissemination of C. difficile spores between households and environment and could contribute to community-relevant sources for C. difficile infection in humans. © 2018 Blackwell Verlag GmbH.
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Alnoman, Maryam; Udompijitkul, Pathima; Banawas, Saeed; Sarker, Mahfuzur R
2018-02-01
Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Kumar, D.; Jaiswal, R. K.
2005-01-01
Application of fertilizers such as urea, diammonium phosphate (DAP) and muriate of potash in soil adversely affected the spore germination of Arthrobotrys dactyloides. Amendment of soil with urea at the concentrations of 1.0%, 0.5% and 0.1% completely inhibited spore germination and direct trap formation on the conidium, whereas muriate of potash delayed and reduced the spore germination even at the lowest concentration. DAP also inhibited spore germination at 1.0% concentration, while at lower concentration the percentage of spore germination was reduced. Application of neem cake at the concentration of 0.5% also inhibited spore germination after 24 h of amendment. The inhibitory effect of neem cake was reduced after 15 days of amendment, while after 30 days after amendment the inhibitory effect was completely lost and the spore germinated by direct trap as in unamended soil. Nematodes were not attracted to ungerminated spores after 24 h of amendment. After 15 days of amendment nematodes were attracted to agar blocks containing fewer germinated spores after 24 h of incubation but after 48 h of incubation large number of nematodes were attracted and trapped by the germinated spores with direct traps. After 30 days of amendment, larger number of nematodes were attracted and trapped by direct traps. PMID:24049500
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Nerandzic, Michelle M; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J
2016-01-01
Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5-2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200-2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin.
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Bryophyte spore germinability is inhibited by peatland substrates
NASA Astrophysics Data System (ADS)
Bu, Zhao-Jun; Li, Zhi; Liu, Li-Jie; Sundberg, Sebastian; Feng, Ya-Min; Yang, Yun-He; Liu, Shuang; Song, Xue; Zhang, Xing-Lin
2017-01-01
Bryophyte substrates and species may affect spore germination through allelopathy. Polytrichum strictum is currently expanding in peatlands in north-eastern China - is this an effect of its superior spore germinability or do its gametophytes have a stronger allelopathic effect than do Sphagnum? We conducted a spore burial experiment to test the effect of species identity, substrate and water table depth (WTD) on spore germinability and bryophyte allelopathic effect with P. strictum and two Sphagnum species (S. palustre and S. magellanicum). After 5 months of burial during a growing season, the spores were tested for germinability. Allelopathic effect of bryophyte substrates was assessed by the difference between spore germinability after being stored inside or outside the substrates. After burial, more than 90% of the spores lost their germinability across all three species due to ageing and allelopathy. Spore germinability differed among species, where the spores in S. palustre had a higher germination frequency than those in P. strictum. The three bryophytes maintained a higher germinability in Sphagnum than in Polytrichum hummocks, probably due to a stronger allelopathic effect of P. strictum. Water table drawdown by 10 cm increased germinability by more than 60% across the three species. The study indicates that P. strictum does not possess an advantage regarding spore germination but rather its gametophytes have a stronger allelopathic effect. Due to the weaker inhibitive effect of Sphagnum gametophytes, P. strictum may have a potential establishment superiority over Sphagnum in peatlands, in addition to a better drought tolerance, which may explain its current expansion.
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Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation
Borch-Pedersen, Kristina; Mellegård, Hilde; Reineke, Kai; Boysen, Preben; Sevenich, Robert; Lindbäck, Toril
2017-01-01
ABSTRACT Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry. PMID:28476768
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Macleod-Glover, Nora; Sadowski, Cheryl
2010-05-01
To review the evidence for the efficacy of products used for environmental or hand cleaning on the rates of Clostridium difficile-associated diarrhea (CDAD). MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews were searched for articles pertinent to the efficacy of cleaning products against C. difficile or studies with outcomes related to rates of CDAD. Evidence was level II. Minimizing the incidence of CDAD in geriatric rehabilitation units is essential to achieving the goals of increasing patient function and independence for discharge into the community. Attention to environmental control of C. difficile and its spores by health care workers and patient visitors is an important secondary prevention strategy. Chlorine-releasing agents are more effective than detergents for killing spores produced by C. difficile. No level I evidence is available to determine if the use of chlorine-releasing agents has an effect on rates of CDAD. Hand-washing is currently the recommended strategy for reducing transmission of C. difficile. Alcohol gels do not inactivate C. difficile spores; however, increased use of alcohol hand gel has not been associated with higher rates of CDAD.
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2015-06-19
animal waste an~ decompositiOn DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. UNCLASSIFIED PR-15-306 Anthrax...influx of water. Ungerminated spore Germination Germinated spore Spore hydratation ~ Non-refractile spore Refractile spore • Fluorescence
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Zhou, Tingting; Dong, Zhiyang; Setlow, Peter; Li, Yong-qing
2013-01-01
Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T lag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T release, and ΔT release (T release – T lag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T lag values. 4) Values of T lag and T release were heterogeneous among individual spores, but ΔT release values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T lag values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species. PMID:24058645
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Slawecki, Richard A.; Ryan, Eileen P.; Young, David H.
2002-01-01
Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC50s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC50s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi. PMID:11823196
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Nerandzic, Michelle M.; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J.
2016-01-01
Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5–2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200–2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin. PMID:26885539
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Nerandzic, Michelle M.; Sunkesula, Venkata C. K.; C., Thriveen Sankar; Setlow, Peter; Donskey, Curtis J.
2015-01-01
Background Due to their efficacy and convenience, alcohol-based hand sanitizers have been widely adopted as the primary method of hand hygiene in healthcare settings. However, alcohols lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We hypothesized that sporicidal activity could be induced in alcohols through alteration of physical or chemical conditions that have been shown to degrade or allow penetration of spore coats. Principal Findings Acidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C. difficile, and to a lesser extent Bacillus thuringiensis and Bacillus subtilis. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature. On skin, sporicidal ethanol formulations were as effective as soap and water hand washing in reducing levels of C. difficile spores. Conclusions These findings demonstrate that novel ethanol-based sporicidal hand hygiene formulations can be developed through alteration of physical and chemical conditions. PMID:26177038
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Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile
Edwards, Adrianne N.; Nawrocki, Kathryn L.
2014-01-01
The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. PMID:25069979
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Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments.
Nagler, Katja; Julius, Christina; Moeller, Ralf
2016-07-01
In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies. Bacillus species-Spores-Germination-High salinity-Salt stress-NaCl-Inhibition. Astrobiology 16, 500-512.
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This document provides an update to the Agency’s interim guidance for the efficacy evaluation of antimicrobial pesticides that are labeled for treating hard non-porous surfaces in healthcare settings contaminated with spores of Clostridium difficile.
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Growth from spores of Clostridium perfringens in the presence of sodium nitrite.
Labbe, R G; Duncan, C L
1970-02-01
The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.
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Reversible Inhibition of Spore Germination by Alcohols 1
Trujillo, Ralph; Laible, Nancy
1970-01-01
Low levels of alcohols have been found to inhibit the process of spore germination. The extent of germination is dependent upon the concentration of alcohol present in the germinating medium. This inhibition is reversible since removal of the alcohol from the spore environment allows germination to proceed. PMID:4993360
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Strategy to inactivate Clostridium perfringens spores in meat products.
Akhtar, Saeed; Paredes-Sabja, Daniel; Torres, J Antonio; Sarker, Mahfuzur R
2009-05-01
The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100-200 MPa, 7 min) and elevated temperature (80 degrees C, 10 min); spore germination at high temperatures (55, 60 or 65 degrees C); and inactivation of germinated spores with elevated temperatures (80 and 90 degrees C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 degrees C, 10 min). Low pressures (100-200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 degrees C, 10 min), and germinated at temperatures lethal for vegetative cells (>or= 55 degrees C) when incubated for 60 min with a mixture of L-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (approximately 4 decimal reduction) in meat by elevated temperatures (80-90 degrees C for 20 min) required a long germination period (55 degrees C for 60 min). However, similar inactivation level was reached with shorter germination period (55 degrees C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 degrees C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 degrees C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 degrees C in about 20 min and further incubation at 55 degrees C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 degrees C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.
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Clostridium difficile Drug Pipeline: Challenges in Discovery and Development of New Agents
2015-01-01
In the past decade Clostridium difficile has become a bacterial pathogen of global significance. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals. In response to the increasing medical burden, a new C. difficile antibiotic, fidaxomicin, was approved in 2011 for the treatment of C. difficile-associated diarrhea. Rudimentary fecal transplants are also being trialed as effective treatments. Despite these advances, therapies that are more effective against C. difficile spores and less damaging to the resident gastrointestinal microbiome and that reduce recurrent disease are still desperately needed. However, bringing a new treatment for C. difficile infection to market involves particular challenges. This review covers the current drug discovery pipeline, including both small molecule and biologic therapies, and highlights the challenges associated with in vitro and in vivo models of C. difficile infection for drug screening and lead optimization. PMID:25760275
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Clostridium difficile infection: molecular pathogenesis and novel therapeutics
Rineh, Ardeshir; Kelso, Michael J; Vatansever, Fatma; Tegos, George P; Hamblin, Michael R
2015-01-01
The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed. PMID:24410618
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Hagerman, A E; Blau, D M; McClure, A L
1985-12-01
A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.
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Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments
NASA Astrophysics Data System (ADS)
Nagler, Katja; Julius, Christina; Moeller, Ralf
2016-07-01
In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies.
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Sokołowska, Barbara; Skapska, Sylwia; Fonberg-Broczek, Monika; Niezgoda, Jolanta; Porebska, Izabela; Dekowska, Agnieszka; Rzoska, Sylwester J
2015-01-01
Given the importance of spoilage caused by Alicyclobacillus acidoterrestris for the fruit juice industry, the objective of this work was to study the germination and inactivation of A. acidoterrestris spores induced by moderate hydrostatic pressure. Hydrostatic pressure treatment can induce the germination and inactivation of A. acidoterrestris spores. At low pH, spore germination of up to 3.59-3.75 log and inactivation of 1.85-2.04 log was observed in a low pressure window (200-300 MPa) applied at 50 degrees C for 20 min. Neutral pH suppressed inactivation, the number of spores inactivated at pH 7.0 was only 0.24-1.06 log. The pressurization temperature significantly affected spore germination and inactivation. The degree of germination in apple juice after pressurization for 30 min with 200 MPa at 20 degrees C was 2.04 log, with only 0.61 log of spores being inactivated, while at 70 degrees C spore germination was 5.94 log and inactivation 4.72 log. This temperature strongly stimulated germination and inactivation under higher (500 MPa) than lower (200 MPa) pressure. When the oscillatory mode was used, the degree of germination and inactivation was slightly higher than at continuous mode. The degree of germination and inactivation was inversely proportional to the soluble solids content and was lowest in concentrated apple juice.
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Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile.
Edwards, Adrianne N; Nawrocki, Kathryn L; McBride, Shonna M
2014-10-01
The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Endotrophic Calcium, Strontium, and Barium Spores of Bacillus megaterium and Bacillus cereus1
Foerster, Harold F.; Foster, J. W.
1966-01-01
Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333–1345. 1966.—Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl2, SrCl2, or BaCl2. Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed “coat fraction” from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH. Images PMID:4956334
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2014-01-01
wild-type spores but ~15-fold higher deltaTrelease values; v ) germination kinetics of wild-type spores given a ? 30 sec 140 MPa HP pulse followed by...15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and ( v ) the germination of wild-type...committed spores, as it does for nutrient-committed spores (14)? ( v ) Can these HP-com- mitted spores be isolated under conditions that do not allow
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Efficacy of cleaning products for C difficile
MacLeod-Glover, Nora; Sadowski, Cheryl
2010-01-01
Abstract OBJECTIVE To review the evidence for the efficacy of products used for environmental or hand cleaning on the rates of Clostridium difficile–associated diarrhea (CDAD). QUALITY OF EVIDENCE MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews were searched for articles pertinent to the efficacy of cleaning products against C difficile or studies with outcomes related to rates of CDAD. Evidence was level II. MAIN MESSAGE Minimizing the incidence of CDAD in geriatric rehabilitation units is essential to achieving the goals of increasing patient function and independence for discharge into the community. Attention to environmental control of C difficile and its spores by health care workers and patient visitors is an important secondary prevention strategy. CONCLUSION Chlorine-releasing agents are more effective than detergents for killing spores produced by C difficile. No level I evidence is available to determine if the use of chlorine-releasing agents has an effect on rates of CDAD. Hand-washing is currently the recommended strategy for reducing transmission of C difficile. Alcohol gels do not inactivate C difficile spores; however, increased use of alcohol hand gel has not been associated with higher rates of CDAD. PMID:20463269
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Etienne-Mesmin, Lucie; Chassaing, Benoit; Adekunle, Oluwaseyi; Mattei, Lisa M; Bushman, Frederic D; Gewirtz, Andrew T
2018-05-01
Clostridium difficile is a toxin-producing bacterium and a leading cause of antibiotic-associated disease. The ability of C. difficile to form spores and infect antibiotic-treated persons at low multiplicity of infection (MOI) underlies its large disease burden. However, C. difficile -induced disease might also result from long-harboured C. difficile that blooms in individuals administered antibiotics. Mice purchased from multiple vendors and repeatedly testing negative for this pathogen by quantitative PCR bloomed C. difficile following antibiotic treatment. This endogenous C. difficile strain, herein termed LEM1, which formed spores and produced toxin, was compared with highly pathogenic C. difficile strain VPI10463. Whole-genome sequencing revealed that LEM1 and VPI10463 shared 95% of their genes, including all known virulence genes. In contrast to VPI10463, LEM1 did not induce overt disease when administered to antibiotic-treated or germ-free mice, even at high doses. Rather, blooms of LEM1 correlated with survival following VPI10463 inoculation, and exogenous administration of LEM1 before or shortly following VPI10463 inoculation prevented C. difficile -induced death. Accordingly, despite similar growth properties in vitro, LEM1 strongly outcompeted VPI10463 in mice even at 100-fold lower inocula. These results highlight the difficulty of determining whether individual cases of C. difficile infection resulted from a bloom of endogenous C. difficile or a new exposure to this pathogen. In addition to impacting the design of studies using mouse models of C. difficile -induced disease, this study identified, isolated and characterised an endogenous murine spore-forming C. difficile strain able to decrease colonisation, associated disease and death induced by a pathogenic C. difficile strain. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
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Mechanisms of Bacterial Spore Germination and Its Heterogeneity
2015-01-10
mathematical model describing spore germination has been developed; 9) much of the work above has been extended to Clostridium spores; and 10) ~90...germination. C) Faeder lab, with Li and Setlow labs. We have developed a mathematical model of bacterial spore germination that accounts for...heterogeneity in both Tlag and commitment times. The model is built from three main mathematical components: a receptor distribution function
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Santoro, T.; Stotzky, G.; Rem, L. T.
1967-01-01
Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores. PMID:6069161
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Wang, Shiwei; Yu, Jing; Suvira, Milomir; Setlow, Peter; Li, Yong-qing
2015-01-01
Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis). PMID:26636757
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Suo, Jinwei; Zhao, Qi; Zhang, Zhengxiu; Chen, Sixue; Cao, Jian'guo; Liu, Guanjun; Wei, Xing; Wang, Tai; Yang, Chuanping; Dai, Shaojun
2015-09-01
Fern spore is a good single-cell model for studying the sophisticated molecular networks in asymmetric cell division, differentiation, and polar growth. Osmunda cinnamomea L. var. asiatica is one of the oldest fern species with typical separate-growing trophophyll and sporophyll. The chlorophyllous spores generated from sporophyll can germinate without dormancy. In this study, the spore ultrastructure, antioxidant enzyme activities, as well as protein and gene expression patterns were analyzed in the course of spore germination at five typical stages (i.e. mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Proteomic analysis revealed 113 differentially expressed proteins, which were mainly involved in photosynthesis, reserve mobilization, energy supplying, protein synthesis and turnover, reactive oxygen species scavenging, signaling, and cell structure modulation. The presence of multiple proteoforms of 25 differentially expressed proteins implies that post-translational modification may play important roles in spore germination. The dynamic patterns of proteins and their encoding genes exhibited specific characteristics in the processes of cell division and rhizoid tip growth, which include heterotrophic and autotrophic metabolisms, de novo protein synthesis and active protein turnover, reactive oxygen species and hormone (brassinosteroid and ethylene) signaling, and vesicle trafficking and cytoskeleton dynamic. In addition, the function skew of proteins in fern spores highlights the unique and common mechanisms when compared with evolutionarily divergent spermatophyte pollen. These findings provide an improved understanding of the typical single-celled asymmetric division and polar growth during fern spore germination. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Kinetics of Germination of Bacillus Spores1
Vary, J. C.; Halvorson, H. O.
1965-01-01
Vary, J. C. (University of Wisconsin, Madison), and H. O. Halvorson. Kinetics or germination of Bacillus spores. J. Bacteriol. 89:1340–1347. 1965.—The kinetics of germination of Bacillus cereus strain T spores was accurately described by McCormick. To study the mechanism of germination, it is necessary to correlate the characteristic changes in a population of germinating spores with the behavior of the individual spores in the same population. Two microscopic events are apparent during germination: microlag, the time interval between the addition of l-alanine to heat-activated spores and the beginning of loss in refractility, and microgermination time, the time for the actual change in refractility to occur. The frequency distributions of both events are skewed, and appear to be independent. The effects of l-alanine concentration, heat activation, and temperature of germination on three parameters, microlag, microgermination, and per cent germination, were microscopically studied. The data are discussed in relation to the mechanism of germination, and a correlation between microlag and microgermination times with the constants of McCormick's equation has been suggested. Images PMID:14293008
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Omotade, T O; Bernhards, R C; Klimko, C P; Matthews, M E; Hill, A J; Hunter, M S; Webster, W M; Bozue, J A; Welkos, S L; Cote, C K
2014-12-01
Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...
2015-07-30
Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less
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Cytological and proteomic analyses of horsetail (Equisetum arvense L.) spore germination
Zhao, Qi; Gao, Jing; Suo, Jinwei; Chen, Sixue; Wang, Tai; Dai, Shaojun
2015-01-01
Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems to understand the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be abundance changed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as proteoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of 14-3-3 protein, small G protein Ran, actin, and caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings lay a foundation toward understanding the molecular mechanisms underlying fern spore asymmetric division and rhizoid polar growth. PMID:26136760
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Peng, Lixin; Chen, De; Setlow, Peter; Li, Yong-qing
2009-01-01
Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade spores’ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased ∼15% and in the second phase ending at Trelease, remaining CaDPA was released rapidly; 2) in L-alanine germination of wild-type spores and spores lacking SleB: a) the ESLI rose ∼2-fold shortly before Tlag at T1; b) following Tlag, the ESLI again rose ∼2-fold at T2 when CaDPA levels had decreased ∼50%; and c) the ESLI reached its maximum value at ∼Trelease and then decreased; 3) in CaDPA germination of wild-type spores: a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and L-alanine germination; b) at Trelease the ESLI again reached its maximum value; 4) in L-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time ΔTrelease (Trelease–Tlag) for excretion of ≥75% of CaDPA was ∼15-fold higher than that for wild-type or sleB spores; and 5) spores lacking only CwlJ exhibited a similar, but not identical ESLI pattern during L-alanine germination to that seen with cwlJ sleB spores, and the high value for ΔTrelease. PMID:19374431
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The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile.
Childress, Kevin O; Edwards, Adrianne N; Nawrocki, Kathryn L; Anderson, Sarah E; Woods, Emily C; McBride, Shonna M
2016-12-01
The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile
Childress, Kevin O.; Edwards, Adrianne N.; Nawrocki, Kathryn L.; Anderson, Sarah E.; Woods, Emily C.
2016-01-01
The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species. PMID:27647869
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NASA Technical Reports Server (NTRS)
Raghavan, V.
1992-01-01
Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.
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Ishikawa, Shu; Yamane, Kunio; Sekiguchi, Junichi
1998-01-01
The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by EςE RNA polymerase. cwlJ-deficient spores responded to both l-alanine and AGFK, the A580 values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A580 and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed. PMID:9515903
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Simmons, Richard J.; Costilow, Ralph N.
1962-01-01
Simmons, R. J. (Michigan State University, East Lansing), and R. N. Costilow. Enzymes of glucose and pyruvate catabolism in cells, spores, and germinated spores of Clostridium botulinum. J. Bacteriol. 84:1274–1281. 1962.—An investigation was made of the enzymes of vegetative cells, spores, and germinated spores of Clostridium botulinum 62-A to elucidate a pathway of glucose metabolism. Manometric studies were conducted with intact cells, and various enzymes and enzyme systems were assayed in cell-free and spore-free extracts by use of spectrophotometric and colorimetric procedures. Glucose fermentation was found to be inducible; glucokinase was the controlling enzyme. All other enzymes of the Embden-Meyerhof-Parnas (EMP) pathway were found in both induced and non-induced cells, but they were in relatively low concentrations in the latter. This, plus the fact that no glucose-6-phosphate dehydrogenase was detected, led to the conclusion that glucose is catabolized primarily by the EMP system. A number of glycolytic enzymes were also found in extracts of spores and germinated spores of this organism, but the activities were extremely low as compared with activities in cell extracts. A phosphoroclastic-type reaction was readily demonstrated in both glucose-adapted and non-adapted cells, but not in spores and germinated spores. However, both acetokinase and phosphotransacetylase, as well as coenzyme A transphorase, were detected in spores and germinated-spore extracts, although at very low activity levels as compared with cell extracts. The specific activity of diaphorase in spore extracts was about one-half that of corresponding cell extracts, and the activity of reduced diphosphopyridine nucleotide (DPNH) oxidase was actually higher in the spore extracts. In addition, the DPNH oxidase in spore extracts was considerably more heat-stable than that in extracts of cells or germinated spores. PMID:13977433
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Rogge, G D; Viana, A M; Randi, A M
2000-01-01
Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores
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Fluorescence-based methods for the detection of pressure-induced spore germination and inactivation
NASA Astrophysics Data System (ADS)
Baier, Daniel; Reineke, Kai; Doehner, Isabel; Mathys, Alexander; Knorr, Dietrich
2011-03-01
The application of high pressure (HP) provides an opportunity for the non-thermal preservation of high-quality foods, whereas highly resistant bacterial endospores play an important role. It is known that the germination of spores can be initiated by the application of HP. Moreover, the resistance properties of spores are highly dependent on their physiological states, which are passed through during the germination. To distinguish between different physiological states and to detect the amount of germinated spores after HP treatments, two fluorescence-based methods were applied. A flow cytometric method using a double staining with SYTO 16 as an indicator for germination and propidium iodide as an indicator for membrane damage was used to detect different physiological states of the spores. During the first step of germination, the spore-specific dipicolinic acid (DPA) is released [P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003), pp. 550-556]. DPA reacts with added terbium to form a distinctive fluorescent complex. After measuring the fluorescence intensity at 270 nm excitation wavelength in a fluorescence spectrophotometer, the amount of germinated spores can be determined. Spores of Bacillus subtilis were treated at pressures from 150 to 600 MPa and temperatures from 37 °C to 60 °C in 0.05 M ACES buffer solution (pH 7) for dwell times of up to 2 h. During the HP treatments, inactivation up to 2log 10 cycles and thermal sensitive populations up to 4log 10 cycles could be detected by plate counts. With an increasing number of thermal sensitive spores, an increased proportion of spores in germinated states was detected by flow cytometry. Also the released amount of DPA increased during the dwell times. Moreover, a clear pressure-temperature-time-dependency was shown by screening different conditions. The fluorescence-based measurement of the released DPA can provide the opportunity of an online monitoring of the germination of spores under HP inside the HP vessel. Implementation can be done using diamond anvil cells, units with inspection glasses or by inserting an optical fiber into the HP vessel. The analytical methods used can help to understand the complex mechanism of germination and inactivation of bacterial spores. Due to its universal, process-independent character, the application of these methods is feasible for established and emerging technologies.
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Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis
2012-11-01
REPORT Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis 14. ABSTRACT 16. SECURITY CLASSIFICATION OF... Bacillus subtilis spores with a gerP mutation triggered spore germination via nutrient germinant receptors (GRs) slowly, although this defect was...gerP, Bacillus subtilis , dipicolinic acid Xuan Y. Butzin, Anthony J. Troiano, William H. Coleman, Keren K. Griffiths, Christopher J. Doona, Florence E
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Porębska, Izabela; Sokołowska, Barbara; Woźniak, Łukasz
2017-03-30
The presence of Alicyclobacillus, a thermoacidophilic and spore-forming bacterium, in acidic fruit juices poses a serious problem for the processing industry. A typical sign of spoilage in contaminated juices is a characteristic phenolic off-flavour associated with the production of guaiacol. Spores are formed in response to starvation and in a natural environment re-access the nutrients, e.g.: L-alanine and AGFK - a mixture of asparagine, glucose, fructose and potassium, triggers germination. The aim of this study was to estimate the impact of L-alanine and AGFK on the germination of the spores of two Alicyclobacillus acidoterrestris strains and to evaluate the relationship of the germination rate with dipicolinic acid (DPA) release. The spores were suspended in apple juice or in buffers at pH 4 and pH 7, followed by the addition of L-alanine and AGFK. Suspensions were or were not subjected, to a temperature of 80°C/10 min and incubated for various periods of time at 45°C. Optical density (OD660) was used to estimate the number of germinated spores. The amount of DPA released was determined using HPLC. The results indicate that the degree of germination of A. acidoterrestris spores depended on the strain and time of incubation and the nutritious compounds used. The data obtained show that the amount of DPA released correlated to the number of A. acidoterrestris spores germinated.
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Resistance to and killing by the sporicidal microbicide peracetic acid.
Leggett, Mark J; Schwarz, J Spencer; Burke, Peter A; Mcdonnell, Gerald; Denyer, Stephen P; Maillard, Jean-Yves
2015-03-01
To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA). Mutants of Bacillus subtilis lacking specific spore structures were used to identify resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination. The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity. PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.
Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less
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Szabo, Jeffrey G; Muhammad, Nur; Heckman, Lee; Rice, Eugene W; Hall, John
2012-04-01
Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone.
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Alvarado, Israel; Margotta, Joseph W; Aoki, Mai M; Flores, Fernando; Agudelo, Fresia; Michel, Guillermo; Elekonich, Michelle M; Abel-Santos, Ernesto
2017-09-01
Paenibacillus larvae, a Gram-positive bacterium, causes American foulbrood (AFB) in honey bee larvae (Apis mellifera Linnaeus [Hymenoptera: Apidae]). P. larvae spores exit dormancy in the gut of bee larvae, the germinated cells proliferate, and ultimately bacteremia kills the host. Hence, spore germination is a required step for establishing AFB disease. We previously found that P. larvae spores germinate in response to l-tyrosine plus uric acid in vitro. Additionally, we determined that indole and phenol blocked spore germination. In this work, we evaluated the antagonistic effect of 35 indole and phenol analogs and identified strong inhibitors of P. larvae spore germination in vitro. We further tested the most promising candidate, 5-chloroindole, and found that it significantly reduced bacterial proliferation. Finally, feeding artificial worker jelly containing anti-germination compounds to AFB-exposed larvae significantly decreased AFB infection in laboratory-reared honey bee larvae. Together, these results suggest that inhibitors of P. larvae spore germination could provide another method to control AFB. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
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Setlow, B; Cabrera-Martinez, R-M; Setlow, P
2004-01-01
To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species. Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH. This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.
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Measuring Total and Germinable Spore Populations
NASA Technical Reports Server (NTRS)
Noell, A.C.; Yung, P.T.; Yang, W.; Lee, C.; Ponce, A.
2011-01-01
It has been shown that bacterial endospores can be enumerated using a microscopy based assay that images the luminescent halos from terbium ions bound to dipicolinic acid, a spore specific chemical marker released upon spore germination. Further development of the instrument has simplified it towards automation while at the same time improving image quality. Enumeration of total spore populations has also been developed allowing measurement of the percentage of viable spores in any population by comparing the germinable/culturable spores to the total. Percentage viability will allow a more quantitative comparison of the ability of spores to survive across a wide range of extreme environments.
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Muhammad, Nur; Heckman, Lee; Rice, Eugene W.; Hall, John
2012-01-01
Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone. PMID:22267659
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2013-01-01
Background Bacillus anthracis is a pathogen that causes life-threatening disease--anthrax. B. anthracis spores are highly resistant to extreme temperatures and harsh chemicals. Inactivation of B. anthracis spores is important to ensure the environmental safety and public health. The 2001 bioterrorism attack involving anthrax spores has brought acute public attention and triggered extensive research on inactivation of B. anthracis spores. Single-walled carbon nanotubes (SWCNTs) as a class of emerging nanomaterial have been reported as a strong antimicrobial agent. In addition, continuous near infrared (NIR) radiation on SWCNTs induces excessive local heating which can enhance SWCNTs’ antimicrobial effect. In this study, we investigated the effects of SWCNTs coupled with NIR treatment on Bacillus anthracis spores. Results and discussion The results showed that the treatment of 10 μg/mL SWCNTs coupled with 20 min NIR significantly improved the antimicrobial effect by doubling the percentage of viable spore number reduction compared with SWCNTs alone treatment (88% vs. 42%). At the same time, SWCNTs-NIR treatment activated the germination of surviving spores and their dipicolinic acid (DPA) release during germination. The results suggested the dual effect of SWCNTs-NIR treatment on B. anthracis spores: enhanced the sporicidal effect and stimulated the germination of surviving spores. Molecular level examination showed that SWCNTs-NIR increased the expression levels (>2-fold) in 3 out of 6 germination related genes tested in this study, which was correlated to the activated germination and DPA release. SWCNTs-NIR treatment either induced or inhibited the expression of 3 regulatory genes detected in this study. When the NIR treatment time was 5 or 25 min, there were 3 out of 7 virulence related genes that showed significant decrease on expression levels (>2 fold decrease). Conclusions The results of this study demonstrated the dual effect of SWCNTs-NIR treatment on B. anthracis spores, which enhanced the sporicidal effect and stimulated the germination of surviving spores. SWCNTs-NIR treatment also altered the expression of germination, regulatory, and virulence-related genes in B. anthracis. PMID:23965258
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Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores
Bernhards, Casey B.
2014-01-01
The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. PMID:25022853
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Ghantoji, Shashank S.; Stibich, Mark; Stachowiak, Julie; Cantu, Sherry; Adachi, Javier A.; Raad, Issam I.
2015-01-01
The standard for Clostridium difficile surface decontamination is bleach solution at a concentration of 10 % of sodium hypochlorite. Pulsed xenon UV light (PX-UV) is a means of quickly producing germicidal UV that has been shown to be effective in reducing environmental contamination by C. difficile spores. The purpose of this study was to investigate whether PX-UV was equivalent to bleach for decontamination of surfaces in C. difficile infection isolation rooms. High-touch surfaces in rooms previously occupied by C. difficile infected patients were sampled after discharge but before and after cleaning using either bleach or non-bleach cleaning followed by 15 min of PX-UV treatment. A total of 298 samples were collected by using a moistened wipe specifically designed for the removal of spores. Prior to disinfection, the mean contamination level was 2.39 c.f.u. for bleach rooms and 22.97 for UV rooms. After disinfection, the mean level of contamination for bleach was 0.71 c.f.u. (P = 0.1380), and 1.19 c.f.u. (P = 0.0017) for PX-UV disinfected rooms. The difference in final contamination levels between the two cleaning protocols was not significantly different (P = 0.9838). PX-UV disinfection appears to be at least equivalent to bleach in the ability to decrease environmental contamination with C. difficile spores. Larger studies are needed to validate this conclusion. PMID:25627208
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Schelkle, Bettina; Choi, Young; Baillie, Leslie W; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa
2017-01-01
Remediation of Bacillus anthracis -contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Undeen, A.H.; Vander Meer, R.K.
Spores of Nosema algerae Vavra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm2, followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm2. Ultraviolet radiation between 1.1 and 3.4 J/cm2 stimulated spores to germinate. However, spores were completelymore » unable to germinate immediately after exposure to dosages above 3.8 J/cm2. Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.« less
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Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto
2010-01-01
Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305
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Bassi, Daniela; Cappa, Fabrizio; Cocconcelli, Pier Sandro
2009-06-01
Clostridium tyrobutyricum is an anaerobic bacterium responsible for late blowing defects during cheese ripening and it is of scientific interest for biological hydrogen production. A scanning electron microscopy (SEM) coating technique and X-ray microanalysis were developed to analyze the architecture and chemical composition of spores upon germination in response to environmental changes. In addition, we investigated the effects of different compounds on this process. Agents and environmental conditions inducing germination were characterized monitoring changes in optical density (OD). Among all tested conditions, the greatest drop in OD(625) (57.4%) was obtained when spores were incubated in l-alanine/l-lactate buffer, pH 4.6. In addition, a carbon-coating SEM technique and X-ray microanalysis were used to observe the architecture of spores and to examine calcium dipicolinate release. Conditions inducing C. tyrobutyricum spore germination were identified and SEM X-ray microanalysis clearly distinguished germinating from dormant spores. We confirmed that calcium dipicolinate release is one of the first events occurring. These microscopy methods could be considered sensitive tools for evaluating morphological and chemical changes in spores of C. tyrobutyricum during the initial phase of germination. Information gathered from this work may provide new data for further research on germination.
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Liu, Hualan; Ray, W Keith; Helm, Richard F; Popham, David L; Melville, Stephen B
2016-06-15
Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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[Survival of Bacillus anthracis spores in various tannery baths].
Mendrycka, M; Mierzejewski, J
2000-01-01
The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B. anthracis spores (Sterne strain) was investigated. The periods and the conditions of this influence were established according to technological process of cow hide tannage. Practically after every bath some part of the spores remained vital. The most effective killing of spores occurred after pickling, liming and deliming. Inversely, the most viable spores remained after bating and retannage process. The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing.
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Tauscher, Courtney; Schuerger, Andrew C; Nicholson, Wayne L
2006-08-01
Bacterial spores have been considered as microbial life that could survive interplanetary transport by natural impact processes or human spaceflight activity. Deposition of terrestrial microbes or their biosignature molecules onto the surface of Mars could negatively impact life detection experiments and planetary protection measures. Simulated Mars solar radiation, particularly the ultraviolet component, has been shown to reduce spore viability, but its effect on spore germination and resulting production of biosignature molecules has not been explored. We examined the survival and germinability of Bacillus subtilis spores exposed to simulated martian conditions that include solar radiation. Spores of B. subtilis that contain luciferase resulting from expression of an sspB-luxAB gene fusion were deposited on aluminum coupons to simulate deposition on spacecraft surfaces and exposed to simulated Mars atmosphere and solar radiation. The equivalent of 42 min of simulated Mars solar radiation exposure reduced spore viability by nearly 3 logs, while germination-induced bioluminescence, a measure of germination metabolism, was reduced by less than 1 log. The data indicate that spores can retain the potential to initiate germination-associated metabolic processes and produce biological signature molecules after being rendered nonviable by exposure to Mars solar radiation.
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A Waking Review: Old and Novel Insights into the Spore Germination in Streptomyces.
Bobek, Jan; Šmídová, Klára; Čihák, Matouš
2017-01-01
The complex development undergone by Streptomyces encompasses transitions from vegetative mycelial forms to reproductive aerial hyphae that differentiate into chains of single-celled spores. Whereas their mycelial life - connected with spore formation and antibiotic production - is deeply investigated, spore germination as the counterpoint in their life cycle has received much less attention. Still, germination represents a system of transformation from metabolic zero point to a new living lap. There are several aspects of germination that may attract our attention: (1) Dormant spores are strikingly well-prepared for the future metabolic restart; they possess stable transcriptome, hydrolytic enzymes, chaperones, and other required macromolecules stabilized in a trehalose milieu; (2) Germination itself is a specific sequence of events leading to a complete morphological remodeling that include spore swelling, cell wall reconstruction, and eventually germ tube emergences; (3) Still not fully unveiled are the strategies that enable the process, including a single cell's signal transduction and gene expression control, as well as intercellular communication and the probability of germination across the whole population. This review summarizes our current knowledge about the germination process in Streptomyces , while focusing on the aforementioned points.
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Fuchs, Felix M; Raguse, Marina; Fiebrandt, Marcel; Madela, Kazimierz; Awakowicz, Peter; Laue, Michael; Stapelmann, Katharina; Moeller, Ralf
2017-11-30
Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.
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Polarity of Spore Germination in Funaria hygrometrica Hedw.
NASA Astrophysics Data System (ADS)
Pundyak, O. I.; Demkiv, O. T.; Khorkavtsiv, O. Ya; Bagrii, B. B.
It is shown that in darkness the spores of moss Funaria hygrometrica Hedw. germinated polarly under the influence of gravity. At the beginning the rhizoids appeared. They grew downwards. Then future chloronematical stolons started to form a germination spore. Usually, they grew upwards. Clinorotation or horizontal placing of Petry dishes could discoordinate such a gravisensitivity.
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Duncan, Charles L.; Foster, E. M.
1968-01-01
The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%. Images Fig. 1 Fig. 2 Fig. 3 PMID:5645423
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Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen.
Awad, Milena M; Johanesen, Priscilla A; Carter, Glen P; Rose, Edward; Lyras, Dena
2014-01-01
The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.
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Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen
Awad, Milena M; Johanesen, Priscilla A; Carter, Glen P; Rose, Edward; Lyras, Dena
2014-01-01
The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions. PMID:25483328
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Sakanoue, Hideyo; Yasugi, Mayo; Miyake, Masami
2018-05-04
Sublethal heating of spores has long been known to stimulate or activate germination, but the underlying mechanisms are not yet fully understood. In this study, we visualized the entire germination-to-outgrowth process of spores from an anaerobic sporeformer, C. perfringens, at single-cell resolution. Quantitative analysis revealed that sublethal heating significantly reduced the time from completion of germination to the beginning of the first cell division. The results indicate that sublethal heating of C. perfringens spores not only sensitizes the responsiveness of germinant receptors but also directly or indirectly facilitates multiple steps during the bacterial regrowth process. © 2018 The Societies and John Wiley & Sons Australia, Ltd.
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NASA Astrophysics Data System (ADS)
Porębska, I.; Rutkowska, M.; Sokołowska, B.
2015-01-01
Alicyclobacillus acidoterrestris is a spore-forming bacterium, causing spoilage of juices. The spores of these bacteria have the ability to survive in the typical conditions used for thermal pasteurization. Therefore, the use of other techniques such as high hydrostatic pressure is considered for their inactivation. The effect of hydrostatic pressure of 200-500 MPa, at temperatures 4-50 °C for 15 min, on the dynamics of germination of A. acidoterrestris spores in apple juice and pH 4 buffer was studied. To estimate the share of germinated spores, the method of determining the optical density at a wavelength of 660 nm (OD660) was used. Parameters of hydrostatic pressure treatment used in this work affected the dynamics of germination of A. acidoterrestris spores in apple juice, and the temperature had the greatest effect. The results indicate that nutrients present in apple juice can promote the germination of A. acidoterrestris spores. This paper was presented at the 8th International Conference on High Pressure Bioscience & Biotechnology (HPBB 2014) in Nantes (France) 15-18 July 2014.
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Schelkle, Bettina; Choi, Young; Baillie, Leslie W.; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa
2018-01-01
Remediation of Bacillus anthracis-contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms. PMID:29379472
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Spore coat architecture of Clostridium novyi NT spores.
Plomp, Marco; McCaffery, J Michael; Cheong, Ian; Huang, Xin; Bettegowda, Chetan; Kinzler, Kenneth W; Zhou, Shibin; Vogelstein, Bert; Malkin, Alexander J
2007-09-01
Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.
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NASA Astrophysics Data System (ADS)
Komarova, A. S.; Likhacheva, A. A.; Lapygina, E. V.; Maksimova, I. A.; Pozdnyakov, A. I.
2010-01-01
The impact of microwaves on the germination of spores of Streptomyces xanthochromogenes in a liquid nutrient medium and in a peat soil was studied. The treatment of inoculums with microwave radiation affected the development of the microorganisms from the stage of spore germination to the stage of the formation of microcolonies of actinomycetes upon the spore cultivation in the liquid medium. Typical hypnum-herbaceous peat was used to study the rate of germination of the actinomycetal spores in soil. The study of the dynamics of the Streptomyces xanthochromogenes population in the control soil (without treatment with microwaves) showed that the most active development of the culture took place in the soil moistened to 60% of the maximum water capacity. When the soil was moistened to the minimum adsorption capacity, the streptomyces did not complete their full cycle of development. The stimulation of the spore germination and mycelium growth with microwaves in the soil medium required a longer period in comparison with that for the liquid medium. The stimulation of the spore germination was observed in the liquid nutrient medium in the case of 30-s treatment and in the soil in the case of 60-s treatment.
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Goda, Hisataka; Yamaoka, Hitoshi; Nakayama-Imaohji, Haruyuki; Kawata, Hiroyuki; Horiuchi, Isanori; Fujita, Yatsuka; Nagao, Tamiko; Tada, Ayano; Terada, Atsushi; Kuwahara, Tomomi
2017-01-01
Sanitation of environmental surfaces with chlorine based-disinfectants is a principal measure to control outbreaks of norovirus or Clostridium difficile. The microbicidal activity of chlorine-based disinfectants depends on the free available chlorine (FAC), but their oxidative potential is rapidly eliminated by organic matter. In this study, the microbicidal activities of weakly acidified chlorous acid water (WACAW) and sodium hypochlorite solution (NaClO) against feline calcivirus (FCV) and C. difficile spores were compared in protein-rich conditions. WACAW inactivated FCV and C. difficile spores better than NaClO under all experimental conditions used in this study. WACAW above 100 ppm FAC decreased FCV >4 log10 within 30 sec in the presence of 0.5% each of bovine serum albumin (BSA), polypeptone or meat extract. Even in the presence of 5% BSA, WACAW at 600 ppm FAC reduced FCV >4 log10 within 30 sec. Polypeptone inhibited the virucidal activity of WACAW against FCV more so than BSA or meat extract. WACAW at 200 ppm FAC decreased C. difficile spores >3 log10 within 1 min in the presence of 0.5% polypeptone. The microbicidal activity of NaClO was extensively diminished in the presence of organic matter. WACAW recovered its FAC to the initial level after partial neutralization by sodium thiosulfate, while no restoration of the FAC was observed in NaClO. These results indicate that WACAW is relatively stable under organic matter-rich conditions and therefore may be useful for treating environmental surfaces contaminated by human excretions. PMID:28472060
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Effects of nifedipine on gravi-dependent germination of moss spores
NASA Astrophysics Data System (ADS)
Khorkavtsiv, O. Y.; Demkiv, O. T.
Influence of gravity on germination of spores and dependence of the generation of a polar axis on a Ca2+ influx were investigated. The germination of spores does not depend on gravity but outgrowth polarity is controlled by light and gravity (Sytnik et al., 1989; Pundiak et al., 2001). We have shown that gravity determines the polarity of germination of spores and development of rhizoid and chloronemal outgrowths in both moss species -- Ceratodon purpureus and Pohlia nutans, the alignment of polar of germinating spores in C. purpureus, however, is less dependent on gravistimulus than in P. nutans. In 48 h after sowing onto culture medium+0,2% glucose in vertically oriented petri dishes in darkness spores of P. nutans germinated positively gravitropic rhizoid at the lower spore side and negatively gravitropic chloronema at the opposite one. The germination of C. purpureus spores is similar but the outgrowths show the lower level of alignment to the gravity vector than that of P. nutans, the dispersion of angles being 8,9 vs. 1,2 respectively. The cellular mechanism by which gravity acts remains unknown. The intracellular signaling Ca2+ ions play a crucial role in gravity perception and ability of a single cell to respond to gravity. We determined relative intensity of Ca2+ luminescence in the spores before their germination and at the early stages of outgrowth formation after treatment with the nifedipine and in a dependence on gravity vector. Gravity determined the position of outgrowth initiation zone and later on the growth direction of spore filaments. Treatment with nifedipine suppressed the gravity-directed calcium channel influx and distrupted polar growth of outgrowths. In experiments with calcium channel blocker sterilized spores were pregerminated on normal Knop's agar one day after were transferred to 50 μ M nifedipine just before emergence of the germ tube. After 48 h on nifedipine treatment, 50% spores did not germinate, 35% grew apolarily and in 15% of spores cell filaments oriented parallely with respect to the gravity vector. Results shown suggest that the endogenic competency of a single-cell spore is necessary condition of gravi- induced initiation of polar axis the competency being realized with Ca2+ movement. The highest level of Ca2+ luminescence was at the bottom of spores. In other sites of the spores the Ca2+ luminescence was about 20-fold lower than at the site of Ca2+ influx. In the 24 h after formation of first outgrowth the new site of Ca2+ influx appeared at the opposite site of spore and the second outgrowth arised. Consequently during the period of gravi-dependent spore development the newly top Ca2+ influx was repeatedly established. The direction of the Ca2+ ions influx correlated with re-orientation of spores with respect to the gravity vector. It is known that the nifedipine partially inhibits polar axis formation (Chatterjee et al., 2000) the latter being formed under the influence Ca2+ gradient (Cove, 2000). Thus, our results confirm that the fast change of Ca2+ influx probably is one of the earliest cell-level responses induced by gravity and it plays a key role in guiding polar events of germinating spores. This research was supported by NASA grant NN-09 (R).
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Brunt, Jason; Carter, Andrew T; Pye, Hannah V; Peck, Michael W
2018-05-04
Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca 2+ -DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.
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Sofos, J N; Busta, F F; Allen, C E
1979-01-01
Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated. PMID:384904
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Barbut, F; Menuet, D; Verachten, M; Girou, E
2009-06-01
To compare a hydrogen peroxide dry-mist system and a 0.5% hypochlorite solution with respect to their ability to disinfect Clostridium difficile-contaminated surfaces in vitro and in situ. Prospective, randomized, before-after trial. Two French hospitals affected by C. difficile. In situ efficacy of disinfectants was assessed in rooms that had housed patients with C. difficile infection. A prospective study was performed at 2 hospitals that involved randomization of disinfection processes. When a patient with C. difficile infection was discharged, environmental contamination in the patient's room was evaluated before and after disinfection. Environmental surfaces were sampled for C. difficile by use of moistened swabs; swab samples were cultured on selective plates and in broth. Both disinfectants were tested in vitro with a spore-carrier test; in this test, 2 types of material, vinyl polychloride (representative of the room's floor) and laminate (representative of the room's furniture), were experimentally contaminated with spores from 3 C. difficile strains, including the epidemic clone ribotype 027-North American pulsed-field gel electrophoresis type 1. There were 748 surface samples collected (360 from rooms treated with hydrogen peroxide and 388 from rooms treated with hypochlorite). Before disinfection, 46 (24%) of 194 samples obtained in the rooms randomized to hypochlorite treatment and 34 (19%) of 180 samples obtained in the rooms randomized to hydrogen peroxide treatment showed environmental contamination. After disinfection, 23 (12%) of 194 samples from hypochlorite-treated rooms and 4 (2%) of 180 samples from hydrogen peroxide treated rooms showed environmental contamination, a decrease in contamination of 50% after hypochlorite decontamination and 91% after hydrogen peroxide decontamination (P < .005). The in vitro activity of 0.5% hypochlorite was time dependent. The mean (+/-SD) reduction in initial log(10) bacterial count was 4.32 +/- 0.35 log(10) colony-forming units after 10 minutes of exposure to hypochlorite and 4.18 +/- 0.8 log(10) colony-forming units after 1 cycle of hydrogen peroxide decontamination. In situ experiments indicate that the hydrogen peroxide dry-mist disinfection system is significantly more effective than 0.5% sodium hypochlorite solution at eradicating C. difficile spores and might represent a new alternative for disinfecting the rooms of patients with C. difficile infection.
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Physico-chemical factors influencing spore germination in cyanobacterium Fischerella muscicola.
Mishra, Biranchi N; Kaushik, Manish S; Abraham, Gerard; Singh, Pawan K
2018-06-19
Spore (akinete) formation in the heterocystous and branched filamentous cyanobacterium Fischerella muscicola involves a significant increase in cell size and formation of several endospores in each of the cells. In present study, the physico-chemical factors (pH, light sources, nutrient deficiency, nitrogen sources, carbon sources, and growth hormones) affecting the germination of spores of F. muscicola were examined. Increase in spore germination frequency was detected above pH 8 with maximum germination (46.04%) recorded at pH 9, whereas a significant decrease in germination was observed at pH 6 when compared to control (pH 7.6). Spore germination was not observed at pH 5. Among light sources germination frequency followed the following order, that is, red light (39.9%) > white light (33.8%) > yellow light (3.4%) > green light (1.3%) whereas germination did not take place in dark and blue light. Ammonium chloride (NH 4 Cl) supported maximum (99.5%) germination frequency followed by calcium nitrate (Ca(NO 3 ) 2 ), potassium nitrate (KNO 3 ), and minimum germination was observed in urea. Nutrient (phosphorus, calcium, and magnesium) deficiency significantly enhanced the germination frequency with maximum increase in magnesium (Mg) deficient condition. Further, supplementation of carbon sources (glucose, fructose, and sodium acetate) and growth hormones (IAA and GA) also enhanced the germination frequency in this cyanobacterium. Therefore, it may be concluded that, those factors supporting higher germination frequency could be considered for successful production and use of this cyanobacterium in biofertilizer and other algal production technologies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Uwamahoro, Marie Christine; Massicotte, Richard; Hurtubise, Yves; Gagné-Bourque, François; Mafu, Akier Assanta; Yahia, L’Hocine
2018-01-01
Spore-forming pathogenic bacteria, such as Clostridium difficile, are associated with nosocomial infection, leading to the increased use of sporicidal disinfectants, which impacts socioeconomic costs. However, C. difficile can be prevented using microorganisms such as Bacillus amyloliquefaciens, a prophylactic agent that has been proven to be effective against it in recent tests or it can be controlled by sporicidal disinfectants. These disinfectants against spores should be evaluated according to a known and recommended standard. Unfortunately, some newly manufactured disinfectants like Bioxy products have not yet been tested. ASTM E2197-11 is a standard test that uses stainless steel disks (1 cm in diameter) as carriers, and the performance of the test formulation is calculated by comparing the number of viable test organisms to that on the control carriers. Surface tests are preferable for evaluating disinfectants with sporicidal effects on hard surfaces. This study applies improved methods, based on the ASTM E2197-11 standard, for evaluating and comparing the sporicidal efficacies of several disinfectants against spores of C. difficile and B. amyloliquefaciens, which are used as the test organisms. With the improved method, all spores were recovered through vortexing and membrane filtration. The results show that chlorine-based products are effective in 5 min and Bioxy products at 5% w/v are effective in 10 min. Although Bioxy products may take longer to prove their effectiveness, their non-harmful effects to hospital surfaces and people have been well established in the literature. PMID:29459891
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Uwamahoro, Marie Christine; Massicotte, Richard; Hurtubise, Yves; Gagné-Bourque, François; Mafu, Akier Assanta; Yahia, L'Hocine
2018-01-01
Spore-forming pathogenic bacteria, such as Clostridium difficile , are associated with nosocomial infection, leading to the increased use of sporicidal disinfectants, which impacts socioeconomic costs. However, C. difficile can be prevented using microorganisms such as Bacillus amyloliquefaciens , a prophylactic agent that has been proven to be effective against it in recent tests or it can be controlled by sporicidal disinfectants. These disinfectants against spores should be evaluated according to a known and recommended standard. Unfortunately, some newly manufactured disinfectants like Bioxy products have not yet been tested. ASTM E2197-11 is a standard test that uses stainless steel disks (1 cm in diameter) as carriers, and the performance of the test formulation is calculated by comparing the number of viable test organisms to that on the control carriers. Surface tests are preferable for evaluating disinfectants with sporicidal effects on hard surfaces. This study applies improved methods, based on the ASTM E2197-11 standard, for evaluating and comparing the sporicidal efficacies of several disinfectants against spores of C. difficile and B. amyloliquefaciens , which are used as the test organisms. With the improved method, all spores were recovered through vortexing and membrane filtration. The results show that chlorine-based products are effective in 5 min and Bioxy products at 5% w/v are effective in 10 min. Although Bioxy products may take longer to prove their effectiveness, their non-harmful effects to hospital surfaces and people have been well established in the literature.
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Theriot, Casey M.; Koenigsknecht, Mark J.; Carlson, Paul E.; Hatton, Gabrielle E.; Nelson, Adam M.; Li, Bo; Huffnagle, Gary B.; Li, Jun; Young, Vincent B.
2014-01-01
Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth. PMID:24445449
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The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.
Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie
2016-01-01
Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.
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The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid
Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie
2016-01-01
Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699
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Vercammen, Anne; Vivijs, Bram; Lurquin, Ine; Michiels, Chris W
2012-01-16
Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60°C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10min at different pressures (100-800MPa) at 40°C. None of these treatments caused any significant inactivation, except perhaps at 800MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300MPa) for A. acidoterrestris and only in a high pressure window (600-800MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800MPa at 25, 40 and 60°C for 10min. At 40°C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60°C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria. Copyright © 2011 Elsevier B.V. All rights reserved.
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Fernández-Miranda, Elena; Majada, Juan; Casares, Abelardo
2017-01-01
The use of spores in applications of ectomycorrhizal fungi requires information regarding spore viability and germination, especially in genera such as Rhizopogon with high rates of spore dormancy. The authors developed a protocol to assess spore viability of Rhizopogon roseolus using four vital stains to quantify spore viability and germination and to optimize storage procedures. They showed that propidium iodide is an excellent stain for quantifying nonviable spores. Observing red fluorescent intravacuolar structures following staining with 2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide (FUN-1) can help identify viable spores that are activated. At 6 mo and 1 y, the spores kept in a water suspension survived better than those left within intact, dry gasterocarps. Our work highlights the importance of temperature, nutrients, and vitamins for maturation and germination of spores of R. roseolus during 1 y of storage.
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1-Octanol, a self-inhibitor of spore germination in Penicillium camemberti.
Gillot, Guillaume; Decourcelle, Nicolas; Dauer, Gaëlle; Barbier, Georges; Coton, Emmanuel; Delmail, David; Mounier, Jérôme
2016-08-01
Penicillium camemberti is a technologically relevant fungus used to manufacture mold-ripened cheeses. This fungal species produces many volatile organic compounds (VOCs) including ammonia, methyl-ketones, alcohols and esters. Although it is now well known that VOCs can act as signaling molecules, nothing is known about their involvement in P. camemberti lifecycle. In this study, spore germination was shown to be self-regulated by quorum sensing in P. camemberti. This phenomenon, also called "crowding effect", is population-dependent (i.e. observed at high population densities). After determining the volatile nature of the compounds involved in this process, 1-octanol was identified as the main compound produced at high-spore density using GC-MS. Its inhibitory effect was confirmed in vitro and 3 mM 1-octanol totally inhibited spore germination while 100 μM only transiently inhibited spore germination. This is the first time that self-inhibition of spore germination is demonstrated in P. camemberti. The obtained results provide interesting perspectives for better control of mold-ripened cheese processes. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Perez, Justo; Springthorpe, V Susan; Sattar, Syed A
2005-08-01
Clostridium difficile is an increasingly common nosocomial pathogen, and its spores are resistant to common environmental surface disinfectants. Many high-level disinfectants (eg, aldehydes) are unsuitable for environmental decontamination because they need several hours of contact to be sporicidal. This study tested the potential of selected oxidative microbicides to inactivate C. difficile spores on hard surfaces in relatively short contact times at room temperature. The spores of a clinical isolate of C. difficile were tested using disks (1 cm diameter) of brushed stainless steel in a quantitative carrier test. The spores of C. sporogenes and Bacillus subtilis, common surrogates for evaluating sporicides, were included for comparison. The clostridia were grown separately in Columbia broth (CB), and B. subtilis was grown in a 1:10 dilution of CB. Each disk received 10 microL test spores with an added soil load, and the inoculum was dried. One disk each was placed in a glass vial and overlaid with 50 microL test formulation; controls received an equivalent volume of normal saline with 0.1% Tween 80. At the end of the contact time the microbicide was neutralized, the inoculum recovered from the disks by vortexing, the eluates were membrane filtered, and the filters placed on plates of recovery medium. The colony-forming units (CFU) on the plates were recorded after 5 days of incubation. The performance criterion was > or = 6 log(10) (> or = 99.9999%) reduction in the viability titer of the spores. The microbicides tested were domestic bleach with free-chlorine (FC) levels of 1000, 3000, and 5000 mg/L; an accelerated hydrogen peroxide (AHP)-based product with 70,000 mg/L H2O2 (Virox STF); chlorine dioxide (600 mg/L FC); and acidified domestic bleach (5000 mg/L FC). Acidified bleach and the highest concentration of regular bleach tested could inactivate all the spores in < or = 10 minutes; Virox STF could do the same in < or = 13 minutes. Regular bleach with 3000 mg/L FC required up to 20 minutes to reduce the viability of the all the spores tested to undetectable levels; chlorine dioxide and the lowest concentration of regular bleach tested needed approximately 30 minutes for the same level of activity. Acidified bleach, Virox STF, and regular bleach (3000-5000 mg/L FC) could inactivate C. difficile spores on hard environmental surfaces in approximately 10 to 15 minutes under ambient conditions. All of these products are strong oxidizers and should be handled with care for protection of staff, but acidified and regular bleach with high levels of FC also release chlorine gas, which can be hazardous if inhaled by staff or patients.
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Proteins YlaJ and YhcN contribute to the efficiency of spore germination in Bacillus subtilis.
Johnson, Christian L; Moir, Anne
2017-04-01
The YlaJ and YhcN spore lipoproteins of Bacillus subtilis contain a common domain, and are of unknown function. Homologues of YlaJ or YhcN are widespread in Bacilli and are also encoded in those Clostridia that use cortex lytic enzymes SleB and CwlJ for cortex hydrolysis during germination. In B. subtilis, we report that single and double mutants lacking YlaJ and/or YhcN show a reduced rate of spore germination in L-alanine, with a delay in loss of heat resistance, release of dipicolinic acid and OD fall. If B. subtilis spores lack the cortex lytic enzyme CwlJ, spore cortex degradation and subsequent outgrowth to form colonies is strictly dependent on the other cortex lytic enzyme SleB, allowing a test of SleB function; in a cwlJ mutant background, the combined loss of both ylaJ and yhcN genes resulted in a spore population in which only 20% of spores germinated and outgrew to form colonies, suggesting that SleB activity is compromised. YlaJ and YhcN have a role in germination that is not yet well defined, but these proteins are likely to contribute, directly or indirectly, to early events in germination, including effective SleB function. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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NASA Technical Reports Server (NTRS)
Scheuerlein, R.; Wayne, R.; Roux, S. J.
1988-01-01
A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21 degrees C. Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R. Using this technique we show for the first time that Ca2+ contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea.
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Highly abundant and stage-specific mRNAs in the obligate pathogen Bremia lactucae.
Judelson, H S; Michelmore, R W
1990-01-01
Germinating spores of the obligate pathogen Bremia lactucae (lettuce downy mildew) contain several unusually abundant species of mRNA. Thirty-nine cDNA clones corresponding to prevalent transcripts were isolated from a library synthesized using poly(A)+ RNA from germinating spores; these clones represented only five distinct classes. Each corresponding mRNA accounted for from 0.4 to 9 percent by mass of poly(A)+ RNA from germinating spores and together represented greater than 20 percent of the mRNA. The expression of the corresponding genes, and a gene encoding Hsp70, was analyzed in spores during germination and during growth in planta. The Hsp70 mRNA and mRNA from one abundant cDNA clone (ham34) were expressed constitutively. Two clones (ham9 and ham12) hybridized only to mRNA from spores and germinating spores. Two clones (ham37 and ham27) showed hybridization specific to germinating spores. Quantification of the number of genes homologous to each cDNA clone indicated that four clones corresponded to one or two copies per haploid genome, and one hybridized to an approximately 11-member family of genes. A sequence of the gene corresponding to ham34 was obtained to investigate its function and to identify sequences conferring high levels of gene expression for use in constructing vectors for the transformation of B. lactucae.
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Global Analysis of the Sporulation Pathway of Clostridium difficile
Fimlaid, Kelly A.; Bond, Jeffrey P.; Schutz, Kristin C.; Putnam, Emily E.; Leung, Jacqueline M.; Lawley, Trevor D.; Shen, Aimee
2013-01-01
The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σF, σE, σG, and σK are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σF, σE, σG, and σK in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σF-dependent, 169 were σE-dependent, 34 were σG-dependent, and 31 were σK-dependent. In contrast with B. subtilis, C. difficile σE was dispensable for σG activation, σG was dispensable for σK activation, and σF was required for post-translationally activating σG. Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. PMID:23950727
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Global analysis of the sporulation pathway of Clostridium difficile.
Fimlaid, Kelly A; Bond, Jeffrey P; Schutz, Kristin C; Putnam, Emily E; Leung, Jacqueline M; Lawley, Trevor D; Shen, Aimee
2013-01-01
The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σ(F), σ(E), σ(G), and σ(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σ(F), σ(E), σ(G), and σ(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σ(F)-dependent, 169 were σ(E)-dependent, 34 were σ(G)-dependent, and 31 were σ(K)-dependent. In contrast with B. subtilis, C. difficile σ(E) was dispensable for σ(G) activation, σ(G) was dispensable for σ(K) activation, and σ(F) was required for post-translationally activating σ(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.
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Doan, L; Forrest, H; Fakis, A; Craig, J; Claxton, L; Khare, M
2012-10-01
Clostridium difficile spores can survive in the environment for months or years, and contaminated environmental surfaces are important sources of nosocomial C. difficile transmission. To compare the clinical and cost effectiveness of eight C. difficile environmental disinfection methods for the terminal cleaning of hospital rooms contaminated with C. difficile spores. This was a novel randomized prospective study undertaken in three phases. Each empty hospital room was disinfected, then contaminated with C. difficile spores and disinfected with one of eight disinfection products: hydrogen peroxide vapour (HPV; Bioquell Q10) 350-700 parts per million (ppm); dry ozone at 25 ppm (Meditrox); 1000 ppm chlorine-releasing agent (Actichlor Plus); microfibre cloths (Vermop) used in combination with and without a chlorine-releasing agent; high temperature over heated dry atomized steam cleaning (Polti steam) in combination with a sanitizing solution (HPMed); steam cleaning (Osprey steam); and peracetic acid wipes (Clinell). Swabs were inoculated on to C. difficile-selective agar and colony counts were performed pre and post disinfection for each method. A cost-effectiveness analysis was also undertaken comparing all methods to the current method of 1000 ppm chlorine-releasing agent (Actichlor Plus). Products were ranked according to the log(10) reduction in colony count from contamination phase to disinfection. The three statistically significant most effective products were hydrogen peroxide (2.303); 1000 ppm chlorine-releasing agent (2.223) and peracetic acid wipes (2.134). The cheaper traditional method of using a chlorine-releasing agent for disinfection was as effective as modern methods. Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
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Voundi, Stève Olugu; Nyegue, Maximilienne; Lazar, Iuliana; Raducanu, Dumitra; Ndoye, Florentine Foe; Marius, Stamate; Etoa, François-Xavier
2015-06-01
The use of essential oils as a food preservative has increased due to their capacity to inhibit vegetative growth of some bacteria. However, only limited data are available on their effect on bacterial spores. The aim of the present study was to evaluate the effect of some essential oils on the growth and germination of three Bacillus species and Geobacillus stearothermophilus. Essential oils were chemically analyzed using gas chromatography and gas chromatography coupled to mass spectrometry. The minimal inhibitory and bactericidal concentrations of vegetative growth and spore germination were assessed using the macrodilution method. Germination inhibitory effect of treated spores with essential oils was evaluated on solid medium, while kinetic growth was followed using spectrophotometry in the presence of essential oils. Essential oil from Drypetes gossweileri mainly composed of benzyl isothiocyanate (86.7%) was the most potent, with minimal inhibitory concentrations ranging from 0.0048 to 0.0097 mg/mL on vegetative cells and 0.001 to 0.002 mg/mL on spore germination. Furthermore, essential oil from D. gossweileri reduced 50% of spore germination after treatment at 1.25 mg/mL, and its combination with other oils improved both bacteriostatic and bactericidal activities with additive or synergistic effects. Concerning the other essential oils, the minimal inhibitory concentration ranged from 5 to 0.63 mg/mL on vegetative growth and from 0.75 to 0.09 mg/mL on the germination of spores. Spectrophotometric evaluation showed an inhibitory effect of essential oils on both germination and outgrowth. From these results, it is concluded that some of the essential oils tested might be a valuable tool for bacteriological control in food industries. Therefore, further research regarding their use as food preservatives should be carried out.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Singh, J.; Devi, S.
1989-07-01
Validity of fern spore germination bioassays for the effects of environmental pollution was established by many researchers. Some workers studied the phytotoxicity of linear alkyl benzene sulfonate (LAS) on the spores of Diplazium esculentum and observed that LAS levels above 0.001% are toxic to fern spores. Water pollution due to synthetic detergents has been increasing continuously during the last few years due to their extensive use in domestic life, agriculture and industry. These detergents are among the most common pollutants responsible for water pollution. In view of this fact, the phytotoxicity of LAS on germination of an aquatic fern Ceratopterismore » thalictroides spores was studied. However, in these studies, only germination pattern was taken as index and no observations were made on the developmental stages.« less
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NASA Technical Reports Server (NTRS)
Nicholson, Wayne L.; Schuerger, Andrew C.
2005-01-01
Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.
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A Novel Regulator Controls Clostridium difficile Sporulation, Motility and Toxin Production
Edwards, Adrianne N.; Tamayo, Rita; McBride, Shonna M.
2016-01-01
SUMMARY Clostridium difficile, is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. PMID:26915493
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A novel regulator controls Clostridium difficile sporulation, motility and toxin production.
Edwards, Adrianne N; Tamayo, Rita; McBride, Shonna M
2016-06-01
Clostridium difficile is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. © 2016 John Wiley & Sons Ltd.
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NASA Technical Reports Server (NTRS)
Campbell, J. E.; Reyes, A. L.; Wehby, A. J.; Crawford, R. G.; Wimsatt, J. C.; Peeler, J. T.
1973-01-01
The mechanism for thermal inactivation of bacterial spores under moist or dry heat was studied. Experimental conditions were established relating to spore loss of heat resistance and loss of optical density as a measure of the rate and extent of germination in spore suspensions. Events occurring during germination were correlated with phase darkening (refractility and non-refractility of spores), stainability characteristics of heat and non-heat treated spores, morphological characteristics, and studies on swelling of spores by an increase in packed cell volume.
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NASA Technical Reports Server (NTRS)
Raghavan, V.
1991-01-01
Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.
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Curran, Harold R.; Pallansch, Michael J.
1963-01-01
Curran, Harold R. (U.S. Department of Agriculture, Washington, D.C.), and Michael J. Pallansch. Incipient germination in heavy suspensions of spores of Bacillus stearothermophilus at subminimal growth temperatures. J. Bacteriol. 86:911–918. 1963.—By use of spore (plate) counts and permeability to stain, labilization was followed periodically in heavy suspensions of washed Bacillus stearothermophilus 1518 spores incubated at different temperatures. Although vegetative proliferation did not occur below 38 C, incipient germination was rapid down to 20 C and much slower and incomplete at 14 C. Dilution of the suspension materially reduced the degree and rate of labilization. The degree of washing and use of deionized water had no appreciable influence upon early development of the spores. The results are discussed from the point of view of the possible origin and nature of the germination stimulant. Images PMID:14080801
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Barkal, Layla J.; Walsh, Naomi M.; Botts, Michael R.; Beebe, David J.; Hull, Christina M.
2016-01-01
Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination. PMID:27026574
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Ghosh, Abhinaba; Manton, James D; Mustafa, Amin R; Gupta, Mudit; Ayuso-Garcia, Alejandro; Rees, Eric J; Christie, Graham
2018-05-04
Germination of Bacillus spores is triggered by certain amino acids and sugar molecules, which permeate through the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here we use the ellipsoid localisation microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex lytic enzymes within the inner coat. We reveal also that the GerPA protein alone can localise in the absence of all other GerP proteins, and that it has an essential role for the localisation of all other GerP proteins within the spore. The latter is also demonstrated to be SafA - but not CotE - dependent for localisation, which is consistent with an inner coat location. GerP null spores are shown also to have reduced permeability to fluorescently labelled dextran molecules compared to wild type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability. Importance The bacterial spore coat comprises a multi-layered proteinaceous structure that influences the distribution, survival and germination properties of spores in the environment. Results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ELM image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, healthcare and environmental sectors. Copyright © 2018 American Society for Microbiology.
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Real time viability detection of bacterial spores
Vanderberg, Laura A.; Herdendorf, Timothy J.; Obiso, Richard J.
2003-07-29
This invention relates to a process for detecting the presence of viable bacterial spores in a sample and to a spore detection system, the process including placing a sample in a germination medium for a period of time sufficient for commitment of any present viable bacterial spores to occur, mixing the sample with a solution of a lanthanide capable of forming a fluorescent complex with dipicolinic acid, and, measuring the sample for the presence of dipicolinic acid, and the system including a germination chamber having inlets from a sample chamber, a germinant chamber and a bleach chamber, the germination chamber further including an outlet through a filtering means, the outlet connected to a detection chamber, the detection chamber having an inlet from a fluorescence promoting metal chamber and the detection chamber including a spectral excitation source and a means of measuring emission spectra from a sample, the detection chamber further connected to a waste chamber. A germination reaction mixture useful for promoting commitment of any viable bacterial spores in a sample including a combination of L-alanine, L-asparagine and D-glucose is also described.
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Zabrocka, L; Langer, K; Michalski, A; Kocik, J; Langer, J J
2015-01-07
A microfluidic device for studies on the germination of bacterial spores (e.g. Bacillus subtilis) based on non-specific interactions on the nanoscale is presented. A decrease in the population of spores during germination followed by the appearance of transition forms and an increase in the number of vegetative cells can be registered directly and simultaneously by using the microfluidic device, which is equipped with a conductive polymer layer (polyaniline) in the form of a nano-network. The lab-on-a-chip-type device, operating in a continuous flow regime, allows monitoring of germination of bacterial spores and analysis of the process in detail. The procedure is fast and accurate enough for quantitative real-time monitoring of the main steps of germination, including final transformation of the spores into vegetative cells. All of this is done without the use of biomarkers or any bio-specific materials, such as enzymes, antibodies and aptamers, and is simply based on an analysis of physicochemical interactions on the nanoscale level.
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Sánchez-Plata, Marcos X; Amézquita, Alejandro; Blankenship, Erin; Burson, Dennis E; Juneja, Vijay; Thippareddi, Harshavardhan
2005-12-01
Spores of foodborne pathogens can survive traditional thermal processing schedules used in the manufacturing of processed meat products. Heat-activated spores can germinate and grow to hazardous levels when these products are improperly chilled. Germination and outgrowth of Clostridium perfringens spores in roast beef during chilling was studied following simulated cooling schedules normally used in the processed-meat industry. Inhibitory effects of organic acid salts on germination and outgrowth of C. perfringens spores during chilling and the survival of vegetative cells and spores under abusive refrigerated storage was also evaluated. Beef top rounds were formulated to contain a marinade (finished product concentrations: 1% salt, 0.2% potassium tetrapyrophosphate, and 0.2% starch) and then ground and mixed with antimicrobials (sodium lactate and sodium lactate plus 2.5% sodium diacetate and buffered sodium citrate and buffered sodium citrate plus 1.3% sodium diacetate). The ground product was inoculated with a three-strain cocktail of C. perfringens spores (NCTC 8238, NCTC 8239, and ATCC 10388), mixed, vacuum packaged, heat shocked for 20 min at 75 degrees C, and chilled exponentially from 54.5 to 7.2 degrees C in 9, 12, 15, 18, or 21 h. C. perfringens populations (total and spore) were enumerated after heat shock, during chilling, and during storage for up to 60 days at 10 degrees C using tryptose-sulfite-cycloserine agar. C. perfringens spores were able to germinate and grow in roast beef (control, without any antimicrobials) from an initial population of ca. 3.1 log CFU/g by 2.00, 3.44, 4.04, 4.86, and 5.72 log CFU/g after 9, 12, 15, 18, and 21 h of exponential chilling. A predictive model was developed to describe sigmoidal C. perfringens growth curves during cooling of roast beef from 54.5 to 7.2 degrees C within 9, 12, 15, 18, and 21 h. Addition of antimicrobials prevented germination and outgrowth of C. perfringens regardless of the chill times. C. perfringens spores could be recovered from samples containing organic acid salts that were stored up to 60 days at 10 degrees C. Extension of chilling time to > or =9 h resulted in >1 log CFU/g growth of C. perfringens under anaerobic conditions in roast beef. Organic acid salts inhibited outgrowth of C. perfringens spores during chilling of roast beef when extended chill rates were followed. Although C. perfringens spore germination is inhibited by the antimicrobials, this inhibition may represent a hazard when such products are incorporated into new products, such as soups and chili, that do not contain these antimicrobials, thus allowing spore germination and outgrowth under conditions of temperature abuse.
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Singh, D. K.; Basha, S. Ameer; Sarma, B. K.; Pandey, V. B.
2006-01-01
Phytoterpenoid isolated from Artabotrytis odoratissimus inhibited spore germination of some plant pathogenic as well as saprophytic fungi e.g. Alternaria alternata, A. solani, Cercospora sp., Curvularia maculans, C. pennisetti, Fusarium udum, Helminthosporium echinochlova, H. frumentacie, H. penniseti and Ustilago cynodontis. In Curvularia maculans and H. frumentacie, spore germination was completely inhibited at 2000 ppm. However, Curvularia maculans and C. pennisetti showed considerable sensitivity to this chemical even at 500 ppm. PMID:24039483
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Lipoxygenase activity accelerates programmed spore germination in Aspergillus fumigatus
Gregory J. Fischer; William Bacon; Jun Yang; Jonathan M. Palmer; Taylor Dagenais; Bruce D. Hammock; Nancy P. Keller
2017-01-01
The opportunistic human pathogen Aspergillus fumigatus initiates invasive growth through a programmed germination process that progresses from dormant spore to swollen spore (SS) to germling (GL) and ultimately invasive hyphal growth. We find a lipoxygenase with considerable homology to human Alox5 and Alox15, LoxB, that impacts the transitions of...
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Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.
Setlow, B; Korza, G; Setlow, P
2016-05-01
This study was to determine the sources, location and identity of α-glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. α-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl-α-d-glucoside (α-MUG) or α-MUG-6-phosphate hydrolysis fluorometrically. While α-MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with α-MUG showed that: (1) the α-glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α-glucosidase activity was only inside vegetative cells; (2) most α-glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti-α-glucosidase antiserum identified ≥2 α-glucosidases in spores and growing cells; (4) α-glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α-glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α-MUG, but glucose inhibited α-MUG uptake. α-MUG hydrolysis by G. stearothermophilus is by α-MUG uptake and hydrolysis by ≥2 α-glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance. © 2016 The Society for Applied Microbiology.
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A role for antimicrobial peptides in intestinal microsporidiosis
Leitch, Gordon J.; Ceballos, Carolina
2009-01-01
SUMMARY Clinical isolates from three microsporidia species, Encephalitozoon intestinalis and Encephalitozoon hellem, and the insect parasite Anncaliia (Brachiola, Nosema) algerae, were used in spore germination and enterocyte-like (C2Bbe1) cell infection assays to determine the effect of a panel of antimicrobial peptides. Spores were incubated with lactoferrin (Lf), lysozyme (Lz), and human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1), alone and in combination with Lz, prior to germination. Of the Encephalitozoon species only E. hellem spore germination was inhibited by HNP1, while A. algerae spore germination was inhibited by Lf, HBD2, HD5 and HNP1, although HBD2 and HD5 inhibition required the presence of Lz. The effects of HBD2 and HD5 on microsporidia enterocyte infection paralleled their effects on spore germination. Lysozyme alone only inhibited infection with A. algerae, while Lf inhibited infection by E. intestinalis and A. algerae. HNP1 significantly reduced enterocyte infection by all three parasite species and a combination of Lf, Lz and HNP1 caused a further reduced infection with A. algerae. These data suggest that intestinal antimicrobial peptides contribute to the defense of the intestine against infection by luminal microsporidia spores and may partially determine which parasite species infects the intestine. PMID:19079820
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2014-01-01
a rapidly growing non thermal food processing technology that ensures the safety of meat, fruit juice and seafood products, extends product shelf...spore germination with nutrient germinants are the sum of values for germination via the GerA GR with L valine and the GerB plus GerK GRs with AGFK...gerKD had no significant effect on mHP germination (Fig. 1d). Complementation of a gerKD strain by introduction of a wild type gerKD gene plus its own
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Li, Lin; Valenzuela-Martinez, Carol; Redondo, Mauricio; Juneja, Vijay K; Burson, Dennis E; Thippareddi, Harshavardhan
2012-11-01
Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV1) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1%, 1.5%, or 2%, w/w), blend of sodium pyro- and poly-phosphates (0.3%), and MoStatin LV1 (0%, 2%, or 2.5%) was inoculated with a 3-strain C. perfringens spore cocktail to achieve final spore population of 2.5 to 3.0 log CFU/g. The inoculated products were heat treated and cooled exponentially from 54.4 to 4.4 °C within 6.5, 9, 12, 15, 18, or 21 h. Cooling of roast beef (2.0% NaCl) within 6.5 and 9 h resulted in <1.0 log CFU/g increase in C. perfringens spore germination and outgrowth, whereas reducing the salt concentration to 1.5% and 1.0% resulted in >1.0 log CFU/g increase for cooling times longer than 9 h (1.1 and 2.2 log CFU/g, respectively). Incorporation of MoStatin LV1 into the roast beef formulation minimized the C. perfringens spore germination and outgrowth to <1.0 log CFU/g, regardless of the salt concentration and the cooling time. Cooked, ready-to-eat meat products should be cooled rapidly to reduce the risk of Clostridium perfringens spore germination and outgrowth. Meat processors are reducing the sodium chloride content of the processed meats as a consequence of the dietary recommendations. Sodium chloride reduces the risk of C. perfringens spore germination and outgrowth in meat products. Antimicrobials that contribute minimally to the sodium content of the product should be incorporated into processed meats to assure food safety. Buffered lemon juice and vinegar can be incorporated into meat product formulations to reduce the risk of C. perfringens spore germination and outgrowth during abusive cooling. Journal of Food Science © 2012 Institute of Food Technologists® No claim to original US government works.
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Inhibition of spore germination of Alternaria tenuis by sulfur dioxide
DOE Office of Scientific and Technical Information (OSTI.GOV)
Couey, H.M.
1962-08-01
As a part of a continuing study of SO/sub 2/ fumigation of table grapes, the effect of SO/sub 2/ on spores of an isolate of A. tenuis Auct. causing decay of table grapes was determined. The amount of SO/sub 2/ required to inhibit completely spore germination depended on availability of moisture and the temperature. At 20/sup 0/C, wet spores required 20-min exposure to 100 ppm SO/sub 2/ to prevent germination, but spores equilibrated at 90% relative humidity (RH) required 10-min exposure to 1000 ppm SO/sub 2/. Dry spores at 60% RH were unaffected by a 20-min exposure to 4000 ppmmore » SO/sub 2/. Increasing the temperature in the range 5-20/sup 0/C increased effectiveness of the SO/sub 2/ treatment. A comparison of Alternaria with Botrytis cinerea Fr. (studied earlier) showed that wet spores of these organisms were about equally sensitive to SO/sub 2/, but that dry Alternaria spores were more resistant to SO/sub 2/ than dry Botrytis spores under comparable conditions.« less
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Grimmond, Terry; Neelakanta, Anu; Miller, Barbara; Saiyed, Asif; Gill, Pam; Cadnum, Jennifer; Olmsted, Russell; Donskey, Curtis; Pate, Kimberly; Miller, Katherine
2018-05-22
A 2015 study matching use of disposable and reusable sharps containers (DSCs, RSCs) with Clostridium difficile infection (CDI) incidence found a decreased incidence with DSCs. We conducted microbiologic samplings and examined the literature and disease-transmission principles to evaluate the scientific feasibility of such an association. (i) 197 RSCs were sampled for C. difficile at processing facilities; (ii) RSCs were challenged with high C. difficile densities to evaluate efficacy of automated decontamination; and (iii) 50 RSCs and 50 DSCs were sampled in CDI patient rooms in 7 hospitals. Results were coupled with epidemiologic studies, clinical requirements, and chain-of-infection principles, and tests of evidence of disease transmission were applied. C. difficile spores were found on 9 of 197 (4.6%) RSCs prior to processing. Processing completely removed C. difficile. In CDI patient rooms, 4 of 50 RSCs (8.0%) and 8 of 50 DSCs (16.0%) had sub-infective counts of C. difficile (P = .27). DSCs were in permanent wall cabinets; RSCs were removed and decontaminated frequently. With C. difficile bioburden being sub-infective on both DSCs and RSCs, sharps containers being no-touch, and glove removal required after sharps disposal, we found 2 links in the chain of infection to be broken and 5 of 7 tests of evidence to be unmet. We conclude that sharps containers pose no risk of C. difficile transmission. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
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Atmospheric ions and germination of uredospores of Puccinia striiformis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sharp, E.L.
1967-06-09
Atmospheric ions, identified by mobility characteristics, were associated with germination of lyophilized uredospores of Puccinia striiformis West. at Bozeman, Montana. Ions of intermediate size were highest in concentration, and percentage germination of spores was lowest during periods conducive to air pollution. In duplicate experiments at an isolated site near Barrow, Alaska, essentially all atmospheric ions were small ions and the fungus spores were consistently germinated near maximum.
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Nerandzic, Michelle M; Cadnum, Jennifer L; Pultz, Michael J; Donskey, Curtis J
2010-07-08
Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against Clostridium difficile spores, but also safe, rapid, and automated. The Tru-D Rapid Room Disinfection device is a mobile, fully-automated room decontamination technology that utilizes ultraviolet-C irradiation to kill pathogens. We examined the efficacy of environmental disinfection using the Tru-D device in the laboratory and in rooms of hospitalized patients. Cultures for C. difficile, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) were collected from commonly touched surfaces before and after use of Tru-D. On inoculated surfaces, application of Tru-D at a reflected dose of 22,000 microWs/cm(2) for approximately 45 minutes consistently reduced recovery of C. difficile spores and MRSA by >2-3 log10 colony forming units (CFU)/cm2 and of VRE by >3-4 log10 CFU/cm(2). Similar killing of MRSA and VRE was achieved in approximately 20 minutes at a reflected dose of 12,000 microWs/cm(2), but killing of C. difficile spores was reduced. Disinfection of hospital rooms with Tru-D reduced the frequency of positive MRSA and VRE cultures by 93% and of C. difficile cultures by 80%. After routine hospital cleaning of the rooms of MRSA carriers, 18% of sites under the edges of bedside tables (i.e., a frequently touched site not easily amenable to manual application of disinfectant) were contaminated with MRSA, versus 0% after Tru-D (P < 0.001). The system required <5 minutes to set up and did not require continuous monitoring. The Tru-D Rapid Room Disinfection device is a novel, automated, and efficient environmental disinfection technology that significantly reduces C. difficile, VRE and MRSA contamination on commonly touched hospital surfaces.
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Fern Spore Longevity in Saline Water: Can Sea Bottom Sediments Maintain a Viable Spore Bank?
de Groot, G. Arjen; During, Heinjo
2013-01-01
Freshwater and marine sediments often harbor reservoirs of plant diaspores, from which germination and establishment may occur whenever the sediment falls dry. Therewith, they form valuable records of historical inter- and intraspecific diversity, and are increasingly exploited to facilitate diversity establishment in new or restored nature areas. Yet, while ferns may constitute a considerable part of a vegetation’s diversity and sediments are known to contain fern spores, little is known about their longevity, which may suffer from inundation and - in sea bottoms - salt stress. We tested the potential of ferns to establish from a sea or lake bottom, using experimental studies on spore survival and gametophyte formation, as well as a spore bank analysis on sediments from a former Dutch inland sea. Our experimental results revealed clear differences among species. For Asplenium scolopendrium and Gymnocarpium dryopteris, spore germination was not affected by inundated storage alone, but decreased with rising salt concentrations. In contrast, for Asplenium trichomanes subsp. quadrivalens germination decreased following inundation, but not in response to salt. Germination rates decreased with time of storage in saline water. Smaller and less viable gametophytes were produced when saline storage lasted for a year. Effects on germination and gametophyte development clearly differed among genotypes of A. scolopendrium. Spore bank analyses detected no viable spores in marine sediment layers. Only two very small gametophytes (identified as Thelypteris palustris via DNA barcoding) emerged from freshwater sediments. Both died before maturation. We conclude that marine, and likely even freshwater sediments, will generally be of little value for long-term storage of fern diversity. The development of any fern vegetation on a former sea floor will depend heavily on the deposition of spores onto the drained land by natural or artificial means of dispersal. PMID:24223951
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Clostridium difficile infection in the twenty-first century
Ghose, Chandrabali
2013-01-01
Clostridium difficile is a spore-forming gram-positive bacillus, and the leading cause of antibiotic-associated nosocomial diarrhea and colitis in the industrialized world. With the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027), the epidemiology of C. difficile infection has rapidly changed in the last decade. C. difficile infection, once thought to be an easy to treat bacterial infection, has evolved into an epidemic that is associated with a high rate of mortality, causing disease in patients thought to be low-risk. In this review, we discuss the changing face of C .difficile infection and the novel treatment and prevention strategies needed to halt this ever growing epidemic. PMID:26038491
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Characteristics of Deoxyribonucleic Acid Polymerase Isolated from Spores of Rhizopus stolonifer1
Gong, Cheng-Shung; Dunkle, Larry D.; Van Etten, James L.
1973-01-01
Deoxyribonucleic acid (DNA)-dependent DNA polymerase was purified several hundredfold from germinated and ungerminated spores of the fungus Rhizopus stolonifer. The partially purified enzymes from both spore stages exhibited identical characteristics; incorporation of [3H]deoxythymidine monophosphate into DNA required Mg2+, DNA, a reducing agent, and the simultaneous presence of deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxyadenosine triphosphate. Heat-denatured and activated DNAs were better templates than were native DNAs. The buoyant density of the radioactive product of the reaction was similar to that of the template DNA. The enzyme is probably composed of a single polypeptide chain with an S value of 5.12 and an estimated molecular weight of 70,000 to 75,000. During the early stages of purification, the enzyme fraction from ungerminated spores required exogenous DNA for maximum activity, whereas the corresponding enzyme fraction from germinated spores did not require added DNA. Apparently DNA polymerase from germinated spores was more tightly bound to endogenous DNA than was the enzyme from ungerminated spores. PMID:4728271
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Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.
Heffron, Jared D; Orsburn, Benjamin; Popham, David L
2009-04-01
The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.
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Cho, Jeong Sub; Seo, Yong Chang; Yim, Tae Bin; Lee, Hyeon Yong
2013-01-01
Nanoencapsulation of thiamine dilauryl sulfate (TDS), a vitamin B1 derivative, was proved to effectively inhibit the spore germination of Fusarium oxysporum f. sp. raphani (F. oxysporum), as well as mycelial growth. The average diameter of nanoparticles was measured as 136 nm by being encapsulated with an edible encapsulant, lecithin, whose encapsulation efficiency was about 55% in containing 200 ppm of TDS concentration: the 100 ppm TDS nanoparticle solution showed a mycelial growth inhibition rate of 59%. These results were about similar or even better than the cases of treating 100 ppm of dazomet, a positive antifungal control (64%). Moreover, kinetic analysis of inhibiting spore germination were estimated as 6.6% reduction of spore germination rates after 24 h treatment, which were 3.3% similar to the case of treating 100 ppm of a positive control (dazomet) for the same treatment time. It was also found that TDS itself could work as an antifungal agent by inhibiting both mycelial growth and spore germination, even though its efficacy was lower than those of nanoparticles. Nanoparticles especially played a more efficient role in limiting the spore germination, due to their easy penetration into hard cell membranes and long resident time on the surface of the spore shell walls. In this work, it was first demonstrated that the nanoparticle of TDS not a harmful chemical can control the growth of F. oxysporum by using a lower dosage than commercial herbicides, as well as the inhibiting mechanism of the TDS. However, field trials of the TDS nanoparticles encapsulated with lecithin should be further studied to be effectively used for field applications. PMID:23429270
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RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.
Mikulík, K; Janda, I; Weiser, J; Stastná, J; Jiránová, A
1984-12-03
Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.
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Effect of synthetic detergents on germination of fern spores
DOE Office of Scientific and Technical Information (OSTI.GOV)
Devi, Y.; Devi, S.
Synthetic detergents constitute one of the most important water pollutants by contaminating the lakes and rivers through domestic and industrial use. Considerable information is now available for the adverse effects of detergents an aquatic fauna including fish, algae, and higher aquatic plants. Marked inhibition of germination in orchids and brinjals and of seedlings growth in raddish suggest that rapidly growing systems could be sensitive to detergent polluted water. The present study of the effect of linear alkyl benzene sulphonate on germination of the spores of a fern, Diplazium esculentum aims at the understanding of the effects of water pollution onmore » pteridophytes and the development of spore germination assay for phytoxicity evaluation.« less
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Omardien, Soraya; Ter Beek, Alexander; Vischer, Norbert; Montijn, Roy; Schuren, Frank; Brul, Stanley
2018-06-14
An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.
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Flavonoids released naturally from alfalfa promote development of symbiotic glomus spores in vitro.
Tsai, S M; Phillips, D A
1991-05-01
Because flavonoids from legumes induce transcription of nodulation genes in symbiotic rhizobial bacteria, it is reasonable to test whether these compounds alter the development of vesicular-arbuscular mycorrhizal (VAM) fungi that infect those plants. Quercetin-3-O-galactoside, the dominant flavonoid released naturally from alfalfa (Medicago sativa L.) seeds, promoted spore germination of Glomus etunicatum and Glomus macrocarpum in vitro. Quercetin produced the maximum increases in spore germination, hyphal elongation, and hyphal branching in G. etunicatum at 1 to 2.5 muM concentrations. Two flavonoids exuded from alfalfa roots, 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone, also enhanced spore germination of this fungal species. Formononetin, an isoflavone that is released from stressed alfalfa roots, inhibited germination of both Glomus species. These in vitro results suggest that plant flavonoids may facilitate or regulate the development of VAM symbioses and offer new hope for developing pure, plant-free cultures of VAM fungi.
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Limited period of graviresponsiveness in germinating spores of Ceratopteris richardii
NASA Technical Reports Server (NTRS)
Edwards, E. S.; Roux, S. J.
1994-01-01
Rhizoids of the fern Ceratopteris richardii Brogn. usually emerge 40 h after germination is initiated by light, and more than 90% of them emerge growing in a downward direction. However, when the spores are germinated on a clinostat, the emerging rhizoids show no preferential orientation. This indicates that under normal 1 g conditions the initial growth direction of rhizoids can be oriented by gravity. If the orientation of the spores is changed 3 h or less after the start of germination, the growth direction of most emerging rhizoids becomes downward relative to the new orientation. However, if the orientation of the spores is changed by 180 degrees 8 h or more after germination is initiated by light, most rhizoids emerge growing upward; i.e., the same direction as if there had been no orientation change. Emerged rhizoids also do not change their direction of growth if their orientation is changed. These results indicate that the growth direction of emerging rhizoids is set by gravity prior to actual emergence, and that the time of full orientation responsiveness is limited to a period ranging from the initiation of germination to about 3-4 h after the start of germination. There is a gravity-oriented nuclear movement beginning at about 13 h after germination, and this movement appears to predict the initial growth direction of rhizoids.
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Limited period of graviresponsiveness in germinating spores of Ceratopteris richardii.
Edwards, E S; Roux, S J
1994-01-01
Rhizoids of the fern Ceratopteris richardii Brogn. usually emerge 40 h after germination is initiated by light, and more than 90% of them emerge growing in a downward direction. However, when the spores are germinated on a clinostat, the emerging rhizoids show no preferential orientation. This indicates that under normal 1 g conditions the initial growth direction of rhizoids can be oriented by gravity. If the orientation of the spores is changed 3 h or less after the start of germination, the growth direction of most emerging rhizoids becomes downward relative to the new orientation. However, if the orientation of the spores is changed by 180 degrees 8 h or more after germination is initiated by light, most rhizoids emerge growing upward; i.e., the same direction as if there had been no orientation change. Emerged rhizoids also do not change their direction of growth if their orientation is changed. These results indicate that the growth direction of emerging rhizoids is set by gravity prior to actual emergence, and that the time of full orientation responsiveness is limited to a period ranging from the initiation of germination to about 3-4 h after the start of germination. There is a gravity-oriented nuclear movement beginning at about 13 h after germination, and this movement appears to predict the initial growth direction of rhizoids.
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Setlow, B; Korza, G; Blatt, K M S; Fey, J P; Setlow, P
2016-01-01
Determine how supercritical CO2 (scCO2 ) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2 -PAA, and if spores inactivated by scCO2 -PAA are truly dead. Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2 -PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2 -PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2 -PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2 -PAA sensitive. These findings suggest that scCO2 -PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO2 -PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2 -PAA resistance only for dry spores. These results provide information on mechanisms of spore inactivation of and resistance to scCO2 -PAA, an agent with increasing use in sterilization applications. © 2015 The Society for Applied Microbiology.
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Shapey, S; Machin, K; Levi, K; Boswell, T C
2008-10-01
Clostridium difficile causes serious healthcare-associated infections. Infection control is difficult, due in part to environmental contamination with C. difficile spores. These spores are relatively resistant to cleaning and disinfection. The activity of a dry mist hydrogen peroxide decontamination system (Sterinis) against environmental C. difficile contamination was assessed in three elderly care wards. Initial sampling for C. difficile was performed in 16 rooms across a variety of wards and specialties, using Brazier's CCEY (cycloserine-cefoxitin-egg yolk) agar. Ten rooms for elderly patients (eight isolation and two sluice rooms) were then resampled following dry mist hydrogen peroxide decontamination. Representative isolates of C. difficile were typed by polymerase chain reaction ribotyping. C. difficile was recovered from 3%, 11% and 26% of samples from low, medium and high risk rooms, respectively. In 10 high risk elderly care rooms, 24% (48/203) of samples were positive for C. difficile, with a mean of 6.8 colony-forming units (cfu) per 10 samples prior to hydrogen peroxide decontamination. Ribotyping identified the presence of the three main UK epidemic strains (ribotypes 001, 027 and 106) and four rooms contained mixed strains. After a single cycle of hydrogen peroxide decontamination, only 3% (7/203) of samples were positive (P<0.001), with a mean of 0.4 cfu per 10 samples ( approximately 94% reduction). The Sterinis hydrogen peroxide system significantly reduced the extent of environmental contamination with C. difficile in these elderly care rooms. This relatively quick and user-friendly technology might be a more reliable method of terminally disinfecting isolation rooms, following detergent cleaning, compared to the manual application of other disinfectants.
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Setlow, Barbara; Korza, George; Blatt, Kelly M.S.; Fey, Julien P.; Setlow, Peter
2015-01-01
Aims Determine how supercritical CO2 (scCO2) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2-PAA, and if spores inactivated by scCO2-PAA are truly dead. Methods and Results Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2-PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2-PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2-PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2-PAA sensitive. Conclusions These findings suggest that scCO2-PAA inactivates spores by damaging spores’ inner membrane. The spore coat provided scCO2-PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2-PAA resistance only for dry spores. Significance and Impact of Study These results provide information on mechanisms of spore inactivation of and resistance to scCO2-PAA, an agent with increasing use in sterilization applications. PMID:26535794
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Ishimori, Takateru; Takahashi, Katsutoshi; Goto, Masato; Nakagawa, Suguru; Kasai, Yoshiaki; Konagaya, Yukifumi; Batori, Hiroshi; Kobayashi, Atsushi
2012-01-01
The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids. PMID:22983975
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NASA Astrophysics Data System (ADS)
Ghormade, Vandana; Deshpande, M. V.
The ability of dimorphism in fungi is conventionally regarded as a reversible change between the two vegetative forms, yeast and mycelium, in response to environmental change. A zygomycetous isolate, Benjaminiella poitrasii, exhibited yeast-mycelium transition in response to the change in temperature (37-28 °C) and decrease in glucose concentration. For the first time the presence of dimorphic response during asexual and sexual spore germination is reported under the dimorphism-triggering conditions in B. poitrasii. The zygospores germinated into budding yeast when subjected to yeast-form supporting conditions. The mycelium-form favoring conditions gave rise to true mycelium. Similarly, the asexual spores displayed a dimorphic response during germination. Our observations suggest that dimorphism is an intrinsic ability present in the vegetative, asexual, and sexual forms of the fungus. As dimorphic fungi are intermediate to the unicellular yeast and the filamentous forms, understanding of the dimorphic character could be useful to trace the evolutionary relationships among taxonomically different fungi. Moreover, the implications of spore germination during the onset of pathogenesis and in drug development for human health care are discussed.
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Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana.
Mwamburi, Lizzy A; Laing, Mark D; Miller, Ray M
2015-03-01
Three non-ionic surfactants: Tween20, Tween80 and Breakthru (®) were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations . Breakthru (®) had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25-30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.
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Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana
Mwamburi, Lizzy A.; Laing, Mark D.; Miller, Ray M.
2015-01-01
Three non-ionic surfactants: Tween20, Tween80 and Breakthru ® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations . Breakthru ® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses. PMID:26221090
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Initiation of sporulation in Clostridium difficile: a twist on the classic model.
Edwards, Adrianne N; McBride, Shonna M
2014-09-01
The formation of dormant endospores is a complex morphological process that permits long-term survival in inhospitable environments for many Gram-positive bacteria. Sporulation for the anaerobic gastrointestinal pathogen Clostridium difficile is necessary for survival outside of the gastrointestinal tract of its host. While the developmental stages of spore formation are largely conserved among endospore-forming bacteria, the genus Clostridium appears to be missing a number of conserved regulators required for efficient sporulation in other spore-forming bacteria. Several recent studies have discovered novel mechanisms and distinct regulatory pathways that control the initiation of sporulation and early-sporulation-specific gene expression. These differences in regulating the decision to undergo sporulation reflects the unique ecological niche and environmental conditions that C. difficile inhabits and encounters within the mammalian host. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
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Permpoonpattana, Patima; Hong, Huynh A.; Phetcharaburanin, Jutarop; Huang, Jen-Min; Cook, Jenny; Fairweather, Neil F.; Cutting, Simon M.
2011-01-01
Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A. PMID:21482682
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In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL
Lambert, Emily A.; Sherry, Nora
2012-01-01
The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified. PMID:22343356
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Beilby, J P; Kidby, D K
1980-08-01
Neutral and polar spore lipids of the vesicular-arbuscular (VA) endophyte Glomus caledonius, were identified and quantitatively determined during spore germination, germ tube growth, and germ tube senescence. There are no previous reports detailing the spore lipid components of any member of the Endogenaceae, which is in the Zygomycotina. The fungus contained 45 to 72% total lipid depending upon its stage of growth. The concentration of neutral lipids decreased during germination while the polar lipids increased. Triacylglycerides were the most abundant neutral lipid, and lesser amounts of diacylglycerides, monoacylglycerides, free fatty acids, bound fatty acids, hydrocarbons, and sterols. The major fatty acids identified by gas--liquid chromatography and mass spectrometry were 16:1, 16:0, and 18:1. The minor fatty acids identified were n-3 and n-6 polyunsaturates. The n-3 polyunsaturated fatty acids have not been reported before in Zygomycetes. The fatty acid composition of the individual lipid classes was examined. The major phospholipids were phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, with smaller amounts of diphosphatidylglycerol and phosphatidic acid. The free sterol fraction was in greater quantity than sterol esters during germination and germ tube elongation. The capacity to synthesize sterols was demonstrated. Approximate net rates of change in the different lipid components were calculated. During spore germination and early germ tube growth, there was a net synthesis of lipids, with a large production of free fatty acids, in the germinating spore. Later in the growth period there was a net degradation of lipid, characterized by a large conversion of free fatty acids to unidentified compounds. During this period net free sterol synthesis ceased and sterol ester synthesis continued using the existing free sterol.
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CodY-Dependent Regulation of Sporulation in Clostridium difficile.
Nawrocki, Kathryn L; Edwards, Adrianne N; Daou, Nadine; Bouillaut, Laurent; McBride, Shonna M
2016-08-01
Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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CodY-Dependent Regulation of Sporulation in Clostridium difficile
Nawrocki, Kathryn L.; Edwards, Adrianne N.; Daou, Nadine; Bouillaut, Laurent
2016-01-01
ABSTRACT Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile. The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile. IMPORTANCE This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile. We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains. PMID:27246573
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Chitosan inhibits enterotoxigenic Clostridium perfringens type A in growth medium and chicken meat.
Alnoman, Maryam; Udompijitkul, Pathima; Sarker, Mahfuzur R
2017-06-01
Clostridium perfringens is a spore-forming bacterium and a major cause of bacterial food-borne illness. In this study, we evaluated the inhibitory effects of chitosan against spore germination, spore outgrowth and vegetative growth of C. perfringens food poisoning (FP) isolates. Chitosan of differing molecular weights inhibited germination of spores of all tested FP isolates in a KCl germinant solution containing 0.1 mg/ml chitosan at pH 4.5. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C. perfringens spores in Tripticase-yeast extract-glucose (TGY) medium. Furthermore, chitosan (1.0 mg/ml) was bacteriostatic against vegetative cells of C. perfringens in TGY medium. Although chitosan showed strong inhibitory activities against C. perfringens in laboratory medium, higher levels (2.0 mg/g) were required to achieve similar inhibition of spores inoculated into chicken meat. In summary, the inhibitory effects of chitosan against C. perfringens FP isolates was concentration dependent, and no major difference was observed when using different molecule weight chitosan as an inhibitor. Our results contribute to a better understanding on the potential application of chitosan in cooked meat products to control C. perfringens-associated disease. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Chea, F P; Chen, Y; Montville, T J; Schaffner, D W
2000-08-01
The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%). Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy. Data collected were used to develop the mathematical models. The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution. Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride. Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.
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NASA Technical Reports Server (NTRS)
Edwards, E. S.; Roux, S. J.
1998-01-01
A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 M sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 M sorbitol layer. Regeneration occurred over a period of 10-24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater.
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Germination Requirements of Bacillus macerans Spores
Sacks, L. E.; Thompson, P. A.
1971-01-01
2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. α-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described. PMID:4251279
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Xiao, Yinghua; van Hijum, Sacha A F T; Abee, Tjakko; Wells-Bennik, Marjon H J
2015-01-01
The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.
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Xiao, Yinghua; van Hijum, Sacha A. F. T.; Abee, Tjakko; Wells-Bennik, Marjon H. J.
2015-01-01
The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies. PMID:25978838
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Georget, Erika; Kapoor, Shobhna; Winter, Roland; Reineke, Kai; Song, Youye; Callanan, Michael; Ananta, Edwin; Heinz, Volker; Mathys, Alexander
2014-08-01
Bacterial spores are a major concern for food safety due to their high resistance to conventional preservation hurdles. Innovative hurdles can trigger bacterial spore germination or inactivate them. In this work, Geobacillus stearothermophilus spore high pressure (HP) germination and inactivation mechanisms were investigated by in situ infrared spectroscopy (FT-IR) and fluorometry. G. stearothermophilus spores' inner membrane (IM) was stained with Laurdan fluorescent dye. Time-dependent FT-IR and fluorescence spectra were recorded in situ under pressure at different temperatures. The Laurdan spectrum is affected by the lipid packing and level of hydration, and provided information on the IM state through the Laurdan generalized polarization. Changes in the -CH2 and -CH3 asymmetric stretching bands, characteristic of lipids, and in the amide I' band region, characteristic of proteins' secondary structure elements, enabled evaluation of the impact of HP on endospores lipid and protein structures. These studies were complemented by ex situ analyses (plate counts and microscopy). The methods applied showed high potential to identify germination mechanisms, particularly associated to the IM. Germination up to 3 log10 was achieved at 200 MPa and 55 °C. A molecular-level understanding of these mechanisms is important for the development and validation of multi-hurdle approaches to achieve commercial sterility. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Asynchronous spore germination in isogenic natural isolates of Saccharomyces paradoxus.
Stelkens, Rike B; Miller, Eric L; Greig, Duncan
2016-05-01
Spores from wild yeast isolates often show great variation in the size of colonies they produce, for largely unknown reasons. Here we measure the colonies produced from single spores from six different wild Saccharomyces paradoxus strains. We found remarkable variation in spore colony sizes, even among spores that were genetically identical. Different strains had different amounts of variation in spore colony sizes, and variation was not affected by the number of preceding meioses, or by spore maturation time. We used time-lapse photography to show that wild strains also have high variation in spore germination timing, providing a likely mechanism for the variation in spore colony sizes. When some spores from a laboratory strain make small colonies, or no colonies, it usually indicates a genetic or meiotic fault. Here, we demonstrate that in wild strains spore colony size variation is normal. We discuss and assess potential adaptive and non-adaptive explanations for this variation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Liu, Han; Li, Mingqian; Cai, Shunfeng; He, Xinyi; Shao, Yongqi; Lu, Xingmeng
2016-11-01
Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Germination and amplification of anthrax spores by soil-dwelling amoebas.
Dey, Rafik; Hoffman, Paul S; Glomski, Ian J
2012-11-01
While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.
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Studying molecular changes during gravity perception and response in a single cell.
Cannon, Ashley E; Salmi, Mari L; Bushart, Thomas J; Roux, Stanley J
2015-01-01
Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spores a valuable model system for the study of how a single cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. Molecular techniques have been developed to evaluate the transcriptomic and proteomic profiles of spores before and after gravity establishes the polarity of their development. Here we describe these techniques, along with protocols for sterilizing the spores, sowing them in a solid or liquid growth media, and evaluating germination.
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Beilby, J P
1983-05-01
The incorporation of isotopically labelled precursors, [1-14C]leucine, [2-14C]uracil and [1-14C]acetate, by germinating spores of the vesicular-arbuscular mycorrhizal fungus Glomus caledonium was investigated after prior incubation with several metabolic inhibitors. Inhibition of isotope incorporation was greatest with ethidium bromide and cycloheximide and least with chloramphenicol and actinomycin D. The incorporation of [1-14C]leucine into protein was reduced by 92% within 60 min of incubation in either ethidium bromide at 5 micrograms/mL or cycloheximide at 100 micrograms/mL. For these studies time zero was coincident with dry quiescent spores being first wet. Lipid synthesis during germination of the spores was detected at 120 min by following the incorporation of [1-14C]acetate. At 51 h, incorporation was greater in the triacylglycerides and diacylglycerides than in the free sterols, phospholipids, or free fatty acid. Spores exposed to cycloheximide for 60 min after imbibition showed very little phospholipid synthesis and no free fatty acid synthesis. The synthesis of RNA after spore imbibition is also discussed.
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Cerf, O
1977-01-01
Spores of Bacillus licheniformis 109-2A0 lost their refractility and absorbancy at 640 nm in the presence of metabolizable molecules (L-alanine). The same occurred with spores treated with 4.4 mol/1 hydrogen peroxide, pH 2.0, at 65 degrees C, even after 5 min of treatment. In addition, these transformations could be promoted after 2 min of treatment by inorganic ions (KI). This possibility occurs following a kinetics of activation. Thermodynamic parameters showed this activation to be combined with a molecular re-organization. Loss of refractility or absorbancy, induced by L-ala or KI, was inhibited by inhibitors of membrane functions or of L-alanine dehydrogenase, enzyme of which a noticeable activity was demonstrated in treated spores. Only 10% of spore calcium leaked during the treatment. Therefore loss of refractility or absorbancy caused by molecules metabolizable or not seemed to correspond to a physiological germination. The first even of the metabolic, as well as or the ionic germination could well be a modification of the spore membrane proton-motive force.
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Relation between germination and mycelium growth of individual fungal spores.
Gougouli, Maria; Koutsoumanis, Konstantinos P
2013-02-15
The relation between germination time and lag time of mycelium growth of individual spores was studied by combining microscopic and macroscopic techniques. The radial growth of a large number (100-200) of Penicillium expansum and Aspergillus niger mycelia originating from single spores was monitored macroscopically at isothermal conditions ranging from 0 to 30°C and 10 to 41.5°C, respectively. The radial growth curve for each mycelium was fitted to a linear model for the estimation of mycelium lag time. The results showed that the lag time varied significantly among single spores. The cumulative frequency distributions of the lag times were fitted to the modified Gompertz model and compared with the respective distributions for the germination time, which were obtained microscopically. The distributions of the measured mycelium lag time were found to be similar to the germination time distributions under the same conditions but shifted in time with the lag times showing a significant delay compared to germination times. A numerical comparison was also performed based on the distribution parameters λ(m) and λ(g), which indicate the time required from the spores to start the germination process and the completion of the lag phase, respectively. The relative differences %(λ(m)-λ(g))/λ(m) were not found to be significantly affected by temperatures tested with mean values of 72.5±5.1 and 60.7±2.1 for P. expansum for A. niger, respectively. In order to investigate the source of the above difference, a time-lapse microscopy method was developed providing videos with the behavior of single fungal spore from germination until mycelium formation. The distances of the apexes of the first germ tubes that emerged from the swollen spore were measured in each frame of the videos and these data were expressed as a function of time. The results showed that in the early hyphal development, the measured radii appear to increase exponentially, until a certain time, where growth becomes linear. The two phases of hyphal development can explain the difference between germination and lag time. Since the lag time is estimated from the extrapolation of the regression line of the linear part of the graph only, its value is significantly higher than the germination time, t(G). The relation of germination and lag time was further investigated by comparing their temperature dependence using the Cardinal Model with Inflection. The estimated values of the cardinal parameters (T(min), T(opt), and T(max)) for 1/λ(g) were found to be very close to the respective values for 1/λ(m), indicating similar temperature dependence between them. Copyright © 2012 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Pasanen, A.-L.; Heinonen-Tanski, H.; Kalliokoski, P.; Jantunen, M. J.
In the subarctic winter, fungal spores are found in indoor air even when outdoor spore levels are very low. The results of this study support an explanation that some indoor airborne fungal spores are derived from unnoticeable fungal microcolonies, which may develop on temporarily wet surfaces. Laboratory experiments on Penicillium verrucosum indicated that the fungus germinated on new wallpaper very quickly (about half an hour) under moist conditions. Hyphal growth and sporulation of the fungus on moist wallpaper occured within one day of incubation. In gravity-settling tape samples from occasionally wet surfaces in a suburban home, large spore aggregates, hyphal fragments with some spores and spores in the germination stage were found, indicating fungal growth. These experiments showed that fungal microcolonies can develop within a week on occasionally wet indoor surfaces.
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Xu Zhou, Ke; Li, Nan; Christie, Graham
2017-01-01
Abstract The adhesion of spores of 3 Bacillus species with distinctive morphologies to stainless steel and borosilicate glass was studied using the fluid dynamic gauging technique. Marked differences were observed between different species of spores, and also between spores of the same species prepared under different sporulation conditions. Spores of the food‐borne pathogen B. cereus were demonstrated to be capable of withstanding shear stresses greater than 1500 Pa when adhered to stainless steel, in contrast to spores of Bacillus subtilis and Bacillus megaterium, which detached in response to lower shear stress. An extended DLVO model was shown to be capable of predicting the relative differences in spore adhesion between spores of different species and different culture conditions, but did not predict absolute values of force of adhesion well. Applying the model to germinating spores showed a significant reduction in adhesion force shortly after triggering germination, indicating a potential strategy to achieve enhanced removal of spores from surfaces in response to shear stress, such as during cleaning‐in‐place procedures. Practical Application Spore‐forming bacteria are a concern to the food industry because they have the potential to cause food‐borne illness and product spoilage, while being strongly adhesive to processing surfaces and resistant to cleaning‐in‐place procedures. This work is of significance to the food processors and manufacturers because it offers insight to the properties of spore adhesion and identifies a potential strategy to facilitate the removal of spores during cleaning procedures. PMID:29125641
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2014-01-01
Background Failures to follow recommendations for reprocessing of surgical instruments may place patients at risk for exposure to pathogenic microorganisms. When such failures occur, medical facilities often face considerable uncertainty and challenges in assessing the actual risks of disease transmission. Methods In 2011, staff at an Ohio hospital determined that surgical instruments inside a Steriset Container had inadvertently been autoclaved on a gravity cycle rather than on the recommended pre-vacuum cycle, potentially exposing 72 patients who underwent surgery with the instruments to risk of infection. To provide an assessment of the level of risk, we tested the effectiveness of the machine washer/disinfector step and of the sterilization process inside the Steriset Container on the gravity cycle for killing of Geobacillus stearothermophilus spores, Clostridium difficile spores, and methicillin-resistant Staphylococcus aureus (MRSA). Based on the test results, the risk of transmission of MRSA by the instruments was calculated and the risk of transmission of hepatitis B virus was estimated. Results The machine washer/disinfector consistently reduced MRSA recovery by a factor of 1:100,000. The sterilization process inside the Steriset Container consistently reduced MRSA concentrations by a factor of >1:10,000,000 and killed 105C. difficile spores and 105G. stearothermophilus spores. The risk of MRSA transmission due to the incident was calculated to be 1 in 100 trillion. Conclusions The risk for transmission of infection due to the failure to follow recommended sterilization processes was negligible based upon complete killing of G. stearothermophilus biological indicator spores, C. difficile spores, and MRSA under conditions that replicated the incident where proper procedures were not followed. Such real-time assessments of the risks associated with specific incidents may provide evidence-based information that can be used to inform decisions regarding disclosure of the incident to patients. PMID:24447336
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Donskey, Curtis J; Yowler, Marian; Falck-Ytter, Yngve; Kundrapu, Sirisha; Salata, Robert A; Rutala, William A
2014-01-21
Failures to follow recommendations for reprocessing of surgical instruments may place patients at risk for exposure to pathogenic microorganisms. When such failures occur, medical facilities often face considerable uncertainty and challenges in assessing the actual risks of disease transmission. In 2011, staff at an Ohio hospital determined that surgical instruments inside a Steriset Container had inadvertently been autoclaved on a gravity cycle rather than on the recommended pre-vacuum cycle, potentially exposing 72 patients who underwent surgery with the instruments to risk of infection. To provide an assessment of the level of risk, we tested the effectiveness of the machine washer/disinfector step and of the sterilization process inside the Steriset Container on the gravity cycle for killing of Geobacillus stearothermophilus spores, Clostridium difficile spores, and methicillin-resistant Staphylococcus aureus (MRSA). Based on the test results, the risk of transmission of MRSA by the instruments was calculated and the risk of transmission of hepatitis B virus was estimated. The machine washer/disinfector consistently reduced MRSA recovery by a factor of 1:100,000. The sterilization process inside the Steriset Container consistently reduced MRSA concentrations by a factor of >1:10,000,000 and killed 105C. difficile spores and 105G. stearothermophilus spores. The risk of MRSA transmission due to the incident was calculated to be 1 in 100 trillion. The risk for transmission of infection due to the failure to follow recommended sterilization processes was negligible based upon complete killing of G. stearothermophilus biological indicator spores, C. difficile spores, and MRSA under conditions that replicated the incident where proper procedures were not followed. Such real-time assessments of the risks associated with specific incidents may provide evidence-based information that can be used to inform decisions regarding disclosure of the incident to patients.
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The role of aquaporins in pH-Dependent germination of Rhizopus delemar spores
USDA-ARS?s Scientific Manuscript database
Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an u...
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Lenz, Christian A.; Reineke, Kai; Knorr, Dietrich; Vogel, Rudi F.
2015-01-01
Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety. PMID:26191048
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Kodama, Takeko; Takamatsu, Hiromu; Asai, Kei; Kobayashi, Kazuo; Ogasawara, Naotake; Watabe, Kazuhito
1999-01-01
The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T2) of sporulation in wild-type cells and in spoIIIG (SigG−) and spoIVCB (SigK−) mutants but not in spoIIAC (SigF−) and spoIIGAB (SigE−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T2 of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in l-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T2 of sporulation and that it is required for the l-alanine-stimulated germination pathway. PMID:10419957
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Ren, Lixuan; Huo, Hongwei; Zhang, Fang; Hao, Wenya; Xiao, Liang; Dong, Caixia; Xu, Guohua
2016-06-02
Watermelon (Citrullus lanatus) is susceptible to wilt disease caused by the fungus Fusarium oxysporum f. sp niveum (FON). Intercropping management of watermelon/aerobic rice (Oryza sativa) alleviates watermelon wilt disease, because some unidentified component(s) in rice root exudates suppress FON sporulation and spore germination. Here, we show that the phenolic acid p-coumaric acid is present in rice root exudates only, and it inhibits FON spore germination and sporulation. We found that exogenously applied p-coumaric acid up-regulated the expression of ClPR3 in roots, as well as increased chitinase activity in leaves. Furthermore, exogenously applied p-coumaric acid increased β-1,3-glucanase activity in watermelon roots. By contrast, we found that ferulic acid was secreted by watermelon roots, but not by rice roots, and that it stimulated spore germination and sporulation of FON. Exogenous application of ferulic acid down-regulated ClPR3 expression and inhibited chitinase activity in watermelon leaves. Salicylic acid was detected in both watermelon and rice root exudates, which stimulated FON spore germination at low concentrations and suppressed spore germination at high concentrations. Exogenously applied salicylic acid did not alter ClPR3 expression, but did increase chitinase and β-1,3-glucanase activities in watermelon leaves. Together, our results show that the root exudates of phenolic acids were different between rice and watermelon, which lead to their special ecological roles on pathogenic fungus and watermelon defense.
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Ren, Lixuan; Huo, Hongwei; Zhang, Fang; Hao, Wenya; Xiao, Liang; Dong, Caixia; Xu, Guohua
2016-01-01
ABSTRACT Watermelon (Citrullus lanatus) is susceptible to wilt disease caused by the fungus Fusarium oxysporum f. sp niveum (FON). Intercropping management of watermelon/aerobic rice (Oryza sativa) alleviates watermelon wilt disease, because some unidentified component(s) in rice root exudates suppress FON sporulation and spore germination. Here, we show that the phenolic acid p-coumaric acid is present in rice root exudates only, and it inhibits FON spore germination and sporulation. We found that exogenously applied p-coumaric acid up-regulated the expression of ClPR3 in roots, as well as increased chitinase activity in leaves. Furthermore, exogenously applied p-coumaric acid increased β-1,3-glucanase activity in watermelon roots. By contrast, we found that ferulic acid was secreted by watermelon roots, but not by rice roots, and that it stimulated spore germination and sporulation of FON. Exogenous application of ferulic acid down-regulated ClPR3 expression and inhibited chitinase activity in watermelon leaves. Salicylic acid was detected in both watermelon and rice root exudates, which stimulated FON spore germination at low concentrations and suppressed spore germination at high concentrations. Exogenously applied salicylic acid did not alter ClPR3 expression, but did increase chitinase and β-1,3-glucanase activities in watermelon leaves. Together, our results show that the root exudates of phenolic acids were different between rice and watermelon, which lead to their special ecological roles on pathogenic fungus and watermelon defense. PMID:27217091
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Biochemical Changes and their Regulation during Spore Formation and Germination.
1980-04-09
r contain a very low level of cyclic GNP (cGMP), but cGMP Is not found in spores and it appears unlikely to be a modulator of sporulation, gemination...obtained that the regulation of this enzyme in vivo is accomplished at leas in part by regulation of levels of free Mn". 8TTatabolism during spore g...and germination, especially with regard to the following questions: 1) what are the levels and oxidation states of these compounds; 2) what are the
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Adenosine Monophosphate-Based Detection of Bacterial Spores
NASA Technical Reports Server (NTRS)
Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya
2009-01-01
A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.
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Clostridium difficile in Food and Animals: A Comprehensive Review.
Rodriguez, C; Taminiau, B; Van Broeck, J; Delmée, M; Daube, G
2016-01-01
Zoonoses are infections or diseases that can be transmitted between animals and humans through direct contact, close proximity or the environment. Clostridium difficile is ubiquitous in the environment, and the bacterium is able to colonise the intestinal tract of both animals and humans. Since domestic and food animals frequently test positive for toxigenic C. difficile, even without showing any signs of disease, it seems plausible that C. difficile could be zoonotic. Therefore, animals could play an essential role as carriers of the bacterium. In addition, the presence of the spores in different meats, fish, fruits and vegetables suggests a risk of foodborne transmission. This review summarises the current available data on C. difficile in animals and foods, from when the bacterium was first described up to the present.
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van Beilen, Johan W A; Brul, Stanley
2013-01-01
The internal pH (pHi) of a living cell is one of its most important physiological parameters. To monitor the pH inside Bacillus subtilis during various stages of its life cycle, we constructed an improved version (IpHluorin) of the ratiometric, pH-sensitive fluorescent protein pHluorin by extending it at the 5' end with the first 24 bp of comGA. The new version, which showed an approximate 40% increase in fluorescence intensity, was expressed from developmental phase-specific, native promoters of B. subtilis that are specifically active during vegetative growth on glucose (PptsG) or during sporulation (PspoIIA, PspoIIID, and PsspE). Our results show strong, compartment-specific expression of IpHluorin that allowed accurate pHi measurements of live cultures during exponential growth, early and late sporulation, spore germination, and during subsequent spore outgrowth. Dormant spores were characterized by an pHi of 6.0 ± 0.3. Upon full germination the pHi rose dependent on the medium to 7.0-7.4. The presence of sorbic acid in the germination medium inhibited a rise in the intracellular pH of germinating spores and inhibited germination. Such effects were absent when acetic was added at identical concentrations.
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Camardo Leggieri, Marco; Mitchell, David; Aldred, David; Battilani, Paola; Magan, Naresh
2014-12-01
The objective was to compare the ability of spores of Aspergillus carbonarius to germinate in vitro, in situ on grape skin and grape flesh in relation to temperature (15-40 °C) and different relative humidities (100-85% RH). Spores were inoculated as a spore suspension (10(6) spores ml(-1)) onto the surface of white organic grapes and directly onto cut grape flesh. For comparison, spores were spread plate onto a synthetic grape juice medium (SGM) modified to the equivalent water activity (a(w)) range of 0.995-0.85. This showed that conidia germinated more rapidly on grape flesh (6 h) followed by that on the SGM medium (9 h) and then grape skin (24 h) under optimal condition of 30-35 °C and 100 % RH. At marginal conditions, such as 15 °C and 85-90% RH, germination was very slow. The time to 5% germination was significantly shorter on grape flesh than in vitro on grape medium and slowest on grape skin. This suggests that damaged grapes provide the main method of infection and contamination of grapes and grape products with ochratoxin A (OTA). The combined effect of temperature and RH on conidial germination of A. carbonarius on SGM and grape skin was described by combining Beta and polynomial equations. The equations developed in this work provided a good fit of the biological processes; they could be integrated in a predictive model for infection and OTA prediction in ripening grapes. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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Infection control implications of the laundering of ambulance staff uniforms and reusable mops.
Mackay, W G; Whitehead, S; Purdue, N; Smith, M; Redhead, N; Williams, C; Wilson, S
2017-05-01
There is a lack of published studies on laundering in ambulance services. We performed bacterial culture on soiled and unsoiled uniforms and reusable mop heads artificially contaminated with Escherichia coli, Staphylococcus aureus, and Clostridium difficile spores. Current laundering processes used for routine cleans in the ambulances appears, from our simulations, to be effective at reducing vegetative pathogenic bacteria to undetectable levels, <3.398log 10 colony-forming units (S. aureus and E. coli). Reduced levels of C. difficile were still detected after laundering but the risk this poses for infection is unknown, as background levels of these spores in the environment are unknown. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
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USDA-ARS?s Scientific Manuscript database
To investigate how B. anthracis Stene spores survive in milk under heat (80 degree C, 10 minutes), pasteurization (72 degree C, 15 seconds) and pasteurization plus microfiltration, the expression levels of genes that related to sporulation and germination were tested using real-time PCR assays. Tw...
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Spore collection and elimination apparatus and method
Czajkowski, Carl [South Jamesport, NY; Warren, Barbara Panessa [Port Jefferson, NY
2007-04-03
The present invention is for a spore collection apparatus and its method of use. The portable spore collection apparatus includes a suction source, a nebulizer, an ionization chamber and a filter canister. The suction source collects the spores from a surface. The spores are activated by heating whereby spore dormancy is broken. Moisture is then applied to the spores to begin germination. The spores are then exposed to alpha particles causing extinction.
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Clostridium difficile Infection in Production Animals and Avian Species: A Review.
Moono, Peter; Foster, Niki F; Hampson, David J; Knight, Daniel R; Bloomfield, Lauren E; Riley, Thomas V
2016-12-01
Clostridium difficile is the leading cause of antibiotic-associated diarrhea and colitis in hospitalized humans. Recently, C. difficile infection (CDI) has been increasingly recognized as a cause of neonatal enteritis in food animals such as pigs, resulting in stunted growth, delays in weaning, and mortality, as well as colitis in large birds such as ostriches. C. difficile is a strictly anaerobic spore-forming bacterium, which produces two toxins A (TcdA) and B (TcdB) as its main virulence factors. The majority of strains isolated from animals produce an additional binary toxin (C. difficile transferase) that is associated with increased virulence. C. difficile is ubiquitous in the environment and has a wide host range. This review summarizes the epidemiology, clinical presentations, risk factors, and laboratory diagnosis of CDI in animals. Increased awareness by veterinarians and animal owners of the significance of clinical disease caused by C. difficile in livestock and avians is needed. Finally, this review provides an overview on methods for controlling environmental contamination and potential therapeutics available.
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Development of actinomycetes in brown semidesert soil under low water pressure
NASA Astrophysics Data System (ADS)
Zvyagintsev, D. G.; Zenova, G. M.; Sudnitsyn, I. I.; Gracheva, T. A.; Lapygina, E. E.; Napol'skaya, K. R.; Sydnitsyna, A. E.
2012-07-01
Under laboratory conditions, the spores of a xerotolerant Streptomyces odorifera strain germinated in brown semidesert soil even at extremely low soil water pressure ( P = -96.4 MPa, -964 atm, a w 0.50); the plantlets increased in length and formed mycelium, on which a new generation of spores was produced (a complete development cycle of the actinomycetes—from a spore to the formation of new spores—passed). The duration of the first cycles of the actinomycetes' development varied from 13 days at P = -27 atm to 57 days at P = -964 atm and was directly proportional to the absolute value of the soil water pressure ( P). In the first cycles of the actinomycetes' development, the rate of increase of the concentration of the germinated spores and mycelium, as well as the logarithms of the mycelium-to-germinated spore concentration ratios, was inversely proportional to the logarithm of P. These relationships indicated that the energy state of the water determined its availability to soil biota and, hence, the activity of its physiological and biochemical processes.
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Differentiation of Dictyostelium discoideum vegetative cells into spores during earth orbit in space
NASA Astrophysics Data System (ADS)
Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.; Ohnishi, T.
2001-01-01
We reported previously that emerged amoebae of Dictyosterium ( D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, γs13, and the parental strain, NC4. In γs13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.
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Zhang, Wen; Cheng, Ying; Du, Pengcheng; Zhang, Yuanyuan; Jia, Hongbing; Li, Xianping; Wang, Jing; Han, Na; Qiang, Yujun; Chen, Chen; Lu, Jinxing
2017-01-01
Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.
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Bernhards, Casey B.; Chen, Yan; Toutkoushian, Hannah
2014-01-01
Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn2+ or Ca2+ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation. PMID:25384476
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USDA-ARS?s Scientific Manuscript database
The inhibition of Bacillus cereus spore germination and outgrowth in cooked rice by nine fruit and vegetable peel powders prepared from store-bought conventional (nonorganic) and organic apples, oranges, and potatoes was investigated. The powders were mixed into rice at 10% (wt/wt) along with heat ...
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USDA-ARS?s Scientific Manuscript database
Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1, 1.5, or 2%, wt/wt), blend of sodium pyro-...
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USDA-ARS?s Scientific Manuscript database
Inhibition of Clostridium perfringens spore germination and outgrowth in ground turkey roast containing minimal ingredients (salt and sugar), by buffered vinegar (MoStatin V) and a blend (buffered) of lemon juice concentrate and vinegar (MoStatin LV) was evaluated. Ground turkey roast was formulat...
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Ali, S; Yui, S; Muzslay, M; Wilson, A P R
2017-10-01
Ultraviolet (UV) light decontamination systems are being used increasingly to supplement terminal disinfection of patient rooms. However, efficacy may not be consistent in the presence of soil, especially against Clostridium difficile spores. To demonstrate in-use efficacy of two whole-room UV decontamination systems against three hospital pathogens with and without soil. For each system, six patient rooms were decontaminated with UV irradiation (enhanced disinfection) following manual terminal cleaning. Total aerobic colony counts of surface contamination were determined by spot-sampling 15 environmental sites before and after terminal disinfection and after UV irradiation. Efficacy against biological indicator coupons (stainless-steel discs) was performed for each system using test bacteria (10 6 cfu EMRSA-15 variant A, carbapenemase-producing Klebsiella pneumoniae) or spores (10 5 cfu C. difficile 027), incorporating low soiling [0.03% bovine serum albumin (BSA)], heavy soiling (10% BSA) or synthetic faeces (C. difficile only) placed at five locations in the room. UV disinfection eliminated contamination after terminal cleaning in 8/14 (57%) and 11/14 (79%) sites. Both systems demonstrated 4-5 log 10 reductions in meticillin-resistant Staphylococcus aureus and K. pneumoniae at low soiling. Lower and more variable log 10 reductions were achieved when heavy soiling was present. Between 0.1 and 4.8 log 10 reductions in C. difficile spores were achieved with low but not heavy soil challenge. Terminal disinfection should be performed on all surfaces prior to UV decontamination. In-house validation studies should be considered to ensure optimal positioning in each room layout and sufficient cycle duration to eliminate target pathogens. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
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Bressuire-Isoard, Christelle; Broussolle, Véronique; Carlin, Frédéric
2018-05-17
Bacterial spores are resistant to physical and chemical insults, which make them a major concern for public health and for industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection, and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination, and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties.
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Spore-to-spore agar culture of the myxomycete Physarum globuliferum.
Liu, Pu; Wang, Qi; Li, Yu
2010-02-01
The ontogeny of the myxomycete Physarum globuliferum was observed on corn meal agar and hanging drop cultures without adding sterile oat flakes, bacteria or other microorganisms. Its complete life cycle including spore germination, myxamoebae, swarm cells, plasmodial development, and maturity of fructifications was demonstrated. Details of spore-to-spore development are described and illustrated.
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van Beilen, Johan W. A.; Brul, Stanley
2013-01-01
The internal pH (pHi) of a living cell is one of its most important physiological parameters. To monitor the pH inside Bacillus subtilis during various stages of its life cycle, we constructed an improved version (IpHluorin) of the ratiometric, pH-sensitive fluorescent protein pHluorin by extending it at the 5′ end with the first 24 bp of comGA. The new version, which showed an approximate 40% increase in fluorescence intensity, was expressed from developmental phase-specific, native promoters of B. subtilis that are specifically active during vegetative growth on glucose (PptsG) or during sporulation (PspoIIA, PspoIIID, and PsspE). Our results show strong, compartment-specific expression of IpHluorin that allowed accurate pHi measurements of live cultures during exponential growth, early and late sporulation, spore germination, and during subsequent spore outgrowth. Dormant spores were characterized by an pHi of 6.0 ± 0.3. Upon full germination the pHi rose dependent on the medium to 7.0–7.4. The presence of sorbic acid in the germination medium inhibited a rise in the intracellular pH of germinating spores and inhibited germination. Such effects were absent when acetic was added at identical concentrations. PMID:23785365
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The Molecular Timeline of a Reviving Bacterial Spore
Sinai, Lior; Rosenberg, Alex; Smith, Yoav; Segev, Einat; Ben-Yehuda, Sigal
2015-01-01
Summary The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens. PMID:25661487
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Oie, Shigeharu; Obayashi, Akiko; Yamasaki, Hirofumi; Furukawa, Hiroyuki; Kenri, Tsuyoshi; Takahashi, Motohide; Kawamoto, Keiko; Makino, Sou-ichi
2011-01-01
To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.
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Phosphorylation of spore coat proteins by a family of atypical protein kinases
DOE Office of Scientific and Technical Information (OSTI.GOV)
Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.
The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less
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Phosphorylation of spore coat proteins by a family of atypical protein kinases
Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...
2016-05-16
The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less
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Spore sensitivity to sunlight and freezing can restrict dispersal in wood-decay fungi
Norros, Veera; Karhu, Elina; Nordén, Jenni; Vähätalo, Anssi V; Ovaskainen, Otso
2015-01-01
Assessment of the costs and benefits of dispersal is central to understanding species' life-history strategies as well as explaining and predicting spatial population dynamics in the changing world. While mortality during active movement has received much attention, few have studied the costs of passive movement such as the airborne transport of fungal spores. Here, we examine the potential of extreme environmental conditions to cause dispersal mortality in wood-decay fungi. These fungi play a key role as decomposers and habitat creators in forest ecosystems and the populations of many species have declined due to habitat loss and fragmentation. We measured the effect of simulated solar radiation (including ultraviolet A and B) and freezing at −25°C on the spore germinability of 17 species. Both treatments but especially sunlight markedly reduced spore germinability in most species, and species with thin-walled spores were particularly light sensitive. Extrapolating the species' laboratory responses to natural irradiance conditions, we predict that sunlight is a relevant source of dispersal mortality at least at larger spatial scales. In addition, we found a positive effect of spore size on spore germinability, suggesting a trade-off between dispersal distance and establishment. We conclude that freezing and particularly sunlight can be important sources of dispersal mortality in wood-decay fungi which can make it difficult for some species to colonize isolated habitat patches and habitat edges. PMID:26380666
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Imaging bacterial spores by soft-x-ray microscopy
DOE Office of Scientific and Technical Information (OSTI.GOV)
Stead, A.D.; Ford, T.W.; Judge, J.
1997-04-01
Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores bymore » soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.« less
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Franco-Ramírez, Alicia; Ferrera-Cerrato, Ronald; Varela-Fregoso, Lucía; Pérez-Moreno, Jesús; Alarcón, Alejandro
2007-10-01
Arbuscular mycorrhizal fungi (AMF) have been hypothesized to enhance plant adaptation and growth in petroleum-contaminated soils. Nevertheless, neither AMF-biodiversity under chronically petroleum-contaminated soils nor spore germination response to petroleum hydrocarbons has been well studied. Chronically petroleum-contaminated rhizosphere soil and roots from Echinochloa polystachya, Citrus aurantifolia and C. aurantium were collected from Activo Cinco Presidentes, Tabasco, Mexico. Root colonization and spore abundance were evaluated. Additionally, rhizosphere soil samples were propagated using Sorghum vulgare L. as a plant trap under greenhouse conditions; subsequently, AMF-spores were identified. AMF-colonization ranged from 63 to 77% while spore number ranged from 715 to 912 in 100 g soil, suggesting that AMF tolerate the presence of petroleum hydrocarbons in the rhizosphere. From grass species, four AMF-morphospecies were identified: Glomus ambisporum, G. sinuosum (previously described as Sclerocystis sinuosum), Acaulospora laevis, and Ambispora gerdermanni. From citrus trees, four AMF-species were also identified: Scutellospora heterogama, G. ambisporum, Acaulospora scrobiculata, and G. citricola. In a second study, it was observed that spore germination and hyphal length of G. mosseae, G. ambisporum, and S. heterogama were significantly reduced by either volatile compounds of crude oil or increased concentrations of benzo[a ]pyrene or phenanthrene in water-agar.
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Banawas, Saeed; Korza, George; Paredes-Sabja, Daniel; Li, Yunfeng; Hao, Bing; Setlow, Peter; Sarker, Mahfuzur R
2015-09-01
The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C. perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Sporulation: how to survive on planet Earth (and beyond).
Huang, Mingwei; Hull, Christina M
2017-10-01
Sporulation is a strategy widely utilized by a wide variety of organisms to adapt to changes in their individual environmental niches and survive in time and/or space until they encounter conditions acceptable for vegetative growth. The spores produced by bacteria have been the subjects of extensive studies, and several systems such as Bacillus subtilis have provided ample opportunities to understand the molecular basis of spore biogenesis and germination. In contrast, the spores of other microbes, such as fungi, are relatively poorly understood. Studies of sporulation in model systems such as Saccharomyces cerevisiae and Aspergillus nidulans have established a basis for investigating eukaryotic spores, but very little is known at the molecular level about how spores function. This is especially true among the spores of human fungal pathogens such as the most common cause of fatal fungal disease, Cryptococcus neoformans. Recent proteomic studies are helping to determine the molecular mechanisms by which pathogenic fungal spores are formed, persist and germinate into actively growing agents of human disease.
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Posada-Flórez, Francisco J
2008-01-01
Two isolates of fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) were grown on cooked rice using diphasic liquid-solid fermentation in plastic bags to produce and harvest spore powder. The cultures were dried and significant differences were found for isolates and time of harvest. The spores were harvested manually and mechanically and after the cultures were dried for nine days, when moisture content was near 10%. After harvesting, spores were submitted to quality control to assess concentration, germination, purity, moisture content, particle size and pathogenicity to the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae). Spore productivity on cooked rice was less than 1×1010 spores/g using both manually and mechanically harvesting methodologies. Germination at 24 hours was over 75% and pathogenicity against H. hampei was over 92.5%. This methodology is suitable for laboratory and field studies, but not for industrial production when a high concentration of spores are required for formulation and field applications.
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Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores
2018-01-01
The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration. PMID:29503973
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Antimicrobial Testing Methods & Procedures: MB-31
Information about ATMP - SOP Quantitative Disk Carrier Test Method (QCT-2) Modified for Testing Antimicrobial Products Against Spores of Clostridium difficile (ATCC 43598) on Inanimate, Hard, Non-porous Surfaces - MB-31-Final
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Efficacy of surotomycin in an in vitro gut model of Clostridium difficile infection.
Chilton, C H; Crowther, G S; Todhunter, S L; Nicholson, S; Freeman, J; Chesnel, L; Wilcox, M H
2014-09-01
We investigated the efficacy of the cyclic lipopeptide surotomycin in treating clindamycin-induced Clostridium difficile infection (CDI) using an in vitro gut model. Two three-stage chemostat gut models were inoculated with human faeces, spiked with C. difficile spores (∼10(7) cfu/mL, PCR ribotype 027 or 001). Clindamycin (33.9 mg/L, four times daily for 7 days) was dosed to induce CDI. Following high-level toxin production, surotomycin (250 mg/L, twice daily for 7 days) was instilled. Microflora populations, C. difficile vegetative cells and spores, cytotoxin titres and antimicrobial levels (LC-MS/MS and bioassay) were determined. The emergence of C. difficile and enterococci with reduced susceptibility to surotomycin was monitored on breakpoint agar (4 × MIC). Counts of viable C. difficile were reduced to near the limit of detection on Days 1 and 3 of surotomycin instillation, and cytotoxin was undetectable on Days 3 and 4 of surotomycin instillation in the 027 and 001 models, respectively. Recurrence of vegetative growth and toxin production occurred 11 days (001 model) and 15 days (027 model) after surotomycin instillation had ceased, and remained for the duration of the experiment. Surotomycin instillation decreased populations of bifidobacteria, clostridia, enterococci and lactobacilli, but was sparing of Bacteroides fragilis group populations. All enumerated organisms had recovered to steady-state levels by 3 weeks post-surotomycin instillation. No evidence of the emergence of reduced susceptibility to surotomycin was observed. Surotomycin successfully reduced C. difficile vegetative cell counts and toxin levels in the gut model and was sparing of B. fragilis group populations. There was no evidence of decreased susceptibility to surotomycin during exposure or post-exposure. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Gebhart, Dana; Lok, Stephen; Clare, Simon; Tomas, Myreen; Stares, Mark; Scholl, Dean; Donskey, Curtis J.; Lawley, Trevor D.
2015-01-01
ABSTRACT Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin (“diffocin”) from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota. PMID:25805733
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Rechenmacher, C; Schmitt, J L; Droste, A
2010-12-01
Cyathea atrovirens (Langsd. & Fisch.) Domin, an intensely exploited tree fern, is found inside forests in several succession stages, as well as in swamps, roadsides and unused fields in the Rio dos Sinos basin, in the state of Rio Grande do Sul, southern Brazil. This study evaluated the in vitro germination and gametophyte development of C. atrovirens under different pH conditions, as well as spore viability after different storage times at 7 ºC. The lowest germination rate of spores was obtained at pH 7.0. At pH 5.0 to 6.5, laminar gametophyte development started at 20 to 30 days of culture. Antheridia and archegonia were first observed at 35 and 128 days, respectively. Storage at 7 ºC did not affect germination rates. The capability of germination at different pH levels may explain the occurrence of the species in a wide range of habitats. The present study contributes to the understanding of the full life-cycle of C. atrovirens and to the analysis of the influence of abiotic components, providing information for the cultivation, management and conservation of the species.
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Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw
2014-01-01
Objective To investigate effect of essential oils on Aspergillus spore germination, growth and mycotoxin production. Method In vitro antifungal and antiaflatoxigenic activity of essential oils was carried out using poisoned food techniques, spore germination assay, agar dilution assay, and aflatoxin arresting assay on toxigenic strains of Aspergillus species. Results Cymbopogon martinii, Foeniculum vulgare and Trachyspermum ammi (T. ammi) essential oils were tested against toxicogenic isolates of Aspergillus species. T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 µl/mL by essential oils of T. ammi. The oil also showed, complete inhibition of spore germination at a concentration of 2 µl/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting aflatoxin production from Aspergillus niger and Aspergillus flavus at 0.5 and 0.75 µl/mL, respectively. Cymbopogon martinii, Foeniculum vulgare and T. ammi oils as antifungal were found superior over synthetic preservative. Moreover, a concentration of 5 336.297 µl/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity and strengthening its traditional reputations. Conclusions In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by storage fungi. PMID:25183114
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Zhang, Kai-Mei; Shi, Lei; Jiang, Chuang-Dao; Li, Zhen-Yu
2008-05-01
Allelopathy of Ageratina adenophora plays an important role in its invasion. However, we have little knowledge of its allelpathic effects on ferns. In Petri dish bioassays, the inhibitory potential of aqueous leachates from roots, stems and leaves of A. adenophora was studied on the spore germination and gametophyte development of Macrothelypteris torresiana. All leachates inhibited the spore germination and growth of the first rhizoid of M. torresiana and inhibitory effects increased with increasing leachate concentrations. Root leachates proved most inhibitory. Gametophyte rhizoids of M. torresiana treated with stem and leaf leachates of A. adenophora were erect, which was similar to those of the control. However, gametophyte rhizoids of M. torresiana treated with root leachates of A. adenophora were erect, but also curving or swollen. Moreover, curving and swollen rhizoids increased with increasing concentrations. As time went by, rhizoids treated with root leachates were not so curved and the swelling almost disappeared. Possible causes are discussed in the present study. The increasing concentrations of leaf leachates also delayed the stages of gametophyte development. With the treatment of root leachates, the delay was more obvious. Thus A. adenophora inhibited the spore germination and gametophyte development of M. torresiana and the root leachates were most inhibitory.
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Antimicrobial Testing Methods & Procedures: MB-31-03
Information about ATMP - SOP Quantitative Disk Carrier Test Method (QCT-2) Modified for Testing Antimicrobial Products Against Spores of Clostridium difficile (ATCC 43598) on Inanimate, Hard, Non-porous Surfaces - MB-31-03
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Fifth international fungus spore conference
DOE Office of Scientific and Technical Information (OSTI.GOV)
Timberlake, W.E.
1993-04-01
This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.
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Kojime, Mineo; Kawakita, Kazuhito; Uritani, Ikuzo
1982-01-01
A factor which agglutinated the spores of Ceratocystis fimbriata in the presence of Ca2+ was purified from sweet potato (Ipomea batatas Lam cv. Norin[1]) root. Element composition of the purified factor was as follows; analysis found: C (29.8%), H (3.97%), O (65.34%), N (0.81%): calculated for C43H69O70N1: C (30.02%), H (4.01%), O (65.15%), N (0.81%). The factor was mainly composed of galacturonic acid (53% of dry weight) and contained arabinose, fucose, and unidentified component as minor components. The factor also agglutinated A-, B-, AB-, and O types of human erythrocytes to almost the same degree in the presence of Ca2+. The differential spore-agglutinating activity of the factor depended on the pH of the assay medium; it agglutinated similarly the germinated spores of sweet potato and coffee strains at pH 7.5 and 5.5, whereas it displayed a distinct differential agglutinating activity at pH 6.5. The factor was assayed for spore-agglutinating activity at pH 6.5, using the germinated and ungerminated spores of seven strains of C. fimbriata; sweet potato, coffee, prune, cacao, oak, taro, and almond strains. The factor agglutinated ungerminated spores of all seven strains similarly, although small differences were observed among strains. On the other hand, a clear differential agglutination was observed among the germinated spores of various strains; sweet potato and almond strains were highly insensitive in comparison with other strains. The growth of the agglutinated spores of C. fimbriata was inhibited. These results are discussed in relation to host-parasite specificity. Images PMID:16662232
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Bushart, Thomas J; Cannon, Ashley E; Ul Haque, Aeraj; San Miguel, Phillip; Mostajeran, Kathy; Clark, Gregory B; Porterfield, D Marshall; Roux, Stanley J
2013-01-01
Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.
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Grapefruit extract activity in the control of rose powdery mildew and black spot.
Wojdyła, A T
2001-01-01
Efficacy of grapefruit extract (a.i. of Biosept 33 SL) in the control of Sphaerotheca pannosa var. rosae and Diplocarpon rosae on roses was investigated during 1998-1999. The extract was applied as plant spray in concentrations from 0.017 to 0.099%. First treatment of rose shrubs was done when visible disease symptoms occurred on leaves and spraying was repeated 3 (in plastic tunnel) or 10-times (in the field) at weekly intervals. In the second experiment roses with visible powdery mildew symptoms were sprayed once with grapefruit extract. Leaves were sampled one or 7 days after the extract application and germination of spores of S. pannosa var. rosae on potato dextrose agar was evaluated. In the next experiment roses grown under plastic tunnel were sprayed once with the tested preparation. After 24 hours leaves were collected and appearance of fungal hyphae and spores of S. pannosa var. rosae was studied in scanning electron microscope. In the control of S. pannosa var. rosae grapefruit extract at conc. 0.066% was as effective as triforine (standard) applied at 0.027%. Reduction of concentration resulted in the decreased efficacy of the tested preparation. Spores of S. pannosa var. rosae collected one day after grapefruit extract application germinated in about 5%. Analyses of spore vitality 6 days letter showed that only about 15% of conidia could germinated on PDA agar. In contrary, spores from untreated leaves germinated in about 95%. Scanning electrone microscope analysis of leaves taken from plants protected with grapefruit extract showed that most of hyphae were separated from leaf surface. Almost all hyphae and spores were degenerated. In the control of D. rosae the preparation in all tested concentrations gave satisfactory results but was less effective than triforine.
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Distribution, dispersal and abundance of hayscented fern spores in mixed hardwood stands
Larry H. McCormick; Kathy A. Penrod
1995-01-01
A study was conducted in 1992 to assess the abundance and distribution of viable hayscented fern spores in the forest floor of central Pennsylvania hardwood stands before and after seasonal spore dispersal. Intact soil samples were collected at various distances and directions from established fern communities and placed in a greenhouse to effect spore germination....
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... spores during processes such as tanning hides and processing wool. Breathing in spores means a person has been exposed to anthrax. But it does not mean the person will have symptoms. The bacterial spores must germinate or sprout (the same way a seed sprouts before a plant grows) before the actual ...
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The inhibitory effects of sorbate and benzoate against Clostridium perfringens type A isolates.
Alnoman, Maryam; Udompijitkul, Pathima; Paredes-Sabja, Daniel; Sarker, Mahfuzur R
2015-06-01
This study evaluated the inhibitory effects of sorbate and benzoate against Clostridium perfringens type A food poisoning (FP) and non-food-borne (NFB) disease isolates. No significant inhibition of germination of spores of both FP and NFB isolates was observed in rich medium (pH 7.0) supplemented with permissive level of sodium sorbate (0.3% ≈ 0.13 mM undissociated sorbic acid) or sodium benzoate (0.1% ≈ 0.01 mM undissociated benzoic acid) used in foods. However, these levels of sorbate and benzoate effectively arrested outgrowth of germinated C. perfringens spores in rich medium. Lowering the pH of the medium increases the inhibitory effects of sorbate and benzoate against germination of spores of NFB isolates, and outgrowth of spores of both FP and NFB isolates. Furthermore, sorbate and benzoate inhibited vegetative growth of C. perfringens isolates. However, the permissible levels of these organic salts could not control the growth of C. perfringens spores in chicken meat stored under extremely abusive conditions. In summary, although sorbate and benzoate showed inhibitory activities against C. perfringens in the rich medium, no such effect was observed in cooked chicken meat. Therefore, caution should be taken when applying these organic salts into meat products to reduce or eliminate C. perfringens spores. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination
2009-12-01
exosporium maturation and assembly and suggest a novel role for the exosporium in germination. During starvation, bacteria of the genus Bacillus...Bacillus subtilis, the outermost struc- ture is a protective layer called the coat, which guards the spore against reactive small molecules, degradative ...analysis. Generation of anti-ExsK antibodies. Recombinant ExsK was generated and purified using the pET expression system (Novagen) according to the
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Shikata, Tomoyuki; Iseki, Mineo; Matsunaga, Shigeru; Higashi, Sho-ichi; Kamei, Yasuhiro; Watanabe, Masakatsu
2011-01-01
Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation. © 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.
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Fifth international fungus spore conference. [Abstracts]: Final technical report
DOE Office of Scientific and Technical Information (OSTI.GOV)
Timberlake, W.E.
1993-04-01
This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.
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Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio
2014-01-01
The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894
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Information about the Current Strain of Clostridium difficile
... rubs may not be as effective against spore-forming bacteria. Contact precautions Environmental cleaning and disinfection strategies. ... Detection of VISA/VRSA S.E.A.R.C.H. Labs Role in the Search and Containment of ...
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Alanazi, Saud; Alnoman, Maryam; Banawas, Saeed; Saito, Ryoichi; Sarker, Mahfuzur R
2018-08-01
C. perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and non-food-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly heat-resistant spores, it is of great interest to develop strategies alternative to thermal processing to inactivate C. perfrinegens. Thus, in this study we evaluated the inhibitory effects of essential oil constituents (EOCs) (cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol) against germination, outgrowth and vegetative growth of spores of C. perfringens FP and NFB disease isolates in laboratory medium and chicken meat. The cinnamaldehyde, eugenol and carvacrol, but not AITC, all at 0.05-0.1%, inhibited the germination of spores of all tested C. perfringens isolates in Tripticase-glucose-yeast extract (TGY) medium. Furthermore, all tested EOCs at 0.05-0.1% arrested the outgrowth and vegetative growth of C. perfringens spores in TGY, with AITC and carvacrol being the most effective. However, among four tested EOCs, only AITC (at 0.5%-2.0%) was able to inhibit the growth of C. perfringens spores in chicken meat and no such inhibitory effect was observed even with a 10-fold higher concentration (5%) of carvacrol. In conclusion, our current work identified AITC as an effective EOC to control spores and vegetative cells of C. perfringens isolates in laboratory medium and chicken meat. Further studies on evaluating the effectiveness of different combination of EOCs against C. perfringens spore growth in different meat products should establish an effective use of EOCs to control the risk of C. perfringens-mediated illnesses. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Winston, Jenessa A; Theriot, Casey M
2016-10-01
Clostridium difficile is an anaerobic, Gram positive, spore-forming bacillus that is the leading cause of nosocomial gastroenteritis. Clostridium difficile infection (CDI) is associated with increasing morbidity and mortality, consequently posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this pathogen. Susceptibility to CDI is associated with alterations in the gut microbiota composition and bile acid metabolome, specifically a loss of microbial derived secondary bile acids. This review aims to summarize in vitro, ex vivo, and in vivo studies done by our group and others that demonstrate how secondary bile acids affect the different stages of the C. difficile life cycle. Understanding the dynamic interplay of C. difficile and microbial derived secondary bile acids within the gastrointestinal tract will shed light on how bile acids play a role in colonization resistance against C. difficile. Rational manipulation of secondary bile acids may prove beneficial as a treatment for patients with CDI. Published by Elsevier Ltd.
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Atypical Presentation of C. Difficile Infection: Report of a Case with Literature Review
Towheed, Arooge; Tul Llah, Sibghat; Bin Abdulhak, Aref; Tilson-Mallett, Nancy R; Salkind, Alan
2016-01-01
Clostridium difficile (C. difficile) is a gram-positive, obligate, anaerobic spore-forming bacillus first reported by Hall and O'Toole in 1935. It occurs mostly after antibiotic use and invariably presents with watery diarrhea. We describe an atypical presentation of C. difficile in a 64-year-old Caucasian female who presented to the our emergency department with abdominal pain, nausea, and vomiting for one day. A complete blood count revealed leukocytosis 30 x 109/L and a subsequent computed tomography (CT) scan of the abdomen and the pelvis, showed fluid filled small bowel loops consistent with enteritis. Her presentation was unusual for lack of diarrhea, the hallmark of C. difficile infection. She was admitted and treated with oral vancomycin. The polymerase chain reaction (PCR) value in the stool for C. difficile was positive. The patient responded very well: her abdominal pain resolved and leukocyte count normalized after a few doses of vancomycin (125 mg po qid). The patient's progress was followed in our clinic for the last three months. PMID:27190728
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USDA-ARS?s Scientific Manuscript database
The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5C for 3 or...
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Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.
Bensman, M D; Mackie, R S; Minter, Z A; Gutting, B W
2012-08-01
The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species. Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria. The data suggested sera can have a significant impact on germination and growth of Sterne bacteria. These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
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Rousseau, Paul; Halvorson, Harlyn O.; Bulla, Lee A.; Julian, Grant St.
1972-01-01
Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation. Images PMID:4551750
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Sayed, W F
2011-06-01
Spores of four Frankia strains, the nitrogen-fixing actinomycete, were exposed to short wavelength UV-C radiation of 254 nm at 1 lux cm(2) (0.24 mw cm2 of energy) for 10 min. The used strains were HFP020203, UGL020604, UGL020602q and ORS021001. Exposure to UV was followed by reactivation with visible white light at 327.4 lux cm(2) for the same period of time. Spore germination percentage, spore protein content, and cell growth were damaged by this treatment. The lower and higher percentages of reduction in spore germination were 32 and 63% and, for the same strains, the recovery by white light was 7.2 and 37%. The lower percentages of UV damage and subsequent low recovery were recorded for strain ORS021001 that is considered more resistant to UV than the other strains. The higher percentages were recorded for strain HFP020203 that is more sensitive to UV but having more efficient repairing mechanisms. All the tested strains showed repairing activity induced by white light as indicated from the increase in their spore germination, protein content and almost restoring the normal shape of Frankia hyphae, after being damaged, as revealed by scanning electron microscope. This is the first evidence that photo-repairing systems are present in Frankia strains although there are variations in their response to both UV-C and photoreactivation by white light.
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Namvar, Azadeh; Warriner, Keith
2007-04-15
The fabrication of Bacillus subtilis endospore imprinted conducting polymer films and subsequent electrochemical detection of bound spores is reported. Imprinted films were prepared by absorbing spores on the surface of glassy carbon electrodes upon which a polypyrrole, followed by a poly(3-methylthiophene), layer were electrochemically deposited. Spore template release was achieved through soaking the modified electrode in DMSO. Binding of endospores to imprinted films could be detected via impedance spectroscopy by monitoring changes in Y'' (susceptance) using Mn(II)Cl2 (0.5M pH 3) as the supporting electrolyte. Here, the change in Y'' could be correlated to spore densities between 10(4) and 10(7)cfu/ml. More sensitive detection of absorbed spores was achieved by following endospore germination via changes in film charge as measured using cyclic voltammetry. Here, imprinted films were submerged in spore suspensions to permit absorption, heat activated at 70 degrees C for 10 min prior to transferring to an electrochemical cell containing germination activators. By using the assay format it was possible to detect 10(2)cfu/ml. The observed changes in film charge could be attributed to the interaction of the supporting conducting polymer with dipicolinic acid (DPA) and other constituents released from the core in the course of germination. In all cases, it was not possible to regenerate the imprinted films without losing electrode response. In summary, the study has provided proof-of-concept for fabricating microbial imprinted films using conducting polymers.
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Octanal inhibits spore germination of Penicillium digitatum involving membrane peroxidation.
Dou, Shiwen; Liu, Shengquan; Xu, Xiaoyong; OuYang, Qiuli; Tao, Nengguo
2017-07-01
Octanal is a potential alternative to chemical fungicides in controlling postharvest disease of citrus fruit. In this study, the antifungal activity and the underlying mechanism of octanal against spore germination of Penicillium digitatum, one of the main postharvest pathogens in citrus, were investigated. Results showed that octanal at different concentrations (0, 0.25, 0.50, 1.00, 2.00 μl/ml) inhibited the growth of P. digitatum spores in a dose-dependent manner. The morphology and the membrane permeability of P. digitatum spores were visibly altered by 0.25 and 2.00 μl/ml of octanal. Meanwhile, octanal decreased the total lipids contents of P. digitatum spores, indicating that the membrane integrity is damaged. Furthermore, octanal apparently induced the massive accumulation of total malonaldehyde (MDA) and the reactive oxygen species (ROS). An increase in the activities of lipoxygenase (LOX), NADH oxidase, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) was also observed. These results suggested that a membrane damage mechanism involving membrane peroxidation might contribute to the antifungal activity of octanal against P. digitatum spores.
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Burke, D G; Harrison, M J; Fleming, C; McCarthy, M; Shortt, C; Sulaiman, I; Murphy, D M; Eustace, J A; Shanahan, F; Hill, C; Stanton, C; Rea, M C; Ross, R P; Plant, B J
2017-03-01
Clostridium difficile is an anaerobic Gram-positive, spore-forming, toxin-producing bacillus transmitted among humans through the faecal-oral route. Despite increasing carriage rates and the presence of C. difficile toxin in stool, patients with CF rarely appear to develop typical manifestations of C. difficile infection (CDI). In this study, we examined the carriage, toxin production, ribotype distribution and antibiotic susceptibility of C. difficile in a cohort of 60 adult patients with CF who were pre-lung transplant. C. difficile was detected in 50% (30/60) of patients with CF by culturing for the bacteria. C. difficile toxin was detected in 63% (19/30) of C. difficile-positive stool samples. All toxin-positive stool samples contained toxigenic C. difficile strains harbouring toxin genes, tcdA and tcdB. Despite the presence of C. difficile and its toxin in patient stool, no acute gastrointestinal symptoms were reported. Ribotyping of C. difficile strains revealed 16 distinct ribotypes (RT), 11 of which are known to be disease-causing including the hyper-virulent RT078. Additionally, strains RT002, RT014, and RT015, which are common in non-CF nosocomial infection were described. All strains were susceptible to vancomycin, metronidazole, fusidic acid and rifampicin. No correlation was observed between carriage of C. difficile or any characteristics of isolated strains and any recorded clinical parameters or treatment received. We demonstrate a high prevalence of hypervirulent, toxigenic strains of C. difficile in asymptomatic patients with CF. This highlights the potential role of asymptomatic patients with CF in nosocomial transmission of C. difficile. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
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Influence of gravity and light on the developmental polarity of Ceratopteris richardii fern spores
NASA Technical Reports Server (NTRS)
Edwards, E. S.; Roux, S. J.
1998-01-01
The polarity of germinating single-celled spores of the fern Ceratopteris richardii Brogn. is influenced by gravity during a time period prior to the first cellular division designated a "polarity-determination window". After this window closes, control of polarity is seen in the downward (with respect to gravity) migration of the nucleus along the proximal face of the spore and the subsequent downward growth of the primary rhizoid. When spores are germinated on a clinostat the direction of nuclear migration and subsequent primary rhizoid growth is random. However, in each case the direction of nuclear migration predicts the direction of rhizoid elongation. Although it is the most obvious movement, the downward migration is not the first movement of the nucleus. During the polarity-determination window, the nucleus moves randomly within a region centered behind the trilete marking. While the polarity of many fern spores has been reported to be controlled by light, spores of C. richardii are the first documented to have their polarity influenced by gravity. Directional white light also affects the polarity of these spores, but this influence is slight and is secondary to that of gravity.
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The Effect of Resin and Monoterpenes on Spore Germination and Growth in Fusarium circinatum.
Slinski, S L; Zakharov, F; Gordon, T R
2015-01-01
Resin obtained from Pinus radiata and five monoterpene components of resin (limonene, α-pinene, β-pinene, camphene, and myrcene) were tested to determine their effects on mycelial growth and germination and survival of spores of Fusarium circinatum, the cause of pitch canker in pine, and F. temperatum, which is interfertile with F. circinatum but not pathogenic to pine. Averaged across all treatments, F. temperatum sustained the greatest reduction in radial growth (16.9±0.02% of control). The greatest reduction in dry weight also occurred in F. temperatum (11.7±0.01% of control), and all isolates of F. circinatum were significantly less affected (P<0.05). Spore germination rates in a saturated atmosphere of monoterpenes were relatively high for all tested isolates but, when placed in direct contact with resin, spore survival was significantly greater for F. circinatum than for F. temperatum. Our results are consistent with the hypothesis that greater tolerance of resin is one factor distinguishing F. circinatum from the nonpathogenic F. temperatum. However, differential tolerance of monoterpene components of resin is not sufficient to explain the observed variation in virulence to pine in F. circinatum.
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The Nature of Cold-induced Dormancy in Urediospores of Puccinia graminis tritici
Maheshwari, Ramesh; Sussman, Alfred S.
1971-01-01
When air-dry urediospores of the wheat stem rust, Puccinia graminis f. sp. tritici, are exposed to temperatures below freezing, their germinability is markedly reduced, even after prolonged thawing at room temperature. Germinability is fully restored by a brief heat-shock or by vapor phase hydration. We have found that this “cold dormancy” cannot be reversed once the spores contact liquid water. Enhanced loss of metabolites occurs immediately upon suspension of cold-dormant urediospores in liquid without a prior heat-shock. Such leakage is two to three times greater than from untreated or heatshocked cold-dormant spores and accounts for up to 70% of the soluble pool of metabolites normally present in germinating urediospores. Respiratory activity of cold-dormant urediospores declines rapidly during incubation in liquid. Incorporation of isotopic carbon into cold-dormant urediospores is only a fraction of that of untreated or heat-activated spores. Thus, cold shock transforms the spores into a state of supersensitivity to liquid water, which is reversed by heat-shock or slow hydration by vapor phase equilibration. The primary cause of damage to cold-dormant cells exposed to liquid water appears to be irreversible permeability damage, followed by metabolic injury. PMID:16657610
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Contamination of ready-to-eat raw vegetables with Clostridium difficile in France.
Eckert, Catherine; Burghoffer, Béatrice; Barbut, Frédéric
2013-09-01
The presence of Clostridium difficile in food like shellfish, vegetables and meat has been reported in several publications during the past few years. The objective of this study was to assess the prevalence of ready-to-eat raw vegetables contaminated with C. difficile in France. One hundred and four ready-to-eat salads and vegetables were studied. Toxigenic C. difficile strains were isolated in three samples (2.9 %): two ready-to-eat salads (one heart of lettuce and one lamb's lettuce salad) and one portion of pea sprouts. The strains belonged to three different PCR ribotypes: 001, 014/020/077 and 015. The detection thresholds for vegetative cells and spores cells varied between 1 and 3 c.f.u. in 20 g salad and between 6 and 15 c.f.u. in 20 g salad, respectively, for the method employed.
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NASA Technical Reports Server (NTRS)
Haas, C. J.; Scheuerlein, R.; Roux, S. J.
1991-01-01
The alkaloid staurosporine, currently known as the most potent inhibitor of protein kinase C, PKC, was tested for its ability to inhibit phytochrome-mediated spore germination in Dryopteris filix-mas L., evaluated by the induction of chlorophyll synthesis. Approximately half-maximal inhibition was obtained at a concentration of 10(-5) M. This effect of staurosporine was phase-specific and was found during the same period in which the presence of extracellular calcium is necessary for realization of the light signal. Furthermore, the ability of staurosporine to prevent progression of a germinated spore into early gametophyte development, evaluated by the accumulation of chlorophyll, was examined. Again, staurosporine (10(-5) M) significantly diminished chlorophyll accumulation, determined quantitatively in vivo by single-cell measurements, in a non-phase specific way. The fact that the phase-specific inhibitory effect of staurosporine in preventing germination was coincident with the phase-specific requirement of Ca2+ suggests that both Ca2+ and staurosporine affect the same step in the signal-transduction chain. A phosphorylation event catalysed by PKC or any Ca2+ -dependent protein kinase is proposed as the target of staurosporine and Ca2+.
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Tzortzakis, Nikos G
2010-08-15
Anthracnose rot (Colletotrichum coccodes) development in vitro or in tomato (Lycopersicon esculentum L.) fruit was evaluated after treatment with absolute ethyl alcohol (AEA), vinegar (VIN), chlorine (CHL) or origanum oil (ORI) and storage at 12 degrees C and 95% relative humidity during or following exposure to the volatiles. Fruit treated with vapours reduced fungal spore germination/production, but in the case of AEA- and VIN-treated fruits, fungal mycelium development was accelerated. Fruit lesion development was suppressed after fruit exposure to pure (100% v/v) AEA or ORI vapours which were accompanied by increased fruit cracking. Exposure to pure VIN-, CHL- and ORI vapours reduced (up to 92%) spore germination in vitro, but no differences were observed in the AEA treatment. The benefits associated with volatiles-enrichment were maintained in fruit pre-exposed to vapours, resulting in suppression in spore germination and spore production. However, studies performed on fungi grown on Potato Dextrose Agar revealed fewer direct effects of volatiles on fungal colony development and spore germination per se, implying that suppression of pathogen development was due in a large part to the impact of volatiles on fruit-pathogen interactions and/or 'memory' effects on fruit tissue. Work is currently focussing on the mechanisms underlying the impacts of volatiles on fruit quality related attributes. The results of this study indicate that volatiles may be considered as an alternative to the traditional postharvest sanitizing techniques. Each commodity needs to be individually assessed, and the volatile concentration and sanitising technique optimised, before the volatile treatment is used commercially. Copyright 2010 Elsevier B.V. All rights reserved.
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Anastomosis behavior differs between asymbiotic and symbiotic hyphae of Rhizophagus clarus.
Purin, Sonia; Morton, Joseph B
2013-01-01
The life history of arbuscular mycorrhizal fungi (AMF, Glomeromycota) consists of a short asymbiotic phase when spores germinate and a longer symbiotic phase where hyphae form a network within roots and subsequently in the rhizosphere. Hyphal anastomosis contributes to colony formation, yet this process has been studied mostly in the asymbiotic phase rather than in mycorrhizal plants because of methodological limitations. We sought to compare patterns of anastomosis during each phase of fungal growth by measuring hyphal fusions in genetically identical and different single spore isolates of Rhizophagus clarus from different environments and geographic locations. These isolates were genotyped with two anonymous markers of microsatellite-flanking regions. Anastomosis of hyphae from germinating spores was examined in axenic Petri dishes. A rhizohyphatron consisting of agar-coated glass slides bridging single or paired mycorrhizal sorghum plants allowed evaluation of anastomosis of symbiotic hyphae. Anastomosis of hyphae within a colony, defined here as a mycelium from an individual germinating spore or from mycorrhizal roots of one plant, occurred with similar frequencies (8-38%). However, anastomosis between paired colonies was observed in germinating spores from either genetically identical or different isolates, but it was never detected in symbiotic hyphae. The frequency of anastomosis in asymbiotic hyphae from paired interactions was low, occurring in fewer than 6% of hyphal contacts. These data suggest that anastomosis is relatively unconstrained when interactions occur within a colony but is confined to asymbiotic hyphae when interactions occur between paired colonies. This pattern of behavior suggests that asymbiotic and symbiotic phases of mycelium development by R. clarus may differ in function. Anastomosis in the asymbiotic phase may provide brief opportunities for gene flow between populations of this and possibly other AMF species.
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Nicholson, Wayne L; Moeller, Ralf; Horneck, Gerda
2012-05-01
Because of their ubiquity and resistance to spacecraft decontamination, bacterial spores are considered likely potential forward contaminants on robotic missions to Mars. Thus, it is important to understand their global responses to long-term exposure to space or martian environments. As part of the PROTECT experiment, spores of B. subtilis 168 were exposed to real space conditions and to simulated martian conditions for 559 days in low-Earth orbit mounted on the EXPOSE-E exposure platform outside the European Columbus module on the International Space Station. Upon return, spores were germinated, total RNA extracted, fluorescently labeled, and used to probe a custom Bacillus subtilis microarray to identify genes preferentially activated or repressed relative to ground control spores. Increased transcript levels were detected for a number of stress-related regulons responding to DNA damage (SOS response, SPβ prophage induction), protein damage (CtsR/Clp system), oxidative stress (PerR regulon), and cell envelope stress (SigV regulon). Spores exposed to space demonstrated a much broader and more severe stress response than spores exposed to simulated martian conditions. The results are discussed in the context of planetary protection for a hypothetical journey of potential forward contaminant spores from Earth to Mars and their subsequent residence on Mars.
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Cuthbertson, Andrew G S; Walters, Keith F A; Deppe, Carola
2005-08-01
The compatibility of the entomopathogenic fungus Lecanicillium muscarium and chemical insecticides used to control the second instar stages of the sweetpotato whitefly, Bemisia tabaci, was investigated. The effect on spore germination of direct exposure for 24 h to the insecticides imidacloprid, buprofezin, teflubenzuron and nicotine was determined. Only exposure to buprofezin was followed by acceptable spore germination. However, all chemicals significantly reduced spore germination when compared to a water control. Infectivity of L. muscarium in the presence of dry residues of buprofezin, teflubenzuron and nicotine (imidacloprid is a systemic pesticide) on foliage were also investigated. No significant detrimental effects on the level of control of B. tabaci was recorded when compared with fungi applied to residue free foliage on either tomato or verbena plants. Fungi in combination with imidacloprid gave higher B. tabaci mortality on verbena foliage compared to either teflubenzuron or nicotine and fungi combinations. Use of these chemical insecticides with L. muscarium in integrated control programmes for B. tabaci is discussed.
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Secondary Metabolites Produced during the Germination of Streptomyces coelicolor.
Čihák, Matouš; Kameník, Zdeněk; Šmídová, Klára; Bergman, Natalie; Benada, Oldřich; Kofroňová, Olga; Petříčková, Kateřina; Bobek, Jan
2017-01-01
Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor . Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer ( quorum sensing ) and/or play a role in competitive microflora repression ( quorum quenching ) in their nature environments.
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Secondary Metabolites Produced during the Germination of Streptomyces coelicolor
Čihák, Matouš; Kameník, Zdeněk; Šmídová, Klára; Bergman, Natalie; Benada, Oldřich; Kofroňová, Olga; Petříčková, Kateřina; Bobek, Jan
2017-01-01
Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments. PMID:29326665
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The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression
Lammers, Peter J.; Jun, Jeongwon; Abubaker, Jehad; Arreola, Raul; Gopalan, Anjali; Bago, Berta; Hernandez-Sebastia, Cinta; Allen, James W.; Douds, David D.; Pfeffer, Philip E.; Shachar-Hill, Yair
2001-01-01
The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of 13C labeling of germinating spores and extraradical mycelium with 13C2-acetate and 13C2-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle. PMID:11706207
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Yang, Wan-Wan; Ponce, Adrian
2011-01-01
A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alanine triggers Clostridium spore germination and the concomitant release of ∼108 molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb3+-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil). PMID:21296951
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Hay-scented fern spore production following clearcutting
Kathy A. Penrod; Larry H. McCormick
1997-01-01
Hay-scented fern is a common forest understory weed native to the Appalachian region. It interferes with oak and other hardwood seedling growth and often leads to regeneration failures. Harvesting is know to increase rates of vegetative expansion, spore germination, and possibly spore production of hay-scented fern. To examine the latter effect, a progressive series of...
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Community Environmental Contamination of Toxigenic Clostridium difficile
Alam, M Jahangir; Walk, Seth T.; Endres, Bradley T.; Basseres, Eugenie; Khaleduzzaman, Mohammed; Amadio, Jonathan; Musick, William L.; Christensen, Jennifer L.; Kuo, Julie; Atmar, Robert L.
2017-01-01
Abstract Background. Clostridium difficile infection is often considered to result from recent acquisition of a C difficile isolate in a healthcare setting. However, C difficile spores can persist for long periods of time, suggesting a potentially large community environmental reservoir. The objectives of this study were to assess community environmental contamination of toxigenic C difficile and to assess strain distribution in environmental versus clinical isolates. Methods. From 2013 to 2015, we collected community environmental swabs from homes and public areas in Houston, Texas to assess C difficile contamination. All positive isolates were tested for C difficile toxins A and B, ribotyped, and compared with clinical C difficile isolates obtained from hospitalized patients in Houston healthcare settings. Results. A total of 2538 environmental samples were collected over the study period. These included samples obtained from homes (n = 1079), parks (n = 491), chain stores (n = 225), fast food restaurants (n = 123), other commercial stores (n = 172), and hospitals (n = 448). Overall, 418 environmental isolates grew toxigenic C difficile (16.5%; P < .001) most commonly from parks (24.6%), followed by homes (17.1%), hospitals (16.5%), commercial stores (8.1%), chain stores (7.6%), and fast food restaurants (6.5%). A similar distribution of ribotypes was observed between clinical and environmental isolates with the exception that ribotype 027 was more common in clinical isolates compared with environmental isolates (P < .001). Conclusions. We identified a high prevalence of toxigenic C difficile from community environs that were similar ribotypes to clinical isolates. These findings suggest that interventions beyond isolation of symptomatic patients should be targeted for prevention of C difficile infection. PMID:28480289
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Patrick, David M.; Mak, Sunny; Jardine, Claire M.; Tang, Patrick; Weese, J. Scott
2014-01-01
Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration. PMID:24317079
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Himsworth, Chelsea G; Patrick, David M; Mak, Sunny; Jardine, Claire M; Tang, Patrick; Weese, J Scott
2014-02-01
Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 "novel" ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.
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[Epidemiology, risk factors and prevention of Clostridium difficile nosocomial infections].
Barbut, F; Petit, J C
2000-10-01
Clostridium difficile is responsible for 10-25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudo-membranous colitis (PMC). This anaerobic spore-forming bacterium has been identified as the leading cause of nosocomial infectious diarrhea in adults. Pathogenesis relies on a disruption of the normal bacterial flora of the colon, a colonization by C. difficile and the release of toxins A and B that cause mucosal damage and inflammation. Incidence of C. difficile intestinal disorders usually varies from one to 40 per thousand patient admissions. Risk factors for C. difficile-associated diarrhea include antimicrobial therapy, older age (> 65 years), antineoplastic chemotherapy, and length of hospital stay. Nosocomial transmission of C. difficile via oro-fecal route occurs in 3-30% of total patient admissions but it remains asymptomatic in more than 66% of cases. Persistent environmental contamination and carrying of the organism on the hands of hospital staff are common. Measures that are effective in reducing cross-infection consist of an accurate and rapid diagnosis, an appropriate treatment, an implementation of enteric precautions for symptomatic patients, a reinforcement of hand-washing and a daily environmental disinfection. C. difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.
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Carvacrol suppresses high pressure high temperature inactivation of Bacillus cereus spores.
Luu-Thi, Hue; Corthouts, Jorinde; Passaris, Ioannis; Grauwet, Tara; Aertsen, Abram; Hendrickx, Marc; Michiels, Chris W
2015-03-16
The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments are conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5 min at 600MPa in the temperature range of 50-100°C, using temperature increments of 5°C. Additionally, we investigated the effect of the natural antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by HPHT was less than about 1 log unit at 50 to 70°C, but gradually increased at higher temperatures up to about 5 log units at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤65°C) than at high (≥70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at ≤65°C and also partly inhibited DPA release at ≥65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 65-90°C but unaffected at 95-100°C. Copyright © 2014 Elsevier B.V. All rights reserved.
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Early development of fern gametophytes in microgravity
NASA Technical Reports Server (NTRS)
Roux, Stanley J.; Chatterjee, Ani; Hillier, Sheila; Cannon, Tom
2003-01-01
Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth. c2002 Published by Elsevier Science Ltd on behalf of COSPAR.
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Fungistatic activity of some perfumes against otomycotic pathogens.
Jain, S K; Agrawal, S C
2002-04-01
The sporostatic effect of five otomycotic pathogens, i.e. Aspergillus niger, A. flavus, Absidia corymbifera, Penicillium nigricans and Candida albicans to nine different perfumes was determined on the basis of their spore germination. These organisms were isolated from patients suffering from fungal infection of the external auditory canal. Volatile vapours emanating from musk, phulwari, jasmine, nagchampa and bela caused approximately 100% inhibition in spore germination of all the test fungi. Volatiles emanating from chandan, khas and hina showed no inhibition for the test pathogens, displaying their resistant character to these perfumes.
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Tompkin, R B; Christiansen, L N; Shaparis, A B
1978-01-01
Perishable canned cured meat inoculated with Clostridium botulinum spores was placed at 4.4 or 10 degrees C after manufacture. Spore germination occurred at 10 degrees C. The germinated cell count remained stable over a period of 16 to 18 weeks. During that time period the inhibitory system and residual nitrite descreased. These factors combine to make perishable canned cured meats more prone to spoilage and potential hazard if they are temperature abused after a period of refrigerated storage. PMID:350155
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Weidenbach, Denise; Jansen, Marcus; Franke, Rochus B; Hensel, Goetz; Weissgerber, Wiebke; Ulferts, Sylvia; Jansen, Irina; Schreiber, Lukas; Korzun, Viktor; Pontzen, Rolf; Kumlehn, Jochen; Pillen, Klaus; Schaffrath, Ulrich
2014-11-01
For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago. © 2014 American Society of Plant Biologists. All Rights Reserved.
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Weidenbach, Denise; Jansen, Marcus; Franke, Rochus B.; Hensel, Goetz; Weissgerber, Wiebke; Ulferts, Sylvia; Jansen, Irina; Schreiber, Lukas; Korzun, Viktor; Pontzen, Rolf; Kumlehn, Jochen; Pillen, Klaus; Schaffrath, Ulrich
2014-01-01
For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago. PMID:25201879
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Release of elicitors from rice blast spores under the action of reactive oxygen species
USDA-ARS?s Scientific Manuscript database
The effects of reactive oxygen species (ROS) on secretion of hypothesized elicitors from spores of rice blast causal fungus Magnaporthe grisea were studied. For spore exposure to exogenous ROS, they were germinated for 5 h in 50 µM H2O2 followed by addition of catalase E.C. 1.11.1.6 (to decompose pe...
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Secco, Danielle Angst; Balassiano, Ilana Teruszkin; Boente, Renata Ferreira; Miranda, Karla Rodrigues; Brazier, Jon; Hall, Val; dos Santos-Filho, Joaquim; Lobo, Leandro Araujo; Nouér, Simone Aranha; Domingues, Regina Maria Cavalcanti Pilotto
2014-08-01
Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 → Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P.
2015-01-01
Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. PMID:26386058
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Tomasula, P M; Mukhopadhyay, S; Datta, N; Porto-Fett, A; Call, J E; Luchansky, J B; Renye, J; Tunick, M
2011-09-01
High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log(10) BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91±0.05 log(10) BA spores/mL of milk and the 1.4-μm membrane removed 4.50±0.35 log(10) BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log(10) spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log(10) BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Gebhart, Dana; Lok, Stephen; Clare, Simon; Tomas, Myreen; Stares, Mark; Scholl, Dean; Donskey, Curtis J; Lawley, Trevor D; Govoni, Gregory R
2015-03-24
Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin ("diffocin") from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota. Treatment and prevention strategies for bacterial diseases rely heavily on traditional antibiotics, which impose strong selection for resistance and disrupt protective microbiota. One consequence has been an upsurge of opportunistic pathogens, such as Clostridium difficile, that exploit antibiotic-induced disruptions in gut microbiota to proliferate and cause life-threatening diseases. We have developed alternative agents that utilize contractile bactericidal protein complexes (R-type bacteriocins) to kill specific C. difficile pathogens. Efficacy in a preclinical animal study indicates these molecules warrant further development as potential prophylactic agents to prevent C. difficile infections in humans. Since these agents do not detectably alter the indigenous gut microbiota or colonization resistance in mice, we believe they will be safe to administer as a prophylactic to block transmission in high-risk environments without rendering patients susceptible to enteric infection after cessation of treatment. Copyright © 2015 Gebhart et al.
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Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J
2014-09-01
Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Applegate, K L; Chipley, J R
1976-01-01
Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A. PMID:938031
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Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores
Rousseau, Paul; Halvorson, Harlyn O.
1973-01-01
After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased. PMID:4570780
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Effects of Surotomycin on Clostridium difficile Viability and Toxin Production In Vitro
Bouillaut, Laurent; McBride, Shonna; Schmidt, Diane J.; Suarez, José M.; Tzipori, Saul; Mascio, Carmela; Chesnel, Laurent
2015-01-01
The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis. PMID:25941230
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Stephenson, Brittany; Lanzas, Cristina; Lenhart, Suzanne; Day, Judy
2017-12-01
The spore-forming, gram-negative bacteria Clostridium difficile can cause severe intestinal illness. A striking increase in the number of cases of C. difficile infection (CDI) among hospitals has highlighted the need to better understand how to prevent the spread of CDI. In our paper, we modify and update a compartmental model of nosocomial C. difficile transmission to include vaccination. We then apply optimal control theory to determine the time-varying optimal vaccination rate that minimizes a combination of disease prevalence and spread in the hospital population as well as cost, in terms of time and money, associated with vaccination. Various hospital scenarios are considered, such as times of increased antibiotic prescription rate and times of outbreak, to see how such scenarios modify the optimal vaccination rate. By comparing the values of the objective functional with constant vaccination rates to those with time-varying optimal vaccination rates, we illustrate the benefits of time-varying controls.
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Impact and Time Course of Clostridium difficile Colonization in Very Low Birth Weight Infants.
Pichler, Karin; Bausenhardt, Benjamin; Huhulescu, Steliana; Lindtner, Claudia; Indra, Alexander; Allerberger, Franz; Berger, Angelika
2018-06-12
Clostridium difficile is a gram-positive, anaerobic spore-forming, toxin-producing bacillus, which is one of the most common causes for health care-associated infections. High colonization rates in clinically asymptomatic neonates and infants have been described, although most studies go back to the early 1980 and 1990s, and were carried out in term and late preterm infants. The aim of our study was to determine both the impact and time course of C. difficile colonization in a cohort of very low birth weight infants (VLBWI) in an era of PCR-based technologies for diagnosis. Stool samples of VLBWI were analyzed for the presence of C. difficile strains in regular intervals during the hospital stay by PCR ribotyping. Analysis was continued throughout the first 2 years of life. A 32% C. difficile colonization rate during the first 2 years of life and an in-hospital colonization rate of 8% was found in a cohort of 190 VLBWI. C. difficile colonization occurred mainly in the first 6 months of life, which was similar to term neonates. In-hospital colonization accounted for only a small percentage of cases with no detection of hypervirulent strains. Also, C. difficile colonization was not related to an adverse outcome in this VLBWI cohort. Oral lactoferrin of bovine origin and treatment with piperacillin/tazobactam were negatively correlated with C. difficile colonization in our study. C. difficile colonization in our cohort of VLBWI was significantly lower than has been described in the literature and was not related to an adverse outcome. © 2018 S. Karger AG, Basel.
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Léonard-Akkari, Lucie; Guégan, Stéphanie; Courand, Fabienne; Couvert, Olivier; Lepage, Jean-François; Rondeau-Mouro, Corinne; Desriac, Noémie; Mathot, Anne-Gabrielle; Leguérinel, Ivan; Coroller, Louis; Decourcelle, Nicolas
2018-07-01
In foodstuffs, physico-chemical interactions and/or physical constraints between spores, inhibitors and food components may exist. Thus, the objective of this study was to investigate such interactions using a model emulsion as a microbial medium in order to improve bacterial spore control with better knowledge of the interactions in the formulation. Emulsions were prepared with hexadecane mixed with nutrient broth using sonication and were stabilized by Tween 80 and Span 80. The hexadecane ratio was either 35% (v/v) or 50% (v/v) and each emulsion was studied in the presence of organic acid (acetic, lactic or hexanoic) at two pH levels (5.5 and 6). Self-diffusion coefficients of emulsion components and the organic acids were measured by Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR). The inhibition effect on the spore germination and cell growth of Bacillus weihenstephanensis KBAB4 was characterized by the measure of the probability of growth using the most probable number methodology, and the measure of the time taken for the cells to germinate and grow using a single cell Bioscreen® method and using flow cytometry. The inhibition of spore germination and growth in the model emulsion depended on the dispersed phase volume fraction and the pH value. The effect of the dispersed phase volume fraction was due to a combination of (i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases and (ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value. The interface phenomena seemed to have a major influence. Future work will focus on the exploration of these phenomena at the interface. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Clostridium difficile in retail baskets, trolleys, conveyor belts, and plastic bags in Saudi Arabia.
Alqumber, Mohammed A
2014-10-01
To determine Clostridium difficile (C. difficile) prevalence on retail surfaces and shoppers plastic bags. From 20 June to 10 August 2011, in a cross-sectional epidemiological study, 17 supermarkets from 2 cities, Albaha and Altaif, Saudi Arabia were sampled. A total of 800 samples, which comprised 200 samples per surveyed surface, were studied. These included baskets, trolleys, conveyer belts, and outgoing shoppers' plastic bags. Clostridium difficile strains were isolated. The isolates were characterized using ribotyping and polymerase chain reaction for the detection of toxin A (tcdA), toxin B (tcdB), binary toxin (cdtB), and toxin C (tcdC) genes. Susceptibility to antibiotics was determined on a Muller-Hinton agar with 5% sheep blood agar using E-tests. Overall, the C. difficile prevalence on sampled surfaces was 0.75%. The highest prevalence was found on retail baskets and trolleys, followed by plastic bags. A total of 5 different ribotypes were identified. Alterations in tcdC were detected in ribotype 027 and BT1. All the identified isolates were susceptible to vancomycin, but resistant to levofloxacin. In this study, C. difficile was present at a rate of 0.75% on supermarket surfaces. Spore disinfection of implicated surfaces may be necessary to control any community-acquired infections caused by this pathogen.
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Takamatsu, Hiromu; Kodama, Takeko; Nakayama, Tatsuo; Watabe, Kazuhito
1999-01-01
Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination. PMID:10438771
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Cooper, Gareth R; Moir, Anne
2011-05-01
The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.
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Environmental Contamination in Households of Patients with Recurrent Clostridium difficile Infection
Bobr, Aleh; Kuskowski, Michael A.; Johnston, Brian D.; Sadowsky, Michael J.; Khoruts, Alexander
2016-01-01
Recurrent Clostridium difficile infection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). Although C. difficile spores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence of C. difficile. Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptive C. difficile isolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping, tcdC gene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detected C. difficile on ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P = 0.025). The most common C. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%). C. difficile was detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multiple C. difficile-positive samples had a single genotype present each. In conclusion, C. difficile was found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective. PMID:26921425
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Sporostatic and sporocidal properties of aqueous formaldehyde.
NASA Technical Reports Server (NTRS)
Trujillo, R.; David, T. J.
1972-01-01
Aqueous formaldehyde is shown to exert both sporostatic and sporocidal effects on Bacillus subtilis spores. The sporostatic effect is a result of the reversible inhibition of spore germination occasioned by aqueous formaldehyde; the sporocidal effect is due to the temperature-dependent inactivation of these spores in aqueous formaldehyde. The physicochemical state of formaldehyde in solution provides a framework with which to interpret both the sporostatic and sporocidal properties of aqueous formaldehyde.
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Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P; Kurtz, Joachim
2015-12-01
Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Redondo-Solano, Mauricio; Valenzuela-Martinez, Carol; Cassada, David A; Snow, Daniel D; Juneja, Vijay K; Burson, Dennis E; Thippareddi, Harshavardhan
2013-09-01
The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 °C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 °C, 20 min) and cooled within 15 h from 54.4 to 7.2 °C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO2 concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Cadnum, Jennifer L; Jencson, Annette L; O'Donnell, Marguerite C; Flannery, Elizabeth R; Nerandzic, Michelle M; Donskey, Curtis J
2017-03-01
BACKGROUND We investigated an increase in the incidence of healthcare-associated Clostridium difficile infection (CDI) that occurred following a change from a bleach disinfectant to a peracetic acid-based disinfectant. OBJECTIVE To evaluate the efficacy of the peracetic acid-based disinfectant. DESIGN Laboratory-based product evaluation. METHODS The commercial peracetic acid-based product is activated on site by mixing a small volume of concentrated hydrogen peroxide and peracetic acid present in a "SmartCap" reservoir with the remaining contents of the container. We measured concentrations of peracetic acid in newly activated and in-use product and determined the stability of nonactivated and activated product. We tested the efficacy of the product against C. difficile spores using the American Society for Testing and Materials standard quantitative carrier disk test method. RESULTS Measured concentrations of peracetic acid (50-800 parts per million [ppm]) were significantly lower than the level stated on the product label (1,500 ppm), and similar results were obtained for containers from multiple lot numbers and from another hospital. Product with peracetic acid levels below 600 ppm had significantly reduced activity against C. difficile spores. Peracetic acid concentrations were reduced markedly after storage of either activated or nonactivated product for several weeks. The Environmental Protection Agency confirmed the finding of low disinfectant levels and ordered discontinuation of sale of the product. CONCLUSION Use of a defective peracetic acid-based surface disinfectant may have contributed to an increase in healthcare-associated CDI. Our findings highlight the importance of evaluating the efficacy of liquid disinfectants in healthcare settings. Infect Control Hosp Epidemiol 2017;38:300-305.
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Clostridium Difficile Infection Due to Pneumonia Treatment: Mortality Risk Models.
Chmielewska, M; Zycinska, K; Lenartowicz, B; Hadzik-Błaszczyk, M; Cieplak, M; Kur, Z; Wardyn, K A
2017-01-01
One of the most common gastrointestinal infection after the antibiotic treatment of community or nosocomial pneumonia is caused by the anaerobic spore Clostridium difficile (C. difficile). The aim of this study was to retrospectively assess mortality due to C. difficile infection (CDI) in patients treated for pneumonia. We identified 94 cases of post-pneumonia CDI out of the 217 patients with CDI. The mortality issue was addressed by creating a mortality risk models using logistic regression and multivariate fractional polynomial analysis. The patients' demographics, clinical features, and laboratory results were taken into consideration. To estimate the influence of the preceding respiratory infection, a pneumonia severity scale was included in the analysis. The analysis showed two statistically significant and clinically relevant mortality models. The model with the highest prognostic strength entailed age, leukocyte count, serum creatinine and urea concentration, hematocrit, coexisting neoplasia or chronic obstructive pulmonary disease. In conclusion, we report on two prognostic models, based on clinically relevant factors, which can be of help in predicting mortality risk in C. difficile infection, secondary to the antibiotic treatment of pneumonia. These models could be useful in preventive tailoring of individual therapy.
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Hampikyan, Hamparsun; Bingol, Enver Baris; Muratoglu, Karlo; Akkaya, Esra; Cetin, Omer; Colak, Hilal
2018-05-01
Clostridium difficile is an anaerobic, spore forming, rod shaped bacterium frequently isolated from butchery animals in recent years. The aim of this study is to evaluate the presence of C. difficile (especially ribotype 027 and 078) in cattle and sheep carcasses and to investigate the antibiotic susceptibility of isolates. The bacterium was isolated in 83 out of 247 (33.6%) cattle and 78 out of 308 (25.3%) sheep carcass samples. 15/83 (18.1%) cattle and 6/78 (7.7%) sheep isolates were identified as ribotype 027, whereas the other hypervirulent isolate ribotype 078 could not be detected among the analysed samples. Almost all isolates were susceptible to amoxicillin-clavulanic acid (98.8%), vancomycin (96.9%) and tetracycline (97.5%), whereas resistant to cefotaxim (97.5%) and imipenem (87.0%). In conclusion, the results demonstrate the presence of toxigenic C. difficile isolates in cattle and sheep carcasses on the slaughter line. As a result, the results of this study demonstrate the presence of toxigenic C. difficile isolates in cattle and sheep carcasses on the slaughter line. Copyright © 2018 Elsevier Ltd. All rights reserved.
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NASA Technical Reports Server (NTRS)
Scheuerlein, R.; Schmidt, K.; Poenie, M.; Roux, S. J.
1991-01-01
Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca2+ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca2+]i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV cm-1, half-life (time constant) 230 microseconds). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca2+ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca2+] (1 mM ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)) by 1 mM CaCl2 caused a fast increase of [Ca2+]i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl2 by EGTA decreased [Ca2+]i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]i according to the Ca2+ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca(2+) -free medium, [Ca2+]i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca(2+) -containing medium and analysed in EGTA, [Ca2+]i was significantly higher (> or = 500 nM). In red-light-irradiated spores, [Ca2+]i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.
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USDA-ARS?s Scientific Manuscript database
Reniform nematodes (Rotylenchulus reniformis) from pot culture were attached with in vitro produced Pasteuria spp. spores using a centrifuge attachment technique that resulted in 40-50% of the vermiform nematodes with spores adhering to their cuticles. Attached nematodes were placed into small plast...
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Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?
Egan, Kevin; Field, Des; Rea, Mary C; Ross, R Paul; Hill, Colin; Cotter, Paul D
2016-01-01
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.
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Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?
Egan, Kevin; Field, Des; Rea, Mary C.; Ross, R. Paul; Hill, Colin; Cotter, Paul D.
2016-01-01
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings. PMID:27092121
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New insights in the bacterial spore resistance to extreme terrestrial and extraterrestrial factors
NASA Astrophysics Data System (ADS)
Moeller, Ralf; Horneck, Gerda; Reitz, Guenther
Based on their unique resistance to various space parameters, Bacillus endospores are one of the model systems used for astrobiological studies. The extremely high resistance of bacterial endospores to environmental stress factors has intrigued researchers since long time and many characteristic spore features, especially those involved in the protection of spore DNA, have already been uncovered. The disclosure of the complete genomic sequence of Bacillus subtilis 168, one of the often used astrobiological model system, and the rapid development of tran-scriptional microarray techniques have opened new opportunities of gaining further insights in the enigma of spore resistance. Spores of B. subtilis were exposed to various extreme ter-restrial and extraterrestrial stressors to reach a better understanding of the DNA protection and repair strategies, which them to cope with the induced DNA damage. Following physical stress factors of environmental importance -either on Earth or in space -were selected for this thesis: (i) mono-and polychromatic UV radiation, (ii) ionizing radiation, (iii) exposure to ultrahigh vacuum; and (iv) high shock pressures simulating meteorite impacts. To reach a most comprehensive understanding of spore resistance to those harsh terrestrial or simulated extraterrestrial conditions, a standardized experimental protocol of the preparation and ana-lyzing methods was established including the determination of the following spore responses: (i) survival, (ii) induced mutations, (iii) DNA damage, (iv) role of different repair pathways by use of a set of repair deficient mutants, and (v) transcriptional responses during spore germi-nation by use of genome-wide transcriptome analyses and confirmation by RT-PCR. From this comprehensive set of data on spore resistance to a variety of environmental stress parameters a model of a "built-in" transcriptional program of bacterial spores in response to DNA damaging treatments to ensure DNA restoration during germination has been developed.
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Schubert, Alyxandria M.; Sinani, Hamide
2015-01-01
ABSTRACT Perturbations to the gut microbiota can result in a loss of colonization resistance against gastrointestinal pathogens such as Clostridium difficile. Although C. difficile infection is commonly associated with antibiotic use, the precise alterations to the microbiota associated with this loss in function are unknown. We used a variety of antibiotic perturbations to generate a diverse array of gut microbiota structures, which were then challenged with C. difficile spores. Across these treatments we observed that C. difficile resistance was never attributable to a single organism, but rather it was the result of multiple microbiota members interacting in a context-dependent manner. Using relative abundance data, we built a machine learning regression model to predict the levels of C. difficile that were found 24 h after challenging the perturbed communities. This model was able to explain 77.2% of the variation in the observed number of C. difficile per gram of feces. This model revealed important bacterial populations within the microbiota, which correlation analysis alone did not detect. Specifically, we observed that populations associated with the Porphyromonadaceae, Lachnospiraceae, Lactobacillus, and Alistipes were protective and populations associated with Escherichia and Streptococcus were associated with high levels of colonization. In addition, a population affiliated with the Akkermansia indicated a strong context dependency on other members of the microbiota. Together, these results indicate that individual bacterial populations do not drive colonization resistance to C. difficile. Rather, multiple diverse assemblages act in concert to mediate colonization resistance. PMID:26173701
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Chilton, C H; Crowther, G S; Baines, S D; Todhunter, S L; Freeman, J; Locher, H H; Athanasiou, A; Wilcox, M H
2014-03-01
We investigated the in vitro activity of cadazolid against 100 Clostridium difficile isolates and its efficacy in a simulated human gut model of C. difficile infection (CDI). MICs of cadazolid, metronidazole, vancomycin, moxifloxacin and linezolid were determined using agar incorporation for 100 C. difficile isolates, including 30 epidemic strains (ribotypes 027, 106 and 001) with reduced metronidazole susceptibility, 2 linezolid-resistant isolates and 2 moxifloxacin-resistant isolates. We evaluated the efficacy of two cadazolid dosing regimens (250 versus 750 mg/L twice daily for 7 days) to treat simulated CDI. Microflora populations, C. difficile total viable counts and spores, cytotoxin titres, possible emergence of cadazolid, linezolid or quinolone resistance, and antimicrobial concentrations were monitored throughout. Cadazolid was active against all (including linezolid- and moxifloxacin-resistant) C. difficile strains (MIC90 0.125, range 0.03-0.25 mg/L). The cadazolid geometric mean MIC was 152-fold, 16-fold, 9-fold and 7-fold lower than those of moxifloxacin, linezolid, metronidazole and vancomycin, respectively. Both cadazolid dosing regimens rapidly reduced C. difficile viable counts and cytotoxin with no evidence of recurrence. Cadazolid levels persisted at 50-100-fold supra-MIC for 14 days post-dosing. Cadazolid inhibition of enumerated gut microflora was limited, with the exception of bifidobacteria; Bacteroides fragilis group and Lactobacillus spp. counts were unaffected. There was no evidence for selection of strains resistant to cadazolid, quinolones or linezolid. Cadazolid activity was greater than other tested antimicrobials against 100 C. difficile strains. Cadazolid effectively treated simulated CDI in a gut model, with limited impact on the enumerated gut microflora and no signs of recurrence or emergence of resistance within the experimental timeframe.
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Photodynamic inactivation of mold fungi spores by newly developed charged corroles.
Preuß, Annegret; Saltsman, Irena; Mahammed, Atif; Pfitzner, Michael; Goldberg, Israel; Gross, Zeev; Röder, Beate
2014-04-05
The photodynamic effect, originally used in photodynamic therapy (PDT) for the treatment of different diseases, e.g. of cancer, has recently been introduced for the inactivation of bacteria. Mold fungi, which provoke health problems like allergies and diseases of the respiratory tract, are even more resistant and their biology is also very different. This study presents the development of four new photosensitizers, which, in combination with low doses of white light, inhibit the germination of mold fungi spores. Two of them even cause lethal damage to the conidia (spores) which are responsible for the spreading of mold fungi. The photoactivity of the newly synthesized corroles was obtained by their application on three different mold fungi: Aspergillus niger, Cladosporium cladosporoides, and Penicillium purpurgenum. To distinguish between inactivation of germination and permanent damage, the fungi were first incubated under illumination for examination of photosensitizer-induced growth inhibition and then left in darkness to test the survival of the conidia. None of the compounds displayed dark toxicity, but all of them attenuated or prevented germination when exposed to light, and the positively charged complexes induced a complete damage of the conidia. Copyright © 2014 Elsevier B.V. All rights reserved.
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Varga, Sandra
2015-03-01
• Arbuscular mycorrhizal fungi usually enhance overall plant performance, yet their effects on seed germination and early plant establishment, crucial steps in plant cycles, are generally overlooked. In gynodioecious species, sexual dimorphism in these traits has been reported, with females producing seeds that germinate at a faster rate than seeds from hermaphrodites.• Using the gynodioecious plant Geranium sylvaticum, I investigated in a greenhouse experiment whether the presence of arbuscular mycorrhizal spores affects seed germination and early plant establishment, examining at the same time whether the sex of the mother producing the seeds also influences these parameters and whether sex-specific interactions between these two factors exist.• The presence of arbuscular mycorrhizal spores in the soil decreased seed germination, did not affect plant survival, but did increase plant growth. Moreover, no significant differences in seed traits were detected between the sexes of the plants producing the seeds.• This study demonstrates that arbuscular mycorrhizal fungi may have contrasting effects for plants during early life stages and that mycorrhizal effects can take place even at the precolonization stage. © 2015 Botanical Society of America, Inc.
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Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile
Hunt, Jonathan J.
2013-01-01
SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade. PMID:24296572
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Vincent, Caroline; Miller, Mark A; Edens, Thaddeus J; Mehrotra, Sudeep; Dewar, Ken; Manges, Amee R
2016-03-14
Clostridium difficile infection (CDI) is the leading infectious cause of nosocomial diarrhea. Hospitalized patients are at increased risk of developing CDI because they are exposed to C. difficile spores through contact with the hospital environment and often receive antibiotics and other medications that can disrupt the integrity of the indigenous intestinal microbiota and impair colonization resistance. Using whole metagenome shotgun sequencing, we examined the diversity and composition of the fecal microbiota in a prospective cohort study of 98 hospitalized patients. Four patients had asymptomatic C. difficile colonization, and four patients developed CDI. We observed dramatic shifts in the structure of the gut microbiota during hospitalization. In contrast to CDI cases, asymptomatic patients exhibited elevated relative abundance of potentially protective bacterial taxa in their gut at the onset of C. difficile colonization. Use of laxatives was associated with significant reductions in the relative abundance of Clostridium and Eubacterium; species within these genera have previously been shown to enhance resistance to CDI via the production of secondary bile acids. Cephalosporin and fluoroquinolone exposure decreased the frequency of Clostridiales Family XI Incertae Sedis, a bacterial family that has been previously associated with decreased CDI risk. This study underscores the detrimental impact of antibiotics as well as other medications, particularly laxatives, on the intestinal microbiota and suggests that co-colonization with key bacterial taxa may prevent C. difficile overgrowth or the transition from asymptomatic C. difficile colonization to CDI.
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Characterization of a Stable, Metronidazole-Resistant Clostridium difficile Clinical Isolate
Lynch, Tarah; Chong, Patrick; Zhang, Jason; Hizon, Romeo; Du, Tim; Graham, Morag R.; Beniac, Daniel R.; Booth, Timothy F.; Kibsey, Pamela; Miller, Mark; Gravel, Denise; Mulvey, Michael R.
2013-01-01
Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. Methodology/Principal Findings Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. Conclusions/Significance This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described. PMID:23349739
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Lactobacillus acidophilus modulates the virulence of Clostridium difficile.
Yun, B; Oh, S; Griffiths, M W
2014-01-01
Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium that colonizes the human gastrointestinal tract. This pathogen causes antibiotic-associated diarrhea and colitis in animals and humans. Antibiotic-associated diseases may be treated with probiotics, and interest is increasing in such uses of probiotics. This study investigated the effect of Lactobacillus strains on the quorum-sensing signals and toxin production of C. difficile. In addition, an in vivo experiment was designed to assess whether Lactobacillus acidophilus GP1B is able to control C. difficile-associated disease. Autoinducer-2 activity was measured for C. difficile using the Vibrio harveyi coupled bioluminescent assay. Cell extract (10μg/mL) of L. acidophilus GP1B exhibited the highest inhibitory activity among 5 to 40μg/mL cell-extract concentrations. Real-time PCR data indicated decreased transcriptional levels in luxS, tcdA, tcdB, and txeR genes in the presence of 10μg/mL of cell extract of L. acidophilus GP1B. Survival rates at 5d for mice given the pathogen alone with L. acidophilus GP1B cell extract or L. acidophilus GP1B were 10, 70, and 80%, respectively. In addition, the lactic acid-produced L. acidophilus GP1B exhibits an inhibitory effect against the growth of C. difficile. Both the L. acidophilus GP1B and GP1B cell extract have significant antipathogenic effects on C. difficile. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Pedraza-Reyes, M; Gutiérrez-Corona, F; Nicholson, W L
1994-01-01
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination Images PMID:8021181
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Xu, Xiangbin; Lei, Huanhuan; Ma, Xiuyan; Lai, Tongfei; Song, Hongmiao; Shi, Xuequn; Li, Jiangkuo
2017-01-16
Anthracnose caused by Colletotrichum gloeosporioides is one of the most important postharvest diseases in mango fruit, often causing huge economic losses. In this study, the effect of 1-methylcyclopropene (1-MCP) against anthracnose in postharvest mango fruit and the mechanisms involved were investigated. 1-MCP induced reactive oxygen species (ROS) generation, damaged the mitochondria and destroyed the integrity of plasma membrane of spores of C. gloeosporioides, significantly suppressing spore germination and mycelial growth of C. gloeosporioides. 1-MCP also decreased the decay incidence and lesion expansion of mango fruit caused by C. gloeosporioides. For the first time this study demonstrated that 1-MCP suppressed anthracnose of postharvest mango fruit by directly inhibiting spore germination and mycelial growth of C. gloeosporioides, thus providing a promising strategy for disease control. Copyright © 2016 Elsevier B.V. All rights reserved.
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Bécard, G; Douds, D D; Pfeffer, P E
1992-03-01
Various flavonoids were tested for their ability to stimulate in vitro growth of germinated spores of vesicular-arbuscular mycorrhizal fungi. Experiments were performed in the presence of 2% CO(2), previously demonstrated to be required for growth of Gigaspora margarita (G. Bécard and Y. Piché, Appl. Environ. Microbiol. 55:2320-2325, 1989). Only the flavonols stimulated fungal growth. The flavones, flavanones, and isoflavones tested were generally inhibitory. Quercetin (10 muM) prolonged hyphal growth from germinated spores of G. margarita from 10 to 42 days. An average of more than 500 mm of hyphal growth and 13 auxiliary cells per spore were obtained. Quercetin also stimulated the growth of Glomus etunicatum. The glycosides of quercetin, rutin, and quercitrin were not stimulatory. The axenic growth of G. margarita achieved here under rigorously defined conditions is the most ever reported for a vesicular-arbuscular mycorrhizal fungus.
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Inoue, Kanako; Tsurumi, Tomohiro; Ishii, Hideo; Park, Pyoyun; Ikeda, Kenichi
2012-01-01
Azoxystrobin (AZ), a strobilurin-derived fungicide, is known to inhibit mitochondrial respiration in fungi by blocking the electron transport chain in the inner mitochondrial membrane. Germination was strongly inhibited when Botrytis cinerea spore suspension was treated with AZ and the alternative oxidase (AOX) inhibitors, salicylhydroxamic acid (SHAM) and n-propyl gallate. However, chemical death indicators trypan blue and propidium iodide showed that those spores were still alive. When the spore suspension in the AZ and SHAM solution was replaced with distilled water, the germination rate almost recovered, at least during the first 2 days of incubation with AZ and SHAM solution. No morphological alteration was detected in the cells treated with AZ and SHAM, especially in mitochondria, using transmission electron microscopy. Therefore, simultaneous application of AZ and AOX inhibitors has a fungistatic, rather than a fungicidal, action. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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Decontamination of Anthrax spores in critical infrastructure and critical assets.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Boucher, Raymond M.; Crown, Kevin K.; Tucker, Mark David
2010-05-01
Decontamination of anthrax spores in critical infrastructure (e.g., subway systems, major airports) and critical assets (e.g., the interior of aircraft) can be challenging because effective decontaminants can damage materials. Current decontamination methods require the use of highly toxic and/or highly corrosive chemical solutions because bacterial spores are very difficult to kill. Bacterial spores such as Bacillus anthracis, the infectious agent of anthrax, are one of the most resistant forms of life and are several orders of magnitude more difficult to kill than their associated vegetative cells. Remediation of facilities and other spaces (e.g., subways, airports, and the interior of aircraft)more » contaminated with anthrax spores currently requires highly toxic and corrosive chemicals such as chlorine dioxide gas, vapor- phase hydrogen peroxide, or high-strength bleach, typically requiring complex deployment methods. We have developed a non-toxic, non-corrosive decontamination method to kill highly resistant bacterial spores in critical infrastructure and critical assets. A chemical solution that triggers the germination process in bacterial spores and causes those spores to rapidly and completely change to much less-resistant vegetative cells that can be easily killed. Vegetative cells are then exposed to mild chemicals (e.g., low concentrations of hydrogen peroxide, quaternary ammonium compounds, alcohols, aldehydes, etc.) or natural elements (e.g., heat, humidity, ultraviolet light, etc.) for complete and rapid kill. Our process employs a novel germination solution consisting of low-cost, non-toxic and non-corrosive chemicals. We are testing both direct surface application and aerosol delivery of the solutions. A key Homeland Security need is to develop the capability to rapidly recover from an attack utilizing biological warfare agents. This project will provide the capability to rapidly and safely decontaminate critical facilities and assets to return them to normal operations as quickly as possible, sparing significant economic damage by re-opening critical facilities more rapidly and safely. Facilities and assets contaminated with Bacillus anthracis (i.e., anthrax) spores can be decontaminated with mild chemicals as compared to the harsh chemicals currently needed. Both the 'germination' solution and the 'kill' solution are constructed of 'off-the-shelf,' inexpensive chemicals. The method can be utilized by directly spraying the solutions onto exposed surfaces or by application of the solutions as aerosols (i.e., small droplets), which can also reach hidden surfaces.« less
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James M. Trappe; Andrew W. Claridge
2010-01-01
Truffles, like mushrooms, are the fruit of fungi. These fleshy organs are temporary reproductive structures that produce spores, which eventually germinate and give rise to new offspring. What sets truffles apart from mushrooms is that their spore-laden fruit forms below ground rather than above. Technically, true truffles are those fungi that belong to the Ascomycota...
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A rapid colorimetric assay for mold spore germination using XTT tetrazolium salt
Carol A. Clausen; Vina W. Yang
2011-01-01
Current laboratory test methods to measure efficacy of new mold inhibitors are time consuming, some require specialized test equipment and ratings are subjective. Rapid, simple quantitative assays to measure the efficacy of mold inhibitors are needed. A quantitative, colorimetric microassay was developed using XTT tetrazolium salt to metabolically assess mold spore...
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The Genetics of Sexual Incompatibility in the Indian Paint Fungus, Echinodontium Tinctorium
A. Dan Wilson
1990-01-01
Basidiocarps of Echinodontium tinctorium were collected from three widely separated localities in Idaho and Arizona. Freezing basidiospore prints at -20 C for ten weeks stimulated germination. The role of low temperatures in breaking spore dormancy is discussed relative to environmental adaptation. Single-spore isolates of a single basidiocarp...
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Singh, K M; Reddy, B; Patel, A K; Panchasara, H; Parmar, N; Patel, A B; Shah, T M; Bhatt, V D; Joshi, C G
2014-12-01
Buffalo rumen microbiome experiences a variety of diet stress and represents reservoir of Dormancy and Sporulation genes. However, the information on genomic responses to such conditions is very limited. The Ion Torrent PGM next generation sequencing technology was used to characterize general microbial diversity and the repertoire of microbial genes present, including genes associated with Dormancy and Sporulation in Mehsani buffalo rumen metagenome. The research findings revealed the abundance of bacteria at the domain level and presence of Dormancy and Sporulation genes which were predominantly associated with the Clostridia and Bacilli taxa belonging to the phyla Firmicutes. Genes associated with Sporulation cluster and Sporulation orphans were increased from 50% to 100% roughage treatment, thereby promoting sporulation all along the treatments. The spore germination is observed to be the highest in the 75% roughage treatment both in the liquid and solid rumen fraction samples with respect to the decrease in the values of the genes associated with spore core dehydration, thereby facilitating spore core hydration which is necessary for spore germination.
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Moradigaravand, Danesh; Gouliouris, Theodore; Ludden, Catherine; Reuter, Sandra; Jamrozy, Dorota; Blane, Beth; Naydenova, Plamena; Judge, Kim; H Aliyu, Sani; F Hadjirin, Nazreen; A Holmes, Mark; Török, Estée; M Brown, Nicholas; Parkhill, Julian; Peacock, Sharon
2018-03-02
There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.
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Moradigaravand, Danesh; Gouliouris, Theodore; Ludden, Catherine; Reuter, Sandra; Jamrozy, Dorota; Blane, Beth; Naydenova, Plamena; Judge, Kim; H. Aliyu, Sani; F. Hadjirin, Nazreen; A. Holmes, Mark; Török, Estée; M. Brown, Nicholas; Parkhill, Julian; Peacock, Sharon
2018-01-01
There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater. PMID:29498619
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Hattingh, M; Alexander, A; Meijering, I; van Reenen, C A; Dicks, L M T
2014-03-03
Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, β-glucan- and arabinoxylan-rich cell walls have to be degraded. β-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced β-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt β-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260 ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on β-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality. Copyright © 2013 Elsevier B.V. All rights reserved.
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Black, Elaine; Owens, Krista; Staub, Richard; Li, Junzhong; Mills, Kristen; Valenstein, Justin; Hilgren, John
2017-01-01
Disinfectants play an important role in controlling microbial contamination on hard surfaces in hospitals. The effectiveness of disinfectants in real life can be predicted by laboratory tests that measure killing of microbes on carriers. The modified Quantitative Disk Carrier Test (QCT-2) is a standard laboratory method that employs American Iron and Steel Institute (AISI) Type 430 stainless steel carriers to measure hospital disinfectant efficacy against Clostridium difficile spores. The formation of a rust-colored precipitate was observed on Type 430 carriers when testing a peracetic acid (PAA)-based disinfectant with the QCT-2 method. It was hypothesized that the precipitate was indicative of corrosion of the Type 430 carrier, and that corrosion could impact efficacy results. The objective of this study was to compare the suitability of AISI Type 430 to Type 304 stainless steel carriers for evaluating PAA-based disinfectants using the QCT-2 method. Type 304 is more corrosion-resistant than Type 430, is ubiquitous in healthcare environments, and is used in other standard methods. Suitability of the carriers was evaluated by comparing their impacts on efficacy results and PAA degradation rates. In efficacy tests with 1376 ppm PAA, reductions of C. difficile spores after 5, 7 and 10 minutes on Type 430 carriers were at least about 1.5 log10 lower than reductions on Type 304 carriers. In conditions simulating a QCT-2 test, PAA concentration with Type 430 carriers was reduced by approximately 80% in 10 minutes, whereas PAA concentration in the presence of Type 304 carriers remained stable. Elemental analyses of residues on each carrier type after efficacy testing were indicative of corrosion on the Type 430 carrier. Use of Type 430 stainless steel carriers for measuring the efficacy of PAA-based disinfectants should be avoided as it can lead to an underestimation of real life sporicidal efficacy. Type 304 stainless steel carriers are recommended as a suitable alternative.
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Owens, Krista; Staub, Richard; Li, Junzhong; Mills, Kristen; Valenstein, Justin; Hilgren, John
2017-01-01
Disinfectants play an important role in controlling microbial contamination on hard surfaces in hospitals. The effectiveness of disinfectants in real life can be predicted by laboratory tests that measure killing of microbes on carriers. The modified Quantitative Disk Carrier Test (QCT-2) is a standard laboratory method that employs American Iron and Steel Institute (AISI) Type 430 stainless steel carriers to measure hospital disinfectant efficacy against Clostridium difficile spores. The formation of a rust-colored precipitate was observed on Type 430 carriers when testing a peracetic acid (PAA)-based disinfectant with the QCT-2 method. It was hypothesized that the precipitate was indicative of corrosion of the Type 430 carrier, and that corrosion could impact efficacy results. The objective of this study was to compare the suitability of AISI Type 430 to Type 304 stainless steel carriers for evaluating PAA-based disinfectants using the QCT-2 method. Type 304 is more corrosion-resistant than Type 430, is ubiquitous in healthcare environments, and is used in other standard methods. Suitability of the carriers was evaluated by comparing their impacts on efficacy results and PAA degradation rates. In efficacy tests with 1376 ppm PAA, reductions of C. difficile spores after 5, 7 and 10 minutes on Type 430 carriers were at least about 1.5 log10 lower than reductions on Type 304 carriers. In conditions simulating a QCT-2 test, PAA concentration with Type 430 carriers was reduced by approximately 80% in 10 minutes, whereas PAA concentration in the presence of Type 304 carriers remained stable. Elemental analyses of residues on each carrier type after efficacy testing were indicative of corrosion on the Type 430 carrier. Use of Type 430 stainless steel carriers for measuring the efficacy of PAA-based disinfectants should be avoided as it can lead to an underestimation of real life sporicidal efficacy. Type 304 stainless steel carriers are recommended as a suitable alternative. PMID:29065168
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Serrano, Mónica; Crawshaw, Adam D.; Dembek, Marcin; Monteiro, João M.; Pereira, Fátima C.; Pinho, Mariana Gomes; Fairweather, Neil F.
2016-01-01
Summary Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc‐binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ‐SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post‐engulfment, forespore and mother cell‐specific gene expression, suggesting a channel‐like function. Both engulfment and a channel‐like function may be ancestral functions of SpoIIQ‐SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. PMID:26690930
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1977-01-01
of Action of Miconazole ; Immunity in Dermatomycoses; Biochemical Studies in Microsporum canis Spore Germination and Mechanism of Cycloheximide Resistance; and Biosynthetic Potentialities of Candida Species.
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Non-gravitational effects on genus penicillium
NASA Technical Reports Server (NTRS)
Loup, Mackenzie
1995-01-01
In September 1994, Shuttle Orbiter Discovery, STS-64, launched into space. Aboard that shuttle was a payload containing Fungi spores, genus Penicillium. With the over looking help of Dr. Audrey Gabel, Associate Professor of Biology at Black Hills State University, investigations on differing media types began. Basis for this experimentation was to determine if there was any differences between the space exposed spores and control spores. Studies concluded that there were differences and those differences were then recorded. It was hypothesized the spores may have been effected causing differences in growth rate, colony size, depth and margins, coloring, germination, and growth on different media.
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Glycerol enhances fungal germination at the water-activity limit for life.
Stevenson, Andrew; Hamill, Philip G; Medina, Ángel; Kminek, Gerhard; Rummel, John D; Dijksterhuis, Jan; Timson, David J; Magan, Naresh; Leong, Su-Lin L; Hallsworth, John E
2017-03-01
For the most-extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0-64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water-activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores -1 ). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water-activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water-activities than previously recorded (0.640, 0.685, 0.651, 0.664 and 0.637 respectively). In addition, the kinetics of germination at low water-activities were substantially faster than those reported previously. Extrapolations indicated theoretical water-activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water-activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease-causing microorganisms; and in biotechnologies that operate at the limits of microbial function. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
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Probiotics in Clostridium difficile infection: reviewing the need for a multistrain probiotic.
Hell, M; Bernhofer, C; Stalzer, P; Kern, J M; Claassen, E
2013-03-01
In the past two years an enormous amount of molecular, genetic, metabolomic and mechanistic data on the host-bacterium interaction, a healthy gut microbiota and a possible role for probiotics in Clostridium difficile infection (CDI) has been accumulated. Also, new hypervirulent strains of C. difficile have emerged. Yet, clinical trials in CDI have been less promising than in antibiotic associated diarrhoea in general, with more meta-analysis than primary papers on CDI-clinical-trials. The fact that C. difficile is a spore former, producing at least three different toxins has not yet been incorporated in the rational design of probiotics for (recurrent) CDI. Here we postulate that the plethora of effects of C. difficile and the vast amount of data on the role of commensal gut residents and probiotics point towards a multistrain mixture of probiotics to reduce CDI, but also to limit (nosocomial) transmission and/or endogenous reinfection. On the basis of a retrospective chart review of a series of ten CDI patients where recurrence was expected, all patients on adjunctive probiotic therapy with multistrain cocktail (Ecologic®AAD/OMNiBiOTiC® 10) showed complete clinical resolution. This result, and recent success in faecal transplants in CDI treatment, are supportive for the rational design of multistrain probiotics for CDI.
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Current knowledge on the laboratory diagnosis of Clostridium difficile infection.
Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira
2017-03-07
Clostridium difficile ( C. difficile ) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.
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Balassiano, I T; Yates, E A; Domingues, R M C P; Ferreira, E O
2012-02-01
Clostridium difficile-associated disease (CDAD) is caused by a spore-forming bacterium and can result in highly variable disease, ranging from mild diarrhoea to severe clinical manifestations. Infections are most commonly seen in hospital settings and are often associated with on-going antibiotic therapy. Incidences of CDAD have shown a sustained increase worldwide over the last ten years and a hypervirulent C. difficile strain, PCR ribotype 027/REA type BI/North American pulsed-field (NAP) type 1 (027/BI/NAP-1), has caused outbreaks in North America and Europe. In contrast, only a few reports of cases in Latin America have been published and the hypervirulent strain 027/BI/NAP-1 has, so far, only been reported in Costa Rica. The potential worldwide spread of this infection calls for epidemiological studies to characterize currently circulating strains and also highlights the need for increased awareness and vigilance among healthcare professionals in currently unaffected areas, such as Latin America. This review attempts to summarize reports of C. difficile infection worldwide, especially in Latin America, and aims to provide an introduction to the problems associated with this pathogen for those countries that might face outbreaks of epidemic strains of C. difficile for the first time in the near future.
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Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M
2015-09-01
Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.
2015-01-01
Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385
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Nuclear dynamics during ascospore germination in Sordaria macrospora.
Teichert, Ines
2017-01-01
The ascomycete Sordaria macrospora has a long history as a model organism for studying fungal sexual development. Starting from an ascospore, sexual fruiting bodies (perithecia) develop within seven days and discharge new ascospores. Sexual development has been studied in detail, revealing genes required for perithecium formation and ascospore germination. However, the germination process per se has not yet been examined. Here I analyze nuclear dynamics during ascospore germination using a fluorescently labeled histone. Live-cell imaging revealed that nuclei are transported into germination vesicles that form on one side of the spore. Polar growth is established from these vesicles. Copyright © 2016 Elsevier Inc. All rights reserved.
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Surface sterilization of hybrid poplar cuttings
Alma M. Waterman
1954-01-01
Fungus diseases of hybrid poplars may be spread by spores that lodge in the resinous coating of buds of dormant cuttings, and in the lenticels. Surface sterilization by dipping the cuttings in fungicides was tested to determine whether such treatment would prevent the germination of spores of the canker-producing fungi Septoria musiva and Dothichiza populea and the...
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William R. Jacobi; Betsy A. Goodrich; Holly S. J. Kearns; Kelly S. Burns; Brian W. Geils
2011-01-01
White pine blister rust occurs when there are compatible interactions between susceptible hosts (white pines and Ribes spp.), inoculum (Cronartium ribicola spores), and local weather conditions during infection. The five spore stages of the white pine blister rust (WPBR) fungus have specific temperature and moisture conditions necessary for production, germination, and...
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Effects of L-Alanine and Inosine Germinants on the Elasticity of Bacillus anthracis Spores
2010-01-22
there was some scanner hysteresis and drift in the detection system, we often observed that the deflection of the free cantilever was not equal to zero...of waste products.5,35 The degradation of the spore coat resulted in a cell that could bemore easily indented by the AFMprobe, as was observed on...changes in the mechanical properties of the spore. In this work, we use atomic force microscopy (AFM) to characterize the mechanical properties of the
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2009-09-01
asexual spores (sporogenic germination), or by sexual fruit bodies (carpogenic germination) (Webster and Weber 2007). Plating of dried microsclerotia...While drying the fungus does not appear to impact efficacy, it is unknown how prolonged storage might affect the viability and virulence of the organism...agar (Table 1). Warm water temperatures (25 ºC ± 1 ºC) and the presence of a host plant may have affected both germination and sporulation of the
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Pleiotropic roles of Clostridium difficile sin locus
Ou, Junjun; Dupuy, Bruno
2018-01-01
Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis. PMID:29529083
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Best, E L; Parnell, P; Thirkell, G; Verity, P; Copland, M; Else, P; Denton, M; Hobson, R P; Wilcox, M H
2014-05-01
Clostridium difficile infection (CDI) remains an infection control challenge, especially when environmental spore contamination and suboptimal cleaning may increase transmission risk. To substantiate the long-term effectiveness throughout a stroke rehabilitation unit (SRU) of deep cleaning and hydrogen peroxide decontamination (HPD), following a high incidence of CDI. Extensive environmental sampling (342 sites on each occasion) for C. difficile using sponge wipes was performed: before and after deep cleaning with detergent/chlorine agent; immediately following HPD; and on two further occasions, 19 days and 20 weeks following HPD. C. difficile isolates underwent polymerase chain reaction ribotyping and multi-locus variable repeat analysis (MLVA). C. difficile was recovered from 10.8%, 6.1%, 0.9%, 0% and 3.5% of sites at baseline, following deep cleaning, immediately after HPD, and 19 days and 20 weeks after HPD, respectively. C. difficile ribotypes recovered after deep cleaning matched those from CDI cases in the SRU during the previous 10 months. Similarly, 10/12 of the positive sites identified at 20 weeks post-HPD harboured the same C. difficile ribotype (002) and MLVA pattern as the isolate from the first post-HPD CDI case. CDI incidence [number of cases on SRU per 10 months (January-October 2011)] declined from 20 before to seven after the intervention. HPD, after deep cleaning with a detergent/chlorine agent, was highly effective for removing environmental C. difficile contamination. Long-term follow-up demonstrated that a CDI symptomatic patient can rapidly recontaminate the immediate environment. Determining a role for HPD should include long-term cost-effectiveness evaluations. Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
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Evaluation of an automated room decontamination device using aerosolized peracetic acid.
Mana, Thriveen S C; Sitzlar, Brett; Cadnum, Jennifer L; Jencson, Annette L; Koganti, Sreelatha; Donskey, Curtis J
2017-03-01
Because manual cleaning is often suboptimal, there is increasing interest in use of automated devices for room decontamination. We demonstrated that an ultrasonic room fogging system that generates submicron droplets of peracetic acid and hydrogen peroxide eliminated Clostridium difficile spores and vegetative pathogens from exposed carriers in hospital rooms and adjacent bathrooms. Published by Elsevier Inc.
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The effects of Fusarium oxysporum on broomrape (Orobanche egyptiaca) seed germination.
Hasannejad, S; Zad, S Javad; Alizade, H Mohamad; Rahymian, H
2006-01-01
Broomrape (Orobanche aegyptiaca L.), one of the most important parasitic weeds in Iran, is a root parasitic plant that can attack several crops such as tobacco, sunflower, tomato and etc. Several methods were used for Orobanche control, however these methods are inefficient and very costly. Biological control is an additional recent tool for the control of parasitic weeds. In order to study of the fungus Fusarium oxysporum (biocontrol agent) effects on broomrape seed germination, two laboratory studies were conducted in Tehran University. In the first experiment, different concentration of GR60 (0, 1, 2 and 5 ppm) as stimulation factor for Orobanche seeds germination were experimented. Results showed that concentrations of GR60 had a significant effect on seed germination. The highest seed germination percent was obtained in 1 ppm. In the second experiment, the effect of Fusarium oxysporum was tested on O. aegyptiaca seeds germination. The fungus Fusarium oxysporum were isolated from infested and juvenile O. aegyptiaca ower stalks in tomato field in karaj. Fungus spores suspension in different concentrations (0 (Control), 10(5) (T1), 10(6) (T2), 10(7) (T3) and 3 x 10(7) (T4)) from potato dextrose agar (PDA) prepared and together with 1ppm of GR60 concentration were tested on O. aegyptiaca seeds. Results show that the highest inhibition of seed germination obtained in 10(5) spores/ml. With increasing of suspension concentrations, inhibition percent was reduced and mortality of seeds germ tube was increased. In this investigation, Fusarium oxysporum can be used to inhibit seed germination, stimulate the "suicidal germination" of seeds and reduce the Orobanche seed bank.
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Activation and injury of Clostridium perfringens spores by alcohols.
Craven, S E; Blankenship, L C
1985-01-01
The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation. PMID:2864897
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Yui, Samuel; Ali, Shanom; Muzslay, Monika; Jeanes, Annette; Wilson, A Peter R
2017-12-01
OBJECTIVE To identify, using a novel enhanced method of recovery, environmental sites where spores of Clostridium difficile persist despite cleaning and hydrogen peroxide aerial decontamination. DESIGN Cohort study. SETTING Tertiary referral center teaching hospital. METHODS In total, 16 sites representing high-frequency contact or difficult-to-clean surfaces in a single-isolation room or bed area in patient bed bays were sampled before and after terminal or hydrogen peroxide disinfection using a sponge swab. In some rooms, individual sites were not present (eg, there were no en-suite rooms in the ICU). Swab contents were homogenized, concentrated by membrane-filtration, and plated onto selective media. Results of C. difficile sampling were used to focus cleaning. RESULTS Over 1 year, 2,529 sites from 146 rooms and 44 bays were sampled. Clostridium difficile was found on 131 of 572 surfaces (22.9%) before terminal cleaning, on 105 of 959 surfaces (10.6%) after terminal cleaning, and on 43 of 967 surfaces (4.4%) after hydrogen peroxide disinfection. Clostridium difficile persisted most frequently on floor corners (97 of 334; 29.0%) after disinfection. Between the first and third quarters, we observed a significant decrease in the number of positive sites (25 of 390 vs 6 of 256). However, no similar change in the number of isolates before terminal cleaning was observed. CONCLUSION Persistence of C. difficile in the clinical environment was widespread. Although feedback of results did not improve the efficacy of manual disinfection, numbers of C. difficile following hydrogen peroxide gradually declined. Infect Control Hosp Epidemiol 2017;38:1487-1492.
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Effects of steam autoclave treatment on Geobacillus stearothermophilus spores.
Huesca-Espitia, L C; Suvira, M; Rosenbeck, K; Korza, G; Setlow, B; Li, W; Wang, S; Li, Y-Q; Setlow, P
2016-11-01
To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs. Spore viability, dipicolinic acid (DPA) release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of DPA and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations. These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability. This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability. © 2016 The Society for Applied Microbiology.
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Predicting and preventing mold spoilage of food products.
Dagnas, Stéphane; Membré, Jeanne-Marie
2013-03-01
This article is a review of how to quantify mold spoilage and consequently shelf life of a food product. Mold spoilage results from having a product contaminated with fungal spores that germinate and form a visible mycelium before the end of the shelf life. The spoilage can be then expressed as the combination of the probability of having a product contaminated and the probability of mold growth (germination and proliferation) up to a visible mycelium before the end of the shelf life. For products packed before being distributed to the retailers, the probability of having a product contaminated is a function of factors strictly linked to the factory design, process, and environment. The in-factory fungal contamination of a product might be controlled by good manufacturing hygiene practices and reduced by particular processing practices such as an adequate air-renewal system. To determine the probability of mold growth, both germination and mycelium proliferation can be mathematically described by primary models. When mold contamination on the product is scarce, the spores are spread on the product and more than a few spores are unlikely to be found at the same spot. In such a case, models applicable for a single spore should be used. Secondary models can be used to describe the effect of intrinsic and extrinsic factors on either the germination or proliferation of molds. Several polynomial models and gamma-type models quantifying the effect of water activity and temperature on mold growth are available. To a lesser extent, the effect of pH, ethanol, heat treatment, addition of preservatives, and modified atmospheres on mold growth also have been quantified. However, mold species variability has not yet been properly addressed, and only a few secondary models have been validated for food products. Once the probability of having mold spoilage is calculated for various shelf lives and product formulations, the model can be implemented as part of a risk management decision tool.
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Cysteine desulfurase IscS2 plays a role in oxygen resistance in Clostridium difficile.
Giordano, Nicole; Hastie, Jessica L; Smith, Ashley D; Foss, Elissa D; Gutierrez-Munoz, Daniela F; Carlson, Paul E
2018-06-04
Clostridium difficile is an anaerobic, spore-forming bacterium capable of colonizing the gastrointestinal tract of humans following disruption of the normal microbiota, typically from antibiotic therapy for an unrelated infection. With approximately 500,000 confirmed infections leading to 29,000 deaths per year in the United States, C. difficile infection (CDI) is an urgent public health threat. We previously determined C. difficile survives in up to 3% oxygen. Low levels of oxygen are present in the intestinal tract with the higher concentrations being associated with the epithelial cell surface. Additionally, antibiotic treatment, the greatest risk factor for CDI, increases intestinal oxygen concentration. Therefore, we hypothesized that the C. difficile genome encodes mechanisms for survival during oxidative stress. Previous data have shown that cysteine desulfurases involved in iron-sulfur cluster assembly are involved in protecting bacteria from oxidative stress. In this study, deletion of a putative cysteine desulfurase ( Cd 630_12790/IscS2) involved in the iron sulfur cluster (Isc) system caused a severe growth defect in the presence of 2% oxygen. Additionally, this mutant delayed colonization in a conventional mouse model of CDI, and failed to colonize in a germ-free model, which has higher intestinal oxygen levels. These data imply an undefined role for this cysteine desulfurase in protecting C. difficile from low levels of oxygen in the gut. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
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Carroll, Thomas M.; Setlow, Peter
2005-01-01
Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582
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Non-gravitational effects on genus penicillium
DOE Office of Scientific and Technical Information (OSTI.GOV)
Loup, M.
1995-09-01
In September 1994, Shuttle Orbiter Discovery, STS-64, launched into space. Aboard that shuttle was a payload containing Fungi spores, genus Penicillium. With the over looking help of Dr. Audrey Gabel, Associate Professor of Biology at Black Hills State University, investigations on differing media types began. Basis for this experimentation was to determine if there was any differences between the space exposed spores and control spores. Studies concluded that there were differences and those differences were then recorded. It was hypothesized the spores may have been effected causing differences in growth rate, colony size, depth and margins, coloring, germination, and growthmore » on different media.« less
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A mother cell-to-forespore channel: current understanding and future challenges.
Crawshaw, Adam D; Serrano, Mónica; Stanley, Will A; Henriques, Adriano O; Salgado, Paula S
2014-09-01
Formation of endospores allows some bacteria to survive extreme nutrient limitation. The resulting dormant cell, the spore, persists in the environment and is highly resistant to physical and chemical stresses. During spore formation, cells divide asymmetrically and the mother cell engulfs the developing spore, encasing it within a double membrane and isolating it from the medium. Communication between mother cell and isolated forespore involves a specialised connection system that allows nurturing of the forespore and continued macromolecular synthesis, required to finalise spore maturation. Here, we review current understanding of this feeding channel formed by a forespore protein, SpoIIQ, and a mother cell protein, SpoIIIAH, in the model organism Bacillus subtilis and the important human pathogen Clostridium difficile. We also analyse the presence of this channel across endospore-forming bacteria and highlight the main questions still remaining. © 2014 The Authors FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.
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Recombinant lipoprotein-based vaccine candidates against C. difficile infections.
Huang, Jui-Hsin; Wu, Chia-Wei; Lien, Shu-Pei; Leng, Chih-Hsiang; Hsiao, Kuang-Nan; Liu, Shih-Jen; Chen, Hsin-Wei; Siu, Leung-Kei; Chong, Pele
2015-08-07
Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli. The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 μg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 μg of TcdA toxoid or 30 μg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin. These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.
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Albert, H; Davies, D J; Woodson, L P; Soper, C J
1998-11-01
The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.
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Survival strategies of Bacillus spores in food.
Stecchini, Mara Lucia; Del Torre, Manuela; Polese, Pierluigi
2013-11-01
Control of bacterial spores is one of the major problem in the food preservation. Spores of Bacillus genus are commonly present in different environments, including soil and the gut of insects and animals and, as a result, they can be spread to all kind of foods. Due to their high resistance properties, their complete inactivation in food is often impossible without changing the product characteristics. Surviving spores can germinate and grow out to vegetative cells, with the consequent great risk of food spoilage and food poisoning after consumption. Spores have evolved various mechanisms, including phenotypic variability, to protect themselves from a wide range of damage resulting from food preservation treatments. Even if the phenotypic heterogeneity contributes to increase the chances of survival of Bacillus spore to conventional preservation treatments, in some specific instances, an homogeneous response could be the result of a strategy adopted by the spores to increase resistance to those treatments.
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Alhmidi, Heba; John, Amrita; Mana, Thriveen C.; Koganti, Sreelatha; Cadnum, Jennifer L.; Shelton, Melissa B.
2017-01-01
Abstract During patient care simulations, cauliflower mosaic virus DNA and bacteriophage MS2 performed similarly as surrogate markers of pathogen dissemination. These markers disseminated to the environment in a manner similar to Clostridium difficile spores but were more frequently detected on skin and clothing of personnel after personal protective equipment removal. PMID:28752103
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Bacillus sphaericus LMG 22257 is physiologically suitable for self-healing concrete.
Wang, Jianyun; Jonkers, Henk M; Boon, Nico; De Belie, Nele
2017-06-01
The suitability of using a spore-forming ureolytic strain, Bacillus sphaericus, was evaluated for self-healing of concrete cracks. The main focus was on alkaline tolerance, calcium tolerance, oxygen dependence, and low-temperature adaptability. Experimental results show that B. sphaericus had a good tolerance. It can grow and germinate in a broad range of alkaline pH. The optimal pH range is 7 ∼ 9. High alkaline conditions (pH 10 ∼ 11) slow down but not stop the growth and germination. Oxygen was strictly needed during bacterial growth and germination, but not an essential factor during bacterial urea decomposition. B. sphaericus also had a good Ca tolerance, especially at a high bacterial concentration of 10 8 cells/mL; no significant influence was observed on bacterial ureolytic activity of the presence of 0.9M Ca 2+ . Furthermore, at a low temperature (10 °C), bacterial spores germinated and revived ureolytic activity with some retardation. However, this retardation can be counteracted by using a higher bacterial concentration and by supplementing yeast extract. It can be concluded that B. sphaericus is a suitable bacterium for application in bacteria-based self-healing concrete.
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Serrano, Mónica; Crawshaw, Adam D; Dembek, Marcin; Monteiro, João M; Pereira, Fátima C; Pinho, Mariana Gomes; Fairweather, Neil F; Salgado, Paula S; Henriques, Adriano O
2016-04-01
Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQ(H120S) interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ(H120S) , binds Zn(2+) , and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQ(H120S) supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
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Dielectric Study of the Physical State of Electrolytes and Water Within Bacillus cereus Spores
Carstensen, Edwin L.; Marquis, Robert E.; Gerhardt, Philipp
1971-01-01
Dielectric measurements revealed that dormant spores of Bacillus cereus have extremely low conductivities at high frequencies (50 MHz) and so must contain remarkably low concentrations of mobile ions both within the core and in the surrounding integuments. Activation, germination, and outgrowth were all accompanied by increases in conductivity of the cells and their suspending medium, and this result indicated that intracellular electrolytes had become ionized and leaked from the spores. High-frequency dielectric constants of spores were consistent with normal states for cell water. These values increased during successive stages of development from dormant spore to vegetative bacillus, and they could be directly related to increases in cell water content. In all, the results refuted a model of the dormant spore involving freely mobile, ionized electrolytes and supported a model involving electrostatically bound electrolytes. PMID:4998245
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Current knowledge on the laboratory diagnosis of Clostridium difficile infection
Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira
2017-01-01
Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI. PMID:28321156
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Davies, A; Pottage, T; Bennett, A; Walker, J
2011-03-01
The recent data for hospital-acquired infections suggest that infection rates for meticillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are beginning to decrease. However, while there is still pressure to maintain this trend, the resistance of C. difficile spores to standard detergents continues to present a problem for many UK hospitals trying to prevent its spread or control outbreaks. Alternative disinfection technologies such as gaseous decontamination are currently being marketed to the healthcare sector as an alternative/supplement to manual disinfection, and have been shown to be effective in reducing environmental contamination. When used correctly, they offer a complementary technology to manual cleaning that increases the probability of an effective reduction in viability and provides a comparatively uniform distribution of disinfectant. Three gaseous decontamination technologies are examined for their suitability in reducing environmental contamination with C. difficile: gaseous hydrogen peroxide, chlorine dioxide and ozone. Air decontamination and UV-based technologies are also briefly described. We conclude that while there is a role to play for these new technologies in the decontamination of ward surfaces contaminated with C. difficile, the requirement for both a preclean before use and the limited 'in vivo' evidence means that extensive field trials are necessary to determine their cost-effectiveness in a healthcare setting. Copyright © 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
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Ajami, N J; Cope, J L; Wong, M C; Petrosino, J F; Chesnel, L
2018-05-01
Clostridium difficile infection (CDI), a common cause of hospital-acquired infections, typically occurs after disruption of the normal gut microbiome by broad-spectrum antibiotics. Fidaxomicin is a narrow-spectrum antibiotic that demonstrates a reduced impact on the normal gut microbiota and is approved for the treatment of CDI. To further explore the benefits of this property, we used a murine model to examine the effects of fidaxomicin versus vancomycin on gut microbiota and susceptibility to C. difficile colonization while tracking microbiota recovery over time. Mice were exposed to fidaxomicin or vancomycin by oral gavage for 3 days and subsequently challenged with C. difficile spores at predetermined time points up to 21 days postexposure to antibiotics. Fecal samples were subsequently collected for analysis. Twenty-four hours postchallenge, mice were euthanized and the colon contents harvested. The microbiota was characterized using 16S rRNA gene sequencing. All fidaxomicin-exposed mice (except for one at day 8) were resistant to C. difficile colonization. However, 9 of 15 vancomycin-exposed mice were susceptible to C. difficile colonization until day 12. All vancomycin-exposed mice recovered colonization resistance by day 16. Bacterial diversity was similar prior to antibiotic exposure in both arms and decreased substantially after exposure. A shift in taxonomic structure and composition occurred after both exposures; however, the shift was greater in vancomycin-exposed than in fidaxomicin-exposed mice. In summary, compared with vancomycin, fidaxomicin exposure had less impact on microbiota composition, promoted faster microbial recovery, and had less impact on the loss of C. difficile colonization resistance. Copyright © 2018 American Society for Microbiology.
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Lister, Michelle; Stevenson, Emma; Heeg, Daniela; Minton, Nigel P; Kuehne, Sarah A
2014-08-01
Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Bettin, K; Clabots, C; Mathie, P; Willard, K; Gerding, D N
1994-11-01
To compare liquid soap versus 4% chlorhexidine gluconate in 4% alcohol for the decontamination of bare or gloved hands inoculated with an epidemic strain of Clostridium difficile. C difficile (6.7 log10 colony-forming units [CFU], 47% spores), was seeded onto bare or latex gloved hands of ten volunteers and allowed to dry. Half the volunteers initially washed with soap and half with chlorhexidine, followed by the other agent 1 week later. Cultures were done with Rodac plates at three sites on the hand: finger/thumbtips, the palmar surfaces of the fingers, and the palm. Statistical comparison was by paired Student's t test. On bare hands, soap and chlorhexidine did not differ in residual bacterial counts on the finger/thumbtips (log10 CFU, 2.0 and 2.1, P = NS) and fingers (log10 CFU, 2.4 and 2.5, P = NS). Counts were too high on bare palms to quantitate. On gloved hands, soap was more effective than chlorhexidine on fingers (log10 CFU 1.3 and 1.7, P < .01) and palms (log10 CFU 1.5 and 2.0, P < .01), but not finger/thumbtips (log10 CFU 1.6 with each, P = NS). Residual C difficile counts were lower on gloved hands than bare hands (P < 0.01 to < 0.0001). The two agents did not differ significantly in residual counts of C difficile on bare hands, but on gloved hands residual counts were lower following soap wash than following chlorhexidine wash. These observations support the use of either soap or chlorhexidine as a handwash for removal of C difficile, but efficacy in the prevention of C difficile transmission must be determined by prospective clinical trials.
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Transmission of Microsporidian Parasites of Mosquitoes.
1983-06-01
aquatic animals in a 19. spore suspension, including ostracods, amphipods, spiders, beetle larvae, and phantom midges, 2) Feeding spores to crayfish...dragonfly larvae, damselfly larvae, water scorpions, beetles , Anopheles larvae, snails, and sosquitofish. In experiment 1, no germination was seen. In... rhinoceros from Tonga. J. Gen. Virol. 47, 431-438. Ohtaki, T. and C.M. Williams. 1970. Inactivation of -- ecdysone and cyas- terone by larvae of the flesh fly
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Joint Service Chemical and Biological Defense Program: FY 06-07 Overview
2006-01-01
Performers Molecular model of human plasma-derived butyryl Electronmicrograph of bacillus spores adhering to cell membrane processes 38866_BATT_TX 11...agents, and radioactive fallout. CPS is integrated with the ship’s Heating, Ventilation, and Air-Conditioning ( HVAC ) systems and provides filtered air...molecules for intervention against protein NTA. • Identify and evaluate effectiveness of spore germination inhibitors. • Expand drug discovery program
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Kanga, Lambert H B; Adamczyk, John; Patt, Joseph; Gracia, Carlos; Cascino, John
2010-12-01
A user-friendly method to deliver Metarhizium spores to honey bee colonies for control of Varroa mites was developed and tested. Patty blend formulations protected the fungal spores at brood nest temperatures and served as an improved delivery system of the fungus to bee hives. Field trials conducted in 2006 in Texas using freshly harvested spores indicated that patty blend formulations of 10 g of conidia per hive (applied twice) significantly reduced the numbers of mites per adult bee, mites in sealed brood cells, and residual mites at the end of the 47-day experimental period. Colony development in terms of adult bee populations and brood production also improved. Field trials conducted in 2007 in Florida using less virulent spores produced mixed results. Patty blends of 10 g of conidia per hive (applied twice) were less successful in significantly reducing the number of mites per adult bee. However, hive survivorship and colony strength were improved, and the numbers of residual mites were significantly reduced at the end of the 42-day experimental period. The overall results from 2003 to 2008 field trials indicated that it was critical to have fungal spores with good germination, pathogenicity and virulence. We determined that fungal spores (1 × 10(10) viable spores per gram) with 98% germination and high pathogenicity (95% mite mortality at day 7) provided successful control of mite populations in established honey bee colonies at 10 g of conidia per hive (applied twice). Overall, microbial control of Varroa mite with M. anisopliae is feasible and could be a useful component of an integrated pest management program.
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PATHWAYS OF GLUCOSE CATABOLISM IN BACILLUS CEREUS1
Goldman, Manuel; Blumenthal, Harold J.
1964-01-01
Goldman, Manuel (The University of Michigan, Ann Arbor), and Harold J. Blumenthal. Pathways of glucose catabolism in Bacillus cereus. J. Bacteriol. 87:377–386. 1964.—Estimates by a radiorespirometric method of the pathways of glucose catabolism of resting-cell suspensions of Bacillus cereus strain terminalis indicate that the Embden-Meyerhof pathway predominates at every stage of development, including the sporogenic and germinative phases. At the filamentous, granular, forespore, and transitional stages, 98% of the glucose was catabolized by the Embden-Meyerhof pathway, and the remainder by the hexose monophosphate oxidative pathway. Estimates of the pathways in resting spore-suspensions arrested at defined stages of development indicate that 20% of the glucose was catabolized through the hexose monophosphate pathway in germinated spores, and 10% in the swollen and elongated stages of postgermination. In cells which had completed the first cell division, the figure fell to about 2%, a level similar to that found for vegetative cells at later stages of development. The key Embden-Meyerhof enzymes, hexokinase, phosphohexoisomerase, phosphofructokinase, and aldolase, as well as several other enzymes, were present at all stages of germination and postgerminative development, supporting the radioisotopic data obtained with whole cells. As indicated by the release of C14O2 from glucose-6-C14, terminal respiration of resting-cell suspensions operates maximally in vegetative cells at the granular, fore-spore, and transitional stages. There was marked inhibition of terminal respiration during the development of spores into vegetative cells. Only slight activity occurred in the earliest vegetative stages, and maximal operation developed after about ten cell divisions. Fumarase was absent in spores until sometime late in the elongation stage. At this point, a weak but definite activity appeared which increased during later stages of development so that, by the end of about the sixth cell division, fumarase had a specific activity about 80 times that observed at elongation. PMID:14151060
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NASA Astrophysics Data System (ADS)
Nicholson, Wayne L.; McCoy, Lashelle E.; Kerney, Krystal R.; Ming, Douglas W.; Golden, D. C.; Schuerger, Andrew C.
2012-08-01
Because Mars is a primary target for life detection and habitability assessment missions, its exploration is also by necessity a Planetary Protection issue. The recent finding of significant levels of perchlorate (ClO4-) in regolith sampled from the Phoenix landing site raises the question of its potential biotoxicity to putative indigenous martian life, microbial forward contaminants from Earth, or future human visitors. To address this issue, an analogue regolith was constructed based on regolith chemistry data from the Phoenix landing site. A Mars Aqueous Regolith Extract (MARE) was prepared from the Phoenix analogue regolith and analyzed by ion chromatography. The MARE contained (mg/L) the cations Na+ (1411 ± 181), Mg2+ (1051 ± 160), Ca2+ (832 ± 125), and K+ (261 ± 29), and the anions SO42-(5911±993), ClO4-(5316±1767), Cl(171±25) and F- (2.0 ± 0.4). Nitrogen-containing species NO3-(773±113) and NO2-(6.9±2.3) were also present as a result of regolith preparation procedures, but their relevance to Mars is at present unknown. The MARE was tested for potential toxic effects on two model spacecraft contaminants, the spore-forming bacteria Bacillus subtilis strain 168 and Bacillus pumilus strain SAFR-032. In B. subtilis, spore germination and initial vegetative growth (up to ˜5 h) was not inhibited in a rich complex medium prepared with the MARE, but growth after 5 h was significantly suppressed in medium prepared using the MARE. Both B. subtilis and B. pumilus exhibited significantly higher rates of spore germination and growth in the MARE vs. DW with no additions (likely due to endogenous spore nutrients), but germination and growth was further stimulated by addition of glucose and a combination of buffered inorganic salts (K2HPO4, KH2PO4, (NH4)2SO4, and MgSO4). The data indicate that the aqueous environment in the regolith from the Phoenix landing site containing high levels of perchlorate does not pose a significant barrier to growth of putative forward contaminants such as B. subtilis and B. pumilus under Earth laboratory conditions.
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Cheon, Wonsu; Kim, Young Soo; Balaraju, Kotnala; Kim, Bong-Su; Lee, Byeong-Ho; Jeon, Yongho
2016-01-01
To study the control of postharvest decay caused by Colletotrichum gloeosporioides and Penicillium expansum, gamma irradiation alone or in combination with fumigation was evaluated to extend the shelf life of apples in South Korea. An irradiation dose of 2.0 kGy resulted in the maximum inhibition of C. gloeosporioides and P. expansum spore germination. The gamma irradiation dose required to reduce the spore germination by 90% was 0.22 and 0.35 kGy for C. gloeosporioides and P. expansum, respectively. Microscopic observations revealed that when the fungal spores were treated with gamma irradiation (4.0 kGy), conidial germination was stopped completely resulting in no germ tube formation in C. gloeosporioides. Treatment with the eco-friendly fumigant ethanedinitrile had a greater antifungal activity against C. gloeosporioides and P. expansum in comparison with the non-treated control under in vitro conditions. The in vitro antifungal effects of the gamma irradiation and fumigation treatments allowed us to further study the effects of the combined treatments to control postharvest decay on stored apples. Interestingly, when apples were treated with gamma irradiation in combined with fumigation, disease inhibition increased more at lower (< 0.4 kGy) than at higher doses of irradiation, suggesting that combined treatments reduced the necessary irradiation dose in phytosanitary irradiation processing under storage conditions. PMID:27721696
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Dhayanithi, N B; Kumar, T T Ajith; Kalaiselvam, M; Balasubramanian, T; Sivakumar, N
2012-08-01
To screen the anti-fungal effects and find out the active metabolites from sponge, Sigmadocia carnosa (S. carnosa) against four dermatophytic fungi. The methanol, ethyl acetate and acetone extract of marine sponge, S. carnosa was examined against Trichophyton mentagrophytes (T. mentagrophytes), Trichophyton rubrum (T. rubrum), Epidermophyton floccosum (E. floccosum) and Microsporum gypseum (M. gypseum) and qualitative analysed to find out the active molecules. The methanol extract of sponge was expressed significant activity than ethyl acetate and acetone. The minimum inhibitory concentration (MIC) of methanol extract of sponge that resulted in complete growth inhibition of T. mentagrophytes, T. rubrum, E. floccosum and M. gypseum were found to 125, 250, 250 and 250 µg/mL respectively. But, 100 % inhibition of fungal spore germination was observed in T. mentagrophytes at 500 µg/mL concentration followed by T. rubrum, E. floccosum and M. gypseum at 1 000 µg/mL concentration. Other two extracts showed weak anti spore germination activity against the tested dermatophytic fungi. Methanol extracts showed presence of terpenoids, steroids, alkaloids, saponins and glycosides. Based on the literature, this is the first study which has conducted to inhibit the growth and spore germination of dermatophytic fungi with S. carnosa. Further research also needs to purify and characterize the secondary metabolites from the sponge, S. carnosa for the valuable source of novel substances for future drug discovery.
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Dhayanithi, NB; Kumar, TT Ajith; Kalaiselvam, M; Balasubramanian, T; Sivakumar, N
2012-01-01
Objective To screen the anti-fungal effects and find out the active metabolites from sponge, Sigmadocia carnosa (S. carnosa) against four dermatophytic fungi. Methods The methanol, ethyl acetate and acetone extract of marine sponge, S. carnosa was examined against Trichophyton mentagrophytes (T. mentagrophytes), Trichophyton rubrum (T. rubrum), Epidermophyton floccosum (E. floccosum) and Microsporum gypseum (M. gypseum) and qualitative analysed to find out the active molecules. Results The methanol extract of sponge was expressed significant activity than ethyl acetate and acetone. The minimum inhibitory concentration (MIC) of methanol extract of sponge that resulted in complete growth inhibition of T. mentagrophytes, T. rubrum, E. floccosum and M. gypseum were found to 125, 250, 250 and 250 µg/mL respectively. But, 100 % inhibition of fungal spore germination was observed in T. mentagrophytes at 500 µg/mL concentration followed by T. rubrum, E. floccosum and M. gypseum at 1 000 µg/mL concentration. Other two extracts showed weak anti spore germination activity against the tested dermatophytic fungi. Methanol extracts showed presence of terpenoids, steroids, alkaloids, saponins and glycosides. Conclusion Based on the literature, this is the first study which has conducted to inhibit the growth and spore germination of dermatophytic fungi with S. carnosa. Further research also needs to purify and characterize the secondary metabolites from the sponge, S. carnosa for the valuable source of novel substances for future drug discovery. PMID:23569985
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Bacillus Coagulans GBI-30 (BC30) improves indices of Clostridium difficile-Induced colitis in mice
2011-01-01
Background Probiotics have beneficial effects in rodent models of Clostridium difficile (C. diffiicle)-induced colitis. The spore forming probiotic strain Bacillus Coagulans GBI-30, 6086 (BC30) has demonstrated anti-inflammatory and immune-modulating effects in vitro. Our goal was to determine if BC30 improved C. difficile-induced colitis in mice. Starting on study day 0, female C57BL/6 mice were dosed by oro-gastric gavage for 15 days with vehicle (saline) or BC30 (2 × 109 CFU per day). Mice in the C. difficile groups received an antibiotic mixture (study days 5 to 8 in the drinking water), and clindamycin (10 mg/kg, i.p., on study day 10). The C. difficile strain VPI 10463 was given by gavage at 104 CFU to induce colitis on day 11. On day 16, stools and colons were collected for further analyses. Results All mice treated with BC30 survived on study day 13, while two mice treated with vehicle did not survive. On day 12, a significant difference (p = 0.0002) in the percentage of mice with normal stools (66.7%) was found in the BC30/C. difficile group, as compared to the vehicle/C. diffcile group (13.0%). On study day 16, 23.8% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0187). On this day, the stool consistency score for the BC30/C. difficile group (1.1 ± 0.2) was significantly lower (p < 0.05) than for the vehicle/C. difficile cohort (1.9 ± 0.2). BC30 modestly attenuated the colonic pathology (crypt damage, edema, leukocyte influx) that was present following C. difficile infection. Colonic MIP-2 chemokine contents (pg/2 cm colon) were: 10.2 ± 0.5 (vehicle/no C. difficile), 24.6 ± 9.5 (vehicle/C. difficile) and 16.3 ± 4.3 (BC30/C. difficle). Conclusion The probiotic BC30 improved some parameters of C. difficile-induced colitis in mice. BC30 prolonged the survival of C. diffiicle infected mice. Particularly, this probiotic improved the stool consistency of mice, in this infectious colitis model. PMID:22014083
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Lall, Sujata; Nataraj, Gita; Mehta, Preeti
2017-01-01
Clostridium difficile is a Gram-positive spore-bearing anaerobic bacillus increasingly associated with both community- and hospital-acquired colitis and diarrhea. It is the most common identifiable bacterial cause of healthcare-associated diarrhea associated with antibiotic use and one of the most common anaerobic infections. The diagnosis of C. difficile infection includes detection of toxin A/B in stool specimens by direct enzyme immunoassay, culture of pathogen from the stool specimens using a selective agar Cycloserine-Cefoxitin fructose agar (CCFA), tissue culture assay, and detection of glutamate dehydrogenase an enzyme produced by C. difficile. With few reports from India on this disease, the present study was planned to throw more light on the prevalence and utility of laboratory diagnostic methods for C. difficile-associated diarrhea (CDAD). After taking approval from the Ethics Committee, 150 patients with antibiotic-associated diarrhea were taken as a study group and fifty patients with exposure to antibiotics but who did not develop diarrhea were taken as controls. Stool specimen was processed for both culture on CCFA and toxin detection by IVD Tox A + B ELISA. Only four specimens were culture positive, whereas 13 were ELISA positive. All culture-positive isolates were toxigenic. C. difficile was neither isolated nor its toxin detected in the control group. Culture- and toxin-based assays may not detect all cases of CDAD. Based on the results of the present study, culture does not provide any additional yield over toxin assay. Better diagnostic modalities would be required to prove CDAD.
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Bacillus spore-based oral carriers loading curcumin for the therapy of colon cancer.
Yin, Liang; Meng, Zhan; Zhang, Yuxiao; Hu, Kaikai; Chen, Wuya; Han, Kaibin; Wu, Bao-Yan; You, Rong; Li, Chu-Hua; Jin, Ying; Guan, Yan-Qing
2018-02-10
Oral drug delivery has attracted substantial attention due to its advantages over other administration routes. Bacillus spores, as oral probiotic agents, are applied widely. In this paper, a novel Bacillus spore-based oral colon targeted carrier loading curcumin was developed for colon cancer treatment. Curcumin was linked covalently with the outer coat of Bacillus spore and folate, respectively (SPORE-CUR-FA). Bacillus spores are capable of delivering drugs to the colon area through gastric barrier, taking the advantage of its tolerance to the harsh conditions and disintegration of the outer coat of spores after germination in the colon. The drug release in vitro and in vivo of SPORE-CUR-FA was investigated. Results showed that SPORE-CUR-FA had the characteristics of colon-targeted drug release. Pharmacokinetic studies confirmed that Bacillus spore-based carriers could efficiently improve the oral bioavailability of curcumin. In vitro and in vivo anti-tumor studies showed that SPORE-CUR-FA had substantial ability for inhibiting colon cancer cells. These findings suggest that this Bacillus spore-based oral drug delivery system has a great potential for the treatment of colon cancer. Copyright © 2017 Elsevier B.V. All rights reserved.
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Ceratopteris richardii: a productive model for revealing secrets of signaling and development
NASA Technical Reports Server (NTRS)
Chatterjee, A.; Roux, S. J.
2000-01-01
Ceratopteris richardii is an aquatic fern grown in tropical and subtropical regions of the world. It is proven to be a productive model system for studies in the genetics, biochemistry, and cell biology of basic biologic processes that occur in early gametophytic development. It provides several advantages to biologists, especially those interested in gravitational biology, polarity development, and in the genetics of sexual development. It is easy to culture, has a relatively short life cycle, and offers an array of attractive features that facilitate genetic studies. The germination and early development of large populations of genetically identical spores are easy to synchronize, and both the direction of polarity development and cell-level gravity responses can be measured and readily manipulated within the first 24 h of spore development. Although there is no reliable transformation system available yet in Ceratopteris, recent studies suggest that the technique of RNA interference can be used to block translation of specific genes in a related fern, Marsilea, and current studies will soon reveal the applicability of this approach, as well as of other transformation approaches, in Ceratopteris. A recently completed expressed sequence tag (EST) sequencing project makes available the partial sequence of more than 2000 cDNAs, representing a significant percentage of the genes being expressed during the first 24 h of spore germination, when many developmentally interesting processes are occurring. A microarray of these ESTs is being constructed, so especially for those scientists interested in basic cellular phenomena that occur early in spore germination, the availability of the ESTs and of the microarray will make Ceratopteris an even more attractive model system.
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Ceratopteris richardii: a productive model for revealing secrets of signaling and development.
Chatterjee, A; Roux, S J
2000-09-01
Ceratopteris richardii is an aquatic fern grown in tropical and subtropical regions of the world. It is proven to be a productive model system for studies in the genetics, biochemistry, and cell biology of basic biologic processes that occur in early gametophytic development. It provides several advantages to biologists, especially those interested in gravitational biology, polarity development, and in the genetics of sexual development. It is easy to culture, has a relatively short life cycle, and offers an array of attractive features that facilitate genetic studies. The germination and early development of large populations of genetically identical spores are easy to synchronize, and both the direction of polarity development and cell-level gravity responses can be measured and readily manipulated within the first 24 h of spore development. Although there is no reliable transformation system available yet in Ceratopteris, recent studies suggest that the technique of RNA interference can be used to block translation of specific genes in a related fern, Marsilea, and current studies will soon reveal the applicability of this approach, as well as of other transformation approaches, in Ceratopteris. A recently completed expressed sequence tag (EST) sequencing project makes available the partial sequence of more than 2000 cDNAs, representing a significant percentage of the genes being expressed during the first 24 h of spore germination, when many developmentally interesting processes are occurring. A microarray of these ESTs is being constructed, so especially for those scientists interested in basic cellular phenomena that occur early in spore germination, the availability of the ESTs and of the microarray will make Ceratopteris an even more attractive model system.
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Mathew, J Itty; Cadnum, Jennifer L; Sankar, Thriveen; Jencson, Annette L; Kundrapu, Sirisha; Donskey, Curtis J
2016-06-01
Mobile handheld devices used in health care settings may become contaminated with health care-associated pathogens. We demonstrated that an enclosed ultraviolet-C radiation device was effective in rapidly reducing methicillin-resistant Staphylococcus aureus, and with longer exposure times, Clostridium difficile spores, on glass slides and reducing contamination on in-use mobile handheld devices. Published by Elsevier Inc.
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Wu, Zhilin; Yin, Xuebin; Bañuelos, Gary S.; Lin, Zhi-Qing; Zhu, Zhu; Liu, Ying; Yuan, Linxi; Li, Miao
2016-01-01
Selenium (Se) has important benefits for crop growth and stress tolerance at low concentrations. However, there is very little information on antimicrobial effect of Se against the economically important fungus Botrytis cinerea. In the present study, using sodium selenite as Se source, we investigated the effect of Se salts on spore germination and mycelial growth of the fungal pathogen in vitro and gray mold control in harvested tomato fruit. Se treatment at 24 mg/L significantly inhibited spore germination of the fungal pathogen and effectively controlled gray mold in harvested tomato fruit. Se treatment at 24 mg/L seems to induce the generation of intracellular reactive oxygen species in the fungal spores. The membrane integrity damage was observed with fluorescence microscopy following staining with propidium iodide after treatment of the spores with Se. These results suggest that Se has the potential for controlling gray mold rot of tomato fruits and might be useful in integrated control against gray mold disease of postharvest fruits and vegetables caused by B. cinerea. The mechanisms by which Se decreased gray mold decay of tomato fruit may be directly related to the severe damage to the conidia plasma membrane and loss of cytoplasmic materials from the hyphae. PMID:26779128
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Pecavar, Verena; Blaschitz, Marion; Hufnagl, Peter; Zeinzinger, Josef; Fiedler, Anita; Allerberger, Franz; Maass, Matthias; Indra, Alexander
2012-06-01
Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.
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Booth, J Leland; Duggan, Elizabeth S; Patel, Vineet I; Langer, Marybeth; Wu, Wenxin; Braun, Armin; Coggeshall, K Mark; Metcalf, Jordan P
2016-10-01
The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur. Published by Elsevier Masson SAS.
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Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L. 1
Templeman, Thomas S.; DeMaggio, Augustus E.; Stetler, David A.
1987-01-01
Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 Mr. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds. Images Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:16665699
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Sundram, Shamala; Meon, Sariah; Seman, Idris Abu; Othman, Radziah
2011-08-01
Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UMPB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.) were tested for their presymbiotic effects on two arbuscular mcorrhizal fungi, Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shriveling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.
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Ecology and Thermal Inactivation of Microbes in and on Interplanetary Space Vehicle Components
NASA Technical Reports Server (NTRS)
Reyes, A. L.; Campbell, J. E.
1975-01-01
Spores of Bacillus subtilis var. niger were heat treated in aqueous suspension at 90 C, and observed for morphological changes and loss of viability. The 5 logs reduction that occurred in broth at 90 min required 210 min in buffered water. Five characteristic changes observed after spores were exposed 120 min at 90 C in buffered water were: (1) 90% loss of spore viability, (2) 5% stainability, (3) 76% increase in spore size (as observed by scanning electron microscopy), (4) 21% of spore areas remaining refractile, and (5) an increase of 77% in packed cell volume (PCV). Stainability and PCV changes were recognized only after secondary exposure in broth. Extended heat exposure (3 h at 90 C) resulted in 99% loss of spore viability and 99% loss of stainability. After 4 hours of heat exposure, 90% of the cells disintegrated. These results suggest that early germinal changes occurr concurrently with the early changes in the heat susceptibility of dormant spores.
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Nally, M C; Pesce, V M; Maturano, Y P; Rodriguez Assaf, L A; Toro, M E; Castellanos de Figueroa, L I; Vazquez, F
2015-07-02
The aim of this study was to determine the putative modes of action of 59 viticultural yeasts (31 Saccharomyces and 28 non-Saccharomyces) that inhibited fungi isolated from sour and grey rot in grapes. Inhibition of fungal mycelial growth by metabolites, enzyme activities (laminarinases, chitinases), antifungal volatiles, competition for nutrients (siderophores, Niche Overlap Index (NOI)), inhibition of fungal spore germination and decreased germinal tube length and induction of resistance were assayed. Biofungicide yeasts were classified into "antifungal patterns", according to their mechanisms of action. Thirty isolates presented at least two of the mechanisms assayed. We propose that inhibition of fungal mycelial growth by metabolites, laminarinases, competition for nutrients, inhibition of fungal spore germination and decreased germinal tube length, and antifungal volatiles by Saccharomyces and non-Saccharomyces viticultural yeasts is used as putative biocontrol mechanisms against phytopathogenic fungi. Twenty-four different antifungal patterns were identified. Siderophore production (N)and a combination of siderophore production and NOI>0.92 (M)were the most frequent antifungal patterns observed in the biofungicide yeasts assayed. Elucidation of these mechanisms could be useful for optimization of an inoculum formulation, resulting in a more consistent control of grey and sour rot with Saccharomyces and non-Saccharomyces biocontrol yeasts. Copyright © 2015 Elsevier B.V. All rights reserved.
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Chilton, C H; Crowther, G S; Freeman, J; Todhunter, S L; Nicholson, S; Longshaw, C M; Wilcox, M H
2014-02-01
Fidaxomicin reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. We investigated fidaxomicin primary or secondary treatment efficacy using a gut model. Four triple-stage chemostat gut models were inoculated with faeces. After clindamycin induction of CDI, fidaxomicin (200 mg/L twice daily), vancomycin (125 mg/L four times daily) or metronidazole (9.3 mg/L three times daily) was administered for 7 days. Following failure/CDI recurrence, fidaxomicin (200 mg/L twice daily, 7 days) was instilled. C. difficile (CD) total viable counts (TVC), spore counts (SP), toxin titres (CYT), gut bacteria counts and antimicrobial concentrations were measured throughout. Fidaxomicin instillation reduced CD TVC/SP and CYT below the limit of detection (LOD) after 2 and 4 days, respectively, with no CDI recurrence. Metronidazole instillation failed to decrease CD TVC or CYT. Vancomycin instillation reduced CD TVC and CYT to LOD by day 4, but SP persisted. Recurrence occurred 13 days after vancomycin instillation; subsequent fidaxomicin instillation reduced CD TVC/SP/CYT below the LOD from day 2. CD was isolated sporadically, with no evidence of spore recrudescence or toxin production. Fidaxomicin had a minimal effect on the microflora, except for bifidobacteria. Fidaxomicin was detected for at least 21 days post-instillation, whereas other antimicrobials were undetectable beyond ∼4 days. Fidaxomicin successfully treated simulated primary and recurrent CDI. Fidaxomicin was superior to metronidazole in reducing CD TVC and SP, and superior to vancomycin in reducing SP without recurrence of vegetative cell growth. Fidaxomicin, but not vancomycin or metronidazole, persisted in the gut model for >20 days after instillation.
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Zidaric, Valerija; Rupnik, Maja
2016-06-01
Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. Copyright © 2016 Elsevier Ltd. All rights reserved.
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USDA-ARS?s Scientific Manuscript database
The objective of this study was to assess the ability of B. cereus spores to germinate and grow in order to determine a safe cooling rate for cooked rice, beans, and pasta, rice/chicken (4:1), rice/chicken/vegetables (3:1:1), rice/beef (4:1), and rice/beef/vegetables (3:1:1). Samples were inoculate...
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Assessment of bacterial endospore viability with fluorescent dyes.
Laflamme, C; Lavigne, S; Ho, J; Duchaine, C
2004-01-01
To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.
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2011-01-01
Background Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. Results A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Conclusions Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust. PMID:21299880
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Vasconcelos, Erico A R; Santana, Celso G; Godoy, Claudia V; Seixas, Claudine D S; Silva, Marilia S; Moreira, Leonora R S; Oliveira-Neto, Osmundo B; Price, Daniel; Fitches, Elaine; Filho, Edivaldo X F; Mehta, Angela; Gatehouse, John A; Grossi-De-Sa, Maria F
2011-02-07
Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.
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Risk factors for the development of Clostridium difficile infection in hospitalized children.
Samady, Waheeda; Pong, Alice; Fisher, Erin
2014-10-01
This article defines the risk factors for Clostridium difficile infection (CDI) in hospitalized children in light of recent studies demonstrating a change in the epidemiology of these infections in both adults and children. Antibiotic exposure within the past 4-12 weeks was noted in a majority of published cases of pediatric CDI, and that remains a key risk factor for infection. Past and/or prolonged hospitalization increase a child's risk for CDI as they increase potential contact with C. difficile spores. Of all CDI, hospital-acquired infection remains more common. Many comorbid conditions have been linked with CDI, with the strongest association existing in children with cancer and inflammatory bowel disease. Severe infections occur infrequently in pediatric patients. Markers established in adults for severe CDI resulting in colectomy or transfer to ICU have not been shown to correlate in pediatric patients. Recent antibiotic exposure and hospitalization remain key risk factors for CDI in the hospitalized pediatric patient. Patients with comorbid conditions such as malignancy and inflammatory bowel disease are at higher risk for CDI. Resistant infections and severe outcomes are not common in the pediatric population.
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Fidaxomicin for the treatment of Clostridium difficile infections.
Whitman, Craig B; Czosnowski, Quinn A
2012-02-01
To evaluate the pharmacology, microbiology, safety, and efficacy of fidaxomicin for treatment of Clostridium difficile infections (CDI). Literature was identified through Ovid MEDLINE (1948-December 2011) and International Pharmaceutical Abstracts (1970-December 2011) using the search terms fidaxomicin, OPT-80, PAR-101, OP-118, difimicin, tiacumicin, lipiarmycin, Clostridium difficile, Clostridium difficile infection, Clostridium difficile-associated diarrhea, and cost. Drug monographs were retrieved from manufacturers' Web pages, and the Red Book component of Micromedex was used for cost information. All pertinent Phase 1, 2, and 3 studies published in English were included. Fidaxomicin is a macrocyclic compound bactericidal against C. difficile and inhibits toxin and spore production. It has poor oral absorption with high fecal concentrations. Available Phase 2 and 3 data with fidaxomicin 200 mg orally every 12 hours demonstrate similar effectiveness in treating CDI compared to oral vancomycin. Fidaxomicin was shown to have less frequency of recurrent infections. Adverse effects are uncommon and occur at similar rates as with oral vancomycin. The most frequently reported adverse effects are gastrointestinal, hematologic, and electrolyte disorders. Available data are lacking in several areas, including the efficacy and safety of fidaxomicin compared to established regimens for mild-to-moderate, life-threatening, and recurrent CDIs. The cost of a 10-day course of fidaxomicin is significantly more than that of metronidazole and vancomycin for treatment of mild-to-moderate CDI. Fidaxomicin appears to be an effective and safe alternative to oral vancomycin for treatment of mild-to-moderate and severe CDI. Data on its use compared to guideline-recommended therapies for mild-to-moderate and life-threatening CDI are needed. Further data assessing the cost-effectiveness of fidaxomicin are needed. Currently, it cannot be recommended over vancomycin for treatment of CDI. However, it may be considered for treatment of recurrent infections.
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Decontamination Of Bacterial Spores by a Peptide-Mimic
2006-11-01
consisting of a thin cell wall and the outer cortex. The cell wall guarantees the maintenance of cellular integrity after germination. Lytic- enzymes ...percent of the water content of the vegetative cell. The enzymes contained in the core become active on germination. All minerals (mainly Ca2+, Mn2+ and...such as amino acids and sugars, by enzymes , by high hydrostatic pressure and by some non-nutrient chemicals such as dodecylamine (see next section
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Couey, H.M.; Uota, M.
1961-12-01
When spores of Botrytis cinerea are exposed to SO/sub 2/ gas, the subsequent reduction in spore germination is quantitatively proportional to the SO/sub 2/ concentration and the exposure time. The toxicity of SO/sub 2/ increases with increasing relative humidity. In an atmosphere of 96% RH, SO/sub 2/ is more than 20 times as effective as at 75% RH. The toxicity also increases about 1.5 times for each 10/sup 0/C rise in temperature between 0/sup 0/ and 30/sup 0/C. 8 references, 4 figures, 1 table.
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A new method to evaluate the biocontrol potential of single spore isolates of fungal entomopathogens
Posada, Francisco J.; Vega, Fernando E.
2005-01-01
Fifty Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) strains isolated from the coffee berry borer were used to develop a novel screening method aimed at selecting strains with the highest biocontrol potential. The screening method is based on percent insect mortality, average survival time, mortality distribution, percent spore germination, fungal life cycle duration, and spore production on the insect. Based on these parameters, only 11 strains merited further study. The use of a sound scientific protocol for the selection of promising fungal entomopathogens should lead to more efficient use of time, labor, and financial resources in biological control programs. PMID:17119619
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Resistant Bacterial Spore Coats and Their Breakdown During Germination
2010-01-01
proteins are released into the outer layers of the spore (SspA, B) or released into the supernatant ( SspE ). 4. Two major proteases of broad...40 30 20 15 10 3.5 N o ve x Sh ar p cw lD ge rm in at io n e xu d at e N o ve x Sh ar p cw lD ge rm in at io n e xu d at e CotA CotQ SodA SspE ...Finally, and unexpectedly, SspE , the major gamma-type SASP (small acid soluble protein) present in the inner cellular compartment of dormant spores, and
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A detailed study of gerJ mutants of Bacillus subtilis.
Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O
1986-08-01
A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.
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Miller, Charles D; Rangel, Drauzio; Braga, Gilberto U L; Flint, Stephan; Kwon, Sun-Il; Messias, Claudio L; Roberts, Donald W; Anderson, Anne J
2004-01-01
Metarhizium anisopliae isolates have a wide insect host range, but an impediment to their commercial use as a biocontrol agent of above-ground insects is the high susceptibility of spores to the near-UV present in solar irradiation. To understand stress responses in M. anisopliae, we initiated studies of enzymes that protect against oxidative stress in two strains selected because their spores differed in sensitivity to UV-B. Spores of the more near-UV resistant strain in M. anisopliae 324 displayed different isozyme profiles for catalase-peroxidase, glutathione reductase, and superoxide dismutase when compared with the less resistant strain 2575. A transient loss in activity of catalase-peroxidase and glutathione reductase was observed during germination of the spores, whereas the intensity of isozymes displaying superoxide dismutase did not change as the mycelium developed. Isozyme composition for catalase-peroxidases and glutathione reductase in germlings changed with growth phase. UV-B exposure from lamps reduced the activity of isozymes displaying catalase-peroxidase and glutathione reductase activities in 2575 more than in 324. The major effect of solar UV-A plus UV-B also was a reduction in catalase-peroxidases isozyme level, a finding confirmed by measurement of catalase specific activity. Impaired growth of M. anisopliae after near-UV exposure may be related to reduced abilities to handle oxidative stress.
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Palaniyandi, S A; Yang, S H; Suh, J-W
2013-07-01
To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.
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Glycerol enhances fungal germination at the water‐activity limit for life
Stevenson, Andrew; Hamill, Philip G.; Medina, Ángel; Kminek, Gerhard; Rummel, John D.; Dijksterhuis, Jan; Timson, David J.; Magan, Naresh; Leong, Su‐Lin L.
2016-01-01
Summary For the most‐extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0–64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water‐activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores−1). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water‐activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water‐activities than previously recorded (0.640, 0.685, 0.651, 0.664 and 0.637 respectively). In addition, the kinetics of germination at low water‐activities were substantially faster than those reported previously. Extrapolations indicated theoretical water‐activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water‐activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease‐causing microorganisms; and in biotechnologies that operate at the limits of microbial function. PMID:27631633
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Kassa, Adane; Stephan, Dietrich; Vidal, Stefan; Zimmermann, Gisbert
2004-01-01
Currently, mycopesticide development for locust and grasshopper control depends on aerial conidia or submerged spores of entomopathogenic fungi. In our study, the production of submerged conidia of Metarhizium anisopliae var. acridum (IMI 330189) was investigated in a liquid medium containing 3% biomalt and 1% yeast extract (BH-medium). The effects of freeze and spray drying techniques on the quality of submerged conidia were determined. The influence of different additives on the viability of fresh submerged conidia and their suitability for oil flowable concentrate formulation development was assessed. In a BH medium maintained at 180 rev min(-1), at 30 degrees C for 72 h, IMI 330189 produced a green pigmented biomass of submerged conidia whereas in Adámek medium it produced a yellowish biomass of submerged spores. The spore concentration was high in both media; however, the size of the spores produced in the BH medium was significantly lower than those produced in Adámek medium (P < 0.001). Submerged conidia can be effectively dried using either freeze or spray drying techniques. The viability and speed of germination were significantly affected by the drying and pulverizing process (P < 0.001). The initial viability was significantly higher for spray-dried submerged conidia than for freeze-dried spores. Pulverizing of freeze-dried submerged conidia reduced the speed of germination and the viability by 63-95%. Dried submerged conidia can be stored over 45 wk at low temperatures (< 10 degrees) without suffering a significant loss in viability. Furthermore, we have identified carriers that are suitable for oil flowable concentrate formulation development.
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NASA Astrophysics Data System (ADS)
Sherry, A.; Bell, E.; Cueto, G.; Suarez-Suarez, A.; Pilloni, G.; Hubert, C. R.
2015-12-01
Reservoir souring is caused by the activity of sulfate-reducing microorganisms (SRM) in subsurface oil reservoirs, and is often induced by seawater injection during secondary oil recovery. Souring can potentially contribute to corrosion of infrastructure, health and safety hazards to the workforce, and reduction in value by increasing refining costs associated with producing the oil resource. Souring causes annual losses in the billions of dollars to the oil industry. Endospore-forming SRM, such as Desulfotomaculum spp., are often suspected culprits in reservoir souring. Endospores can survive unfavourable conditions for long periods, yet remain poised to germinate and become active if conditions become more favourable. Factors governing endospore germination are poorly understood, but are thought to include availability of nutrients, possibly metabolic by products of other anaerobic bioprocesses, and/or variations in temperature. Most research has focused on aerobic Bacillus spp., with very few studies dedicated to spore germination among anaerobes (order Clostridiales) including the sulfate-reducing Desulfotomaculum found in anoxic subsurface petroleum reservoirs. For Desulfotomaculum spores in deep hot oil reservoirs, cold seawater introduction during secondary oil recovery may create thermal viability zones for sulfate reduction near the injection wellbore. To evaluate these processes, sulfate-containing microcosms were prepared with different marine sediments as a source of spores, and amended with organic substrates in the presence or absence of oil. Incubation at 80°C for six days was followed by a down-shift in temperature to 60°C to mimic cold seawater injection into a hot reservoir. Souring did not occur at 80°C, but commenced within hours at 60°C. Microcosms were monitored for sulfate reduction and organic acids in combination with next generation sequencing of 16S rRNA genes (Ion Torrent, Illumina MiSeq). Through a combination of high-throughput microbial DNA sequencing and geochemical process analyses we show that altered conditions in oil reservoirs during seawater flooding activates dormant Desulfotomaculum endospores, which leads to reservoir souring, and provide insights on the factors governing the germination of endospores in the deep hot biosphere.
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Permeability of bacterial spores. IV. Water content, uptake, and distribution.
BLACK, S H; GERHARDT, P
1962-05-01
Black, S. H. (The University of Michigan, Ann Arbor) and Philipp Gerhardt. Permeability of bacterial spores. IV. Water content, uptake, and distribution. J. Bacteriol. 83:960-967. 1962.-Dormant and germinated spores of Bacillus cereus strain terminalis were examined for water properties. Respectively, they exhibited a mean density of 1.28 and 1.11 g/ml, a water content of 64.8 and 73.0%, and a total water uptake of 66.6 and 75.6%, based on spore weight, or 86.0 and 83.9%, based on spore volume. The results confirmed a previous report that internal and external water are in virtually complete equilibrium, but refuted a prevailing hypothesis that heat resistance is attributable to a dry core. A model of spore ultrastructure that evolved from the cumulative results pictures a moist, dense, heteroporous core. A new hypothesis is formulated as an explanation for thermostability in spores and possibly in other instances; it postulates the occurrence of an insolubly gelled core with cross-linking between macromolecules through stable but reversible bonds so as to form a high-polymer matrix with entrapped free water.
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Germination of penicillium paneum Conidia is regulated by 1-octen-3-ol, a volatile self-inhibitor.
Chitarra, Gilma S; Abee, Tjakko; Rombouts, Frank M; Posthumus, Maarten A; Dijksterhuis, Jan
2004-05-01
Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25 degrees C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.
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Germination of Penicillium paneum Conidia Is Regulated by 1-Octen-3-ol, a Volatile Self-Inhibitor
Chitarra, Gilma S.; Abee, Tjakko; Rombouts, Frank M.; Posthumus, Maarten A.; Dijksterhuis, Jan
2004-01-01
Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 109 conidia ml−1. By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25°C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development. PMID:15128538
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Chlorogenic acid is a fungicide active against phytopathogenic fungi.
Martínez, Guadalupe; Regente, Mariana; Jacobi, Santiago; Del Rio, Marianela; Pinedo, Marcela; de la Canal, Laura
2017-08-01
Plants synthesize diverse types of secondary metabolites and some of them participate in plant protection against pathogen attack. These compounds are biodegradable and renewable alternatives, which may be envisaged for the control of plant pests and diseases. Chlorogenic acid (CGA) is a phenolic secondary metabolite which accumulates in diverse plant tissues and can be found in several agro-industrial by-products and waste. The aim of this work was to determine whether CGA could control the growth of various plant pathogenic fungi, gaining insight into its mechanism of action. Microscopic analysis showed the complete inhibition of spore germination or reduction of mycelial growth for Sclerotinia sclerotiorum, Fusarium solani, Verticillium dahliae, Botrytis cinerea and Cercospora sojina. CGA concentrations that did not completely abolish spore germination were able to produce a partial inhibition of mycelial growth. Viability tests and vital dye staining demonstrate that CGA induces fungal cell lysis. Its fungicidal activity involves an early membrane permeabilization of the spores. These results show the antifungal activity of CGA against phytopathogenic fungi relevant in horticulture and agriculture highlighting the potential of CGA-enriched wastes and by-products to be used as biofungicides. Copyright © 2017 Elsevier Inc. All rights reserved.
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Strong environmental tolerance of moss Venturiella under very high pressure
NASA Astrophysics Data System (ADS)
Ono, F.; Mori, Y.; Takarabe, K.; Nishihira, N.; Shindo, A.; Saigusa, M.; Matsushima, Y.; Saini, N. L.; Yamashita, M.
2010-03-01
It was shown by the present authors group that tardigrade can survive under high pressure of 7.5 GPa. In the case of land plants, however, no result of such experiment has been reported. We have extended our experiments to moss searching for lives under very high pressure. Spore placentas of moss Venturiella were sealed in a small Teflon capsule together with a liquid pressure medium. The capsule was put in the center of a pyrophillite cube, and the maximum pressure of 7.5 GPa was applied using a two-stage cubic anvil press. The pressure was kept constant at the maximum pressure for12, 24, 72 and 144 hours. After the pressure was released, the spores were seeded on a ager medium, and incubated for one week and more longer at 25°C with white light of 2000 lux. It was proved that 70-90% of the spores were alive and germinated after exposed to the maximum pressure of 7.5 GPa for up to 72 hours. However, after exposed to 7.5 GPa for 6 days, only 4 individuals in a hundred were germinated. The pressure tolerance of moss Venturiella is found to be stronger than a small animal, tardigrade.
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Yu, Sumei; Teng, Chunying; Liang, Jinsong; Song, Tao; Dong, Liying; Bai, Xin; Jin, Yu; Qu, Juanjuan
2017-11-01
In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 μg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl 3 ·6H 2 O. Spore germination was completely inhibited by 50 μl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.
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Growth and sporulation of some pathogenic fungi in the presence of grapefruit extract.
Orlikowski, Leszek B; Skrzypczak, Cz; Jaworska-Marosz, A
2002-01-01
Development of Fusarium oxysporum f. sp. cyclaminis, F. oxysporum f. sp. dianthi, Botrytis cinerea and B. elliptica in the presence of grapefruit extract (GE) was evaluated. Amendment of potato-dextrose agar with 40 micrograms of GE/cm3 inhibited linear growth of tested species at least in 50%. Addition of 40 micrograms of GE/cm3 of medium inhibited spore germination of F. oxysporum f.sp. cyclaminis about 34% whereas germ tube growth was suppressed in 87%. In case of Botrytis species, B. cinerea spores were more susceptible on GE than B. elliptica. They did not germinate, however, at 200 micrograms of GE/cm3. Drenching of peat, artificially infested with F. oxysporum f. sp. dianthi, with GE at conc. 165 micrograms/cm3 resulted in drastical decrease of colony forming unit numbers and this suppressive effect was observed at least 35 days.
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Narbutaite, V; Fernandez, A; Horn, N; Juodeikiene, G; Narbad, A
2008-12-01
To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin-like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.
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Porphyra yezoensis bei Helgoland — eine entwicklungsgeschichtliche Studie
NASA Astrophysics Data System (ADS)
Kornmann, P.
1986-09-01
A Porphyropsis-like epiphytic specimen found in the harbour of Helgoland was grown in culture and proved to be identical with the Japanese Porphyra yezoensis. Life history studies on this economically important alga resulted in some interesting and hitherto unknown details. The variability of the adult frond is fundamentally determined by the pattern of spore germination. Settled on Chaetomorpha filaments, monospores elongate within 20 minutes; the epiphytic germlings are attached to the substrate by a typical basal cell and give rise exclusively to elongated fronds provided with a cuneate base. Unattached spores, however, germinate into buds with rhizoids; they develop into elongated elliptical to oval fronds provided with round or cordate bases. Only plants with male areas were observed in the cultures, but Conchocelis was abundantly produced from cells of aged thalli. Grown in mussel-shells, the filamentous phase liberated conchospores for a long time.
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NASA Astrophysics Data System (ADS)
Wang, Lei; Elliott, Brad; Jin, Xixuan; Zeng, Ling; Chen, Jian
2015-12-01
The antimicrobial property of volatiles produced by red imported fire ants, Solenopsis invicta, against Beauveria bassiana, a common entomopathogenic fungus, was demonstrated. The germination rate of B. bassiana spores was significantly reduced after they were exposed to volatiles within an artificial ant nest. Since the air that contained the same level of O2 and CO2 as that in artificial fire ant nests did not suppress the germination rate of B. bassiana, the observed reduction of germination rate must be caused by the toxicity of nest volatiles. Nest fumigation may be an important component of the social immune system in S. invicta.
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Wang, Lei; Elliott, Brad; Jin, Xixuan; Zeng, Ling; Chen, Jian
2015-12-01
The antimicrobial property of volatiles produced by red imported fire ants, Solenopsis invicta, against Beauveria bassiana, a common entomopathogenic fungus, was demonstrated. The germination rate of B. bassiana spores was significantly reduced after they were exposed to volatiles within an artificial ant nest. Since the air that contained the same level of O2 and CO2 as that in artificial fire ant nests did not suppress the germination rate of B. bassiana, the observed reduction of germination rate must be caused by the toxicity of nest volatiles. Nest fumigation may be an important component of the social immune system in S. invicta.
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Effect of gamma radiation on growth and survival of common seed-borne fungi in India
NASA Astrophysics Data System (ADS)
Maity, J. P.; Chakraborty, A.; Chanda, S.; Santra, S. C.
2008-07-01
The present work describes radiation-induced effects of major seeds like Oryza sativa Cv-2233, Oryza sativa Cv-Shankar, Cicer arietinum Cv-local and seed-borne fungi like Alternaria sp., Aspergillus sp., Trichoderma sp. and Curvularia sp. 60Co gamma source at 25 °C emitting gamma ray at 1173 and 1332 keV energy was used for irradiation. Dose of gamma irradiation up to 3 kGy (0.12 kGy/h) was applied for exposing the seed and fungal spores. Significant depletion of the fungal population was noted with irradiation at 1-2 kGy, whereas germinating potential of the treated grain did not alter significantly. However, significant differential radiation response in delayed seed germination, colony formation of the fungal spores and their depletion of growth were noticed in a dose-dependent manner. The depletion of the fungal viability (germination) was noted within the irradiation dose range of 1-2 kGy for Alternaria sp. and Aspergillus sp., while 0.5-1 kGy for Trichoderma sp. and Curvularia sp. However, complete inhibition of all the selected fungi was observed above 2.5 kGy.
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Navazio, Lorella; Moscatiello, Roberto; Genre, Andrea; Novero, Mara; Baldan, Barbara; Bonfante, Paola; Mariani, Paola
2007-01-01
The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca2+ indicator aequorin to detect intracellular Ca2+ changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca2+ response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis. PMID:17142489
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Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9-5.
DeFilippi, Stefanie; Groulx, Emma; Megalla, Merna; Mohamed, Rowida; Avis, Tyler J
2018-04-01
Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9-5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9-5 were quantified over the entire period of microbial interaction. B. subtilis B9-5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens' overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9-5 protecting its ecological niche against the ingress of these pathogens.
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Biles, Charles L; Wright, Desiree; Fuego, Marianni; Guinn, Angela; Cluck, Terry; Young, Jennifer; Martin, Markie; Biles, Josiah; Poudyal, Shubhra
2012-12-01
Chaetomium globosum Kunze:Fr is a dermatophytic, dematiaceous fungus that is ubiquitous in soils, grows readily on cellulolytic materials, and is commonly found on water-damaged building materials. Chlorate affects nitrogen metabolism in fungi and is used to study compatibility among anamorphic fungi by inducing nit mutants. The effect of chlorate toxicity on C. globosum was investigated by amending a modified malt extract agar (MEA), oat agar, and carboxymethyl cellulose agar (CMC) with various levels of potassium chlorate (KClO(3)). C. globosum perithecia production was almost completely inhibited (90-100 %) at low levels of KClO(3) (0.1 mM) in amended MEA. Inhibition of perithecia production was also observed on oat agar and CMC at 1 and 10 mM, respectively. However, hyphal growth in MEA was only inhibited 20 % by 0.1-100 mM KClO(3) concentrations. Hyphal growth was never completely inhibited at the highest levels tested (200 mM). Higher levels of KClO(3) were needed on gypsum board to inhibit perithecia synthesis. In additional experiments, KClO(3) did not inhibit C. globosum, Fusarium oxysporum, Aspergillus niger, Penicillum expansum, and airborne fungal spore germination. The various fungal spores were not inhibited by KClO(3) at 1-100 mM levels. These results suggest that C. globosum perithecia synthesis is more sensitive to chlorate toxicity than are hyphal growth and spore germination. This research provides basic information that furthers our understanding about perithecia formation and may help in developing control methods for fungal growth on building materials.
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Immunoproteomics Approach for Screening of Vaccine Candidates against Intestinal Botulism.
Sharma, Arti; Rani, Sarita; Alam, Syed Imteyaz; Ponmariappan, Sarkaraisamy
2017-01-01
Intestinal botulism is an infectious form of botulism in which disease results from ingesting spores, which is followed by spore germination and intraluminal production of botulinum neurotoxins over an extended period. Botulinum neurotoxin is produced by endospore forming bacteria called C. botulinum. Immunoproteomic study was used to screen the cross reactive immunogenic proteins of Clostridium botulinum type B using C. botulinum type B live spore antiserum. The whole cell proteins were separated by two dimensional gel electrophoresis and transferred to polyvinylidene difluoride membranes. Further, the Western blotting was performed with mouse pups immune serum against C. botulinum type B live spores. Eight predominant cross immunoreactive proteins were identified by mass spectrometry. These immunogenic proteins might be used to develop novel subunit vaccine candidates against the intestinal botulism. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Zhang, Pengfei; Kong, Lingbo; Wang, Guiwen; Setlow, Peter; Li, Yong-qing
2011-01-01
Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time Tlag, the levels of spore nucleic acids remained nearly unchanged, and the Tlag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before Tlag and reached maximum at a time slightly later than Trelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment. PMID:21602365
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Peng, Zhong; Liu, Sidi; Meng, Xiujuan; Liang, Wan; Xu, Zhuofei; Tang, Biao; Wang, Yuanguo; Duan, Juping; Fu, Chenchao; Wu, Bin; Wu, Anhua; Li, Chunhui
2017-01-01
Clostridium difficile is an anaerobic Gram-positive spore-forming gut pathogen that causes antibiotic-associated diarrhea worldwide. A small number of C. difficile strains express the binary toxin (CDT), which is generally found in C. difficile 027 (ST1) and/or 078 (ST11) in clinic. However, we isolated a binary toxin-positive non-027, non-078 C. difficile LC693 that is associated with severe diarrhea in China. The genotype of this strain was determined as ST201. To understand the pathogenesis-basis of C. difficile ST201, the strain LC693 was chosen for whole genome sequencing, and its genome sequence was analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. The project finally generated an estimated genome size of approximately 4.07 Mbp for strain LC693. Genome size of the three ST201 strains ranged from 4.07 to 4.16 Mb, with an average GC content between 28.5 and 28.9%. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance. The ST201 strains contained a larger PaLoc with a Tn6218 element inserted than the 027/ST1 and 078/ST11 strains, and encoded a truncated TcdC. In addition, the ST201 strains contained intact binary toxin coding and regulation genes which are highly homologous to the 027/ST1 strain. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The presence of those genes also contributes to the pathogenesis of the ST201 strains. In this study, the genomic characterization of three binary toxin-positive C. difficile ST201 strains in clade 3 was discussed and compared to the genomes of the epidemic 027 and the 078 strains. Our analysis identified a number fitness and virulence associated genes/loci in the ST201 genomes that contribute to the pathogenesis of C. difficile ST201.
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Evaluation of Clostridium novyi-NT spores in dogs with naturally occurring tumors.
Krick, Erika L; Sorenmo, Karin U; Rankin, Shelley C; Cheong, Ian; Kobrin, Barry; Thornton, Katherine; Kinzler, Kenneth W; Vogelstein, Bert; Zhou, Shibin; Diaz, Luis A
2012-01-01
To establish the maximum tolerated dose of Clostridium novyi-NT spores in tumor-bearing dogs and evaluate spore germination within tumors and tumor response. 6 client-owned dogs. A standard dose-escalation study was planned, with maximum tolerated dose defined as the highest dose at which 0 or 1 of 6 dogs had dose-limiting toxicoses (DLT). Dogs received 1 dose of C. novyi-NT spores i.v.. Toxicoses were graded and interventions performed according to specific guidelines. Grade 3 or higher toxicosis or any toxicosis combination that substantially affected patient status was considered DLT. Clinical response was measured by use of response evaluation criteria in solid tumors at 28 days. The first 2 dogs had DLT. The dose was decreased. Two of the next 4 dogs had DLT; therefore, dose administration was stopped because the study endpoint had been reached. The most common toxicosis was fever (n = 6 dogs). Two dogs developed abscesses (1 within a nasal carcinoma and 1 splenic abscess) attributable to C. novyi-NT infection; both required surgical intervention. Clostridium novyi-NT was cultured from 1 of 6 tumors. Five dogs were available for response assessment (4 had stable disease; 1 had progressive disease). Results indicated that C. novyi-NT can germinate within tumors of dogs. Toxicosis, although common and sometimes severe, was manageable with treatment. Further studies in dogs with superficial tumors may allow for continued dose escalation and provide information for use in clinical trials in veterinary and human oncology.
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Direct mechanical dispersion and in vitro culture of fusiform rust fungus single basidiospores
Alex M. Diner
1999-01-01
Single basidiospores of Cronartium quercuum f. sp. fusiforme were cast from telia suspended over a solidified nutritional medium affixed to an operating orbital shaker. Spores thus mechanically dispersed and isolated, germinated to develop single-genotype colonies.
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Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.
Stiles, Bradley G; Pradhan, Kisha; Fleming, Jodie M; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R
2014-09-05
Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.
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Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being
Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.
2014-01-01
Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129
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Mandeel, Qaher A
2006-03-01
In earlier studies, biological control of Fusarium wilt of cucumber induced by Fusarium oxysporum f. sp. cucumerinum was demonstrated using nonpathogenic strains C5 and C14 of Fusarium oxysporum. Strain C14 induced resistance and competed for infection sites whether roots were wounded or intact, whereas strain C5 required wounds to achieve biocontrol. In the current work, additional attributes involved in enhanced resistance by nonpathogenic biocontrol agents strains to Fusarium wilt of cucumber and pea were further investigated. In pre-penetration assays, pathogenic formae specials exhibited a significantly higher percentage of spore germination in 4-day-old root exudates of cucumber and pea than nonpathogens. Also, strain C5 exhibited the lowest significant reduction in spore germination in contrast to strain C14 or control. One-day-old cucumber roots injected with strain C14 resulted in significant reduction in germ tube orientation towards the root surface, 48-96 h after inoculation with F. o. cucumerinum spores, whereas strain C5 induced significantly lower spore orientation of the pathogen and only at 72 and 96 h after inoculation. In post-penetration tests, passive transport of microconidia of pathogenic and nonpathogens in stems from base to apex were examined when severed plant roots were immersed in spore suspension. In repeated trials, strain C5, F. o. cucumerinum and F. o. pisi were consistently isolated from stem tissues of both cucumber and pea at increasing heights over a 17 days incubation period. Strain C14 however, was recovered at a maximum translocation distance of 4.6 cm at day 6 and later height of isolation significantly declined thereafter to 1.2 cm at day 17. In pea stem, the decline was even less. Significant induction of resistance to challenge inoculation by the pathogen in cucumber occurred 72 and 96 h after pre-inoculation with biocontrol agents. Nonetheless, strain C14 induced protection as early as 48 h and the maximum resistance was reached at 96 h. The presented data confirm the previous findings that attributes important for nonpathogenic fusaria to induce resistant are: rapid spore germination and orientation in response to root exudate; active root penetration and passive conidia transport in stem to initiate defence reaction without pathogenicity and enough lag period between induction and challenge inoculation. Strain C14 possesses all these qualifications and hence its ability to enhance host resistance is superior than strain C5.
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Juneja, Vijay K; Baker, David A; Thippareddi, H; Snyder, O Peter; Mohr, Tim B
2013-01-01
The ability of Clostridium perfringens to germinate and grow in acidified ground beef as well as in 10 commercially prepared acidified beef, pork, and poultry products was assessed. The pH of ground beef was adjusted with organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commercial products ranged from 4.74 to 6.35. Products were inoculated with a three-strain cocktail of C. perfringens spores to achieve ca. 2-log (low) or 4-log (high) inoculum levels, vacuum packaged, and cooled exponentially from 54.4 to 7.2°C for 6, 9, 12, 15, 18, or 21 h to simulate abusive cooling; the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) recommends a cooling time of 6.5 h. Total germinated C. perfringens populations were determined after plating on tryptose-sulfite-cycloserine agar and incubating the plates anaerobically at 37°C for 48 h. In addition, C. perfringens growth from spores was assessed at an isothermal temperature of 44°C. Growth from spores was inhibited in ground beef with a pH of 5.5 or below, even during extended cooling from 54.4 to 7.2°C in 21 h. In ground beef with a pH of 5.6, the growth was >1 log after 18 h of cooling from 54.4 to 7.2°C. However, 15 h of cooling controlled the growth to <1 log, regardless of the inoculum level. In addition, no growth was observed in any product with a pH ranging from 4.74 to 5.17, both during exponential abusive cooling periods of up to 21 h and during storage for 21 h at 44°C. While <1-log growth of C. perfringens from spores was observed in the pH 5.63 product cooled exponentially from 54.4 to 7.2°C in 15 h or less, the pH 6.35 product supported growth, even after 6 h of cooling from 54.4 to 7.2°C. These challenge tests demonstrate that adjustment of ground beef to pH of 5.5 or less and of barbeque products to pH of 5.63 or less inhibits C. perfringens spore germination and outgrowth during extended cooling periods from 54.4 to 7.2°C up to 15 h. Therefore, safe cooling periods for products with homogeneous, lower pHs can be substantially longer.
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Gao, Nan; Zhang, Yunfang; Huang, Pengcheng; Xiang, Zhehao; Wu, Fang-Ying; Mao, Lanqun
2018-06-05
Lanthanide-based luminescent sensors have been widely used for the detection of the anthrax biomarker dipicolinic acid (DPA). However, mainly based on DPA sensitization to the lanthanide core, most of them failed to realize robust detection of DPA in bacterial spores. We proposed a new strategy for reliable detection of DPA by perturbing a tandem energy transfer in heterobinuclear lanthanide coordination polymer nanoparticles simply constructed by two kinds of lanthanide ions, Tb 3+ and Eu 3+ , and guanosine 5'-monophosphate. This smart luminescent probe was demonstrated to exhibit highly sensitive and selective visual luminescence color change upon exposure to DPA, enabling accurate detection of DPA in complex biosystems such as bacterial spores. DPA release from bacterial spores on physiological germination was also successfully monitored in real time by confocal imaging. This probe is thus expected to be a powerful tool for efficient detection of bacterial spores in responding to anthrax threats.
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Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J
2015-02-01
The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.
Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan
2014-05-19
Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Mosci, D; Marmo, G W; Sciolino, L; Zaccaro, C; Antonellini, R; Accogli, L; Lazzarotto, T; Mongardi, M; Landini, M P
2017-10-01
New technologies for automated disinfection have been developed, including the use of hydrogen peroxide atomized by specific equipment, with associated silver compounds. To compare the effectiveness of an automated disinfection system with hydrogen peroxide <8% and silver ion versus a manual method with 0.5% sodium hypochlorite solution when evaluating the reduction of microbial mesophilic contamination and Clostridium difficile presence; and to evaluate the time required for both of these processes. This was a randomized multicentre trial performed in different hospital wards that had been occupied previously by patients with Clostridium difficile infection. When patients were discharged their rooms were randomized to one of two decontamination arms. The surfaces where sampled using swabs, before and after disinfection. Swab samples were cultured for quantitative detection of microbial mesophilic contamination and qualitative detection of C. difficile. Before disinfection, 13% of surfaces decontaminated with hydrogen peroxide and silver ions and 20% of surfaces decontaminated with sodium hypochlorite showed presence of C. difficile spores. After disinfection, the samples containing C. difficile were 0% (P < 0.001) in the group decontaminated with hydrogen peroxide and silver ions, and were 3% (P < 0.001) in the group decontaminated with sodium hypochlorite. This difference was not statistically significant; nor was the difference in the reduction of the microbial mesophilic contamination. The differences between the groups were not statistically significant; however, the disinfection with hydrogen peroxide and silver ions is preferable due to less dependence on operators. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Not Available
1970-01-01
Crown rust, the most destructive disease of oats, was suppressed in laboratory fumigation chambers by ozone air pollution levels commonly surpassed in many areas. Whether the effects of air pollution on crown rust are of economic importance under field conditions is yet to be determined. Crown rust, caused by the fungus Puccinia coronata, is particularly destructive in Southern and North Central States, often reducing yields 20 percent or more. Rust pustules on oats were significantly smaller when plants were exposed to 10 parts per hundred million ozone for 6 hours in the light on the 10 days after infection. Aboutmore » half as many rust spores were produced in the ozone chamber as in one protected by carbon filters. Exposure to 10 pphm ozone did not affect viability of spores. Spores produced on exposed plants germinated and penetrated stomates of oat leaves as well as spores produced on unexposed leaves.« less
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Park, Hee Jin; Park, Keun-Cheol; Yoon, Ki Sun
2014-12-01
This study investigated the antimicrobial effects of rooibos (tea extract), potassium lactate (PL) and sodium diacetate (SDA) mixture alone or in combinations on the growth of Clostridium perfringens vegetative cell and spore in ready-to-eat (RTE) Jokbal (pig's trotters). Addition of a combination of 10% rooibos and 4% PL + SDA inhibit growth of C. perfringens vegetative cell in Jokbal at 24 °C and 36 °C. The significant inhibition on germination and growth of C. perfringens spores was also observed in Jokbal with a combination of 10% rooibos and 4% PL + SDA (PL: 2.24%, SDA: 0.16%) at 24 °C. The Jokbal treated with 10% rooibos and 4% PL + SDA mixture had significantly (P < 0.05) lower TBARS values than the control at 10 and 24 °C. The lipid oxidation inhibition effect was the highest (P < 0.05) in anaerobic packed Jokbal with 10% rooibos. The addition of a combination of 10% rooibos and 4% PL + SDA during the processing of Jokbal prevented the growth of C. perfringens and the germination and growth of C. perfringens spores at room temperature. This study shows rooibos tea as a valuable natural food preservative in meat products. © 2014 Institute of Food Technologists®
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Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi.
Palma-Guerrero, J; Jansson, H-B; Salinas, J; Lopez-Llorca, L V
2008-02-01
To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis-lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine-labelled chitosan enters rapidly into conidia of both fungi, in an energy-dependent process. Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.
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Sathishkumar, Yesupatham; Velmurugan, Natarajan; Lee, Hyun Mi; Rajagopal, Kalyanaraman; Im, Chan Ki; Lee, Yang Soo
2014-08-01
Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.
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Zhao, Shengming; Han, Jinzhi; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia
2016-04-06
Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.
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Seasonal Trends in Airborne Fungal Spores in Coastal California Ecosystems
NASA Astrophysics Data System (ADS)
Morfin, J.; Crandall, S. G.; Gilbert, G. S.
2014-12-01
Airborne fungal spores cause disease in plants and animals and may trigger respiratory illnesses in humans. In terrestrial systems, fungal sporulation, germination, and persistence are strongly regulated by local meteorological conditions. However, few studies investigate how microclimate affects the spatio-temporal dynamics of airborne spores. We measured fungal aerospora abundance and microclimate at varying spatial and time scales in coastal California in three habitat-types: coast redwood forest, mixed-evergreen forest, and maritime chaparral. We asked: 1) is there a difference in total airborne spore concentration between habitats, 2) when do we see peak spore counts, and 3) do spore densities correlate with microclimate conditions? Fungal spores were caught from the air with a volumetric vacuum air spore trap during the wet season (January - March) in 2013 and 2014, as well as monthly in 2014. Initial results suggest that mixed-evergreen forests exhibit the highest amounts of spore abundance in both years compared to the other habitats. This may be due to either a higher diversity of host plants in mixed-evergreen forests or a rich leaf litter layer that may harbor a greater abundance of saprotrophic fungi. Based on pilot data, we predict that temperature and to a lesser degree, relative humidity, will be important microclimate predictors for high spore densities. These data are important for understanding when and under what weather conditions we can expect to see high levels of fungal spores in the air; this can be useful information for managers who are interested in treating diseased plants with fungicides.
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Infection of hamsters with historical and epidemic BI types of Clostridium difficile.
Razaq, Nadia; Sambol, Susan; Nagaro, Kristin; Zukowski, Walter; Cheknis, Adam; Johnson, Stuart; Gerding, Dale N
2007-12-15
North American and European hospitals have reported outbreaks of Clostridium difficile-associated disease with unexpectedly high mortality caused by a newly recognized group of C. difficile strains, group BI. Our objective was to compare, in hamsters, the virulence of a historical nonepidemic BI type, BI1, with that of 2 recent epidemic BI types, BI6 and BI17, and with that of 2 standard toxigenic strains, K14 and 630. For each strain, 10 hamsters were given 1 dose of clindamycin, followed 5 days later with 100 C. difficile spores administered by gastric inoculation. Outcomes were recorded. The hamster model demonstrated variations in mean times from inoculation to death (for BI6, 40 h; for BI1, 48 h; for K14, 49 h; for BI17, 69 h; for 630, 102 h; for BI6, BI1, and K14 vs. 630, P< .01; for BI17 vs. 630, P< .05) and from colonization to death (for BI1, 7 h; for BI17, 13 h; for BI6, 16 h; for K14, 17 h; for 630, 52 h; for BI1, BI17, BI6, and K14 vs. 630, P< .01). Group BI strains were not more rapidly fatal than the standard toxinotype 0 strain K14 but were more rapidly fatal than the standard toxinotype 0 strain 630. BI6, the most common BI type in our collection, was particularly virulent in hamsters, consistently causing death within 48 h of inoculation.
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Sensitivity of spore germination and germ tube elongation of Saccharina japonica to metal exposure.
Han, Taejun; Kong, Jeong-Ae; Kang, Hee-Gyu; Kim, Seon-Jin; Jin, Gyo-Sun; Choi, Hoon; Brown, Murray T
2011-11-01
The sensitivity of early life stages of the brown seaweed Saccharina japonica to six metals (Cd, Cu, Hg, Ni, Pb, Zn) and two waste-water samples were investigated and a new toxicity bioassay developed. The two endpoints used were spore germination and germ tube elongation with an exposure time of 24 h. Optimal test conditions determined for photon irradiance, pH, salinity and temperature were darkness, pH 8, 35‰ and 15°C, respectively. The toxicity ranking of five metals was: Hg (EC(50) of 41 and 42 μg l(-1)) > Cu (120 and 81 μg l(-1)) > Ni (2,009 and 1,360 μg l(-1)) > Zn (3,024 and 3,897 μg l(-1)) > Pb (4,760 and 4,429 μg l(-1)) > Cd (15,052 and 7,541 μg l(-1)) for germination and germ tube elongation, respectively. The sensitivities to Cd, Cu and Ni were greater in germ tube elongation than in germination process. When tested against two different waste-water samples (processed animal and printed circuit board waste-water) values of EC(50) were between 21.29 and 32.02% for germination and between 5.33 and 8.98% for germ tube elongation. Despite differences in their chemical composition, the toxic effects of waste-water samples, as indicated by EC(50) values, did not differ significantly for the same endpoints. The CV range for both germination and germ tube elongation was between 4.61 and 37.69%, indicating high levels of precision of the tests. The results compare favourably with those from more established test procedures employing micro- and macroalgae. The advantages and potential limitations of the bioassay for the assessment of anthropogenic impacts on coastal ecosystems and commercial cultivation areas in near-shore environments are discussed.
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de Rezende Ramos, Alessandra; Lüdke Falcão, Loeni; Salviano Barbosa, Guilherme; Helena Marcellino, Lucilia; Silvano Gander, Eugen
2007-01-01
Witches' broom and pod rot are the two most devastating diseases of cocoa in South America and Africa, respectively. Their control by means of phytosanitation and chemical fungicides is labor-intensive, costly and, in many cases, environmentally undesirable. Therefore efforts are made in order to identify alternative, environmentally safe and cost-efficient methods for the control of these pathogens. Promising candidates are components of the neem tree (Azadirachta indica), that have been used for centuries in Asia as insecticides, fungicides, anticonceptionals in popular medicine. Here we report about tests on the effect of various concentrations of extracts from neem leaves on growth of mycelia of Crinipellis and Phytophthora and on germination of spores of Crinipellis. We show a 35% growth reduction of mycelia of Phytophthora on neem leaf extract media, whereas growth of mycelia of Crinipellis was not affected, even at the highest concentration of neem leaf extracts used (35%). However, the most dramatic effect of neem leaf extracts is observed on Crinipellis spore germination, here the extracts (20-35%) reduced germination almost completely. Based on these results, we believe that the neem tree might be a source for the production, on small and medium scale, of an effective and cheap formulation for the control of Crinipellis and Phytophthora.
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Nathues, Eva; Jörgens, Cordula; Lorenz, Nicole; Tudzynski, Paul
2007-09-01
SUMMARY Histidine kinases are important mediators for adaptation of bacteria and plants to environmental signals. Genome analyses of filamentous fungi have revealed the presence of a high number of potential hybrid histidine kinase (HK)-encoding genes; the role of most of these potential sensors is so far unclear, though some members of the class III histidine kinases were shown to be involved in osmostress responses. Here we present a functional analysis of cphk2, a histidine kinase-encoding gene in the biotrophic grass pathogen Claviceps purpurea. The putative product of cphk2 (CpHK2) was shown to group within family X of fungal HKs and it had high homology to the oxidative stress sensors SpMAK2/3 of Schizosaccharomyces pombe. Analysis of a cphk2 deletion mutant indicated that this histidine kinase is involved in spore germination, sensitivity to oxidative stress and fungicide resistance. In addition, virulence of the Dcphk2 mutant on rye was significantly reduced compared with the wild-type strain, even if the conidial titre was adjusted to the lower germination rate. This is the first report of a role for a class X histidine kinase in a filamentous fungus.
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Federal Register 2010, 2011, 2012, 2013, 2014
2012-12-21
.... The microsclerotia are composed of melanaized fungal hyphae and can be dried to a moisture content of... microsclerotia germinate hyphally within 24 hours and sprorogenically within 72 hours. The hyphae and the spores...
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Lazar, E E; Wills, R B H; Ho, B T; Harris, A M; Spohr, L J
2008-06-01
To evaluate the antifungal activity of nitric oxide (NO) against the growth of the postharvest horticulture pathogens Aspergillus niger, Monilinia fructicola and Penicillium italicum under in vitro conditions. Different volumes of NO gas were injected into the Petri dish headspace to obtain the desired concentrations of 50-500 microl l(-1). The growth of the fungi was measured for 8 days of incubation in air at 25 degrees C. All concentrations of NO were found to produce an antifungal effect on spore germination, sporulation and mycelial growth of the three fungi, with the most effective concentration for A. niger and P. italicum being 100 and 500 microl l(-1) for M. fructicola. Short-term exposure to a low concentration of NO gas was able to inhibit the subsequent growth of A. niger, M. fructicola and P. italicum. NO gas has potential use as a natural fungicide to inhibit microbial growth on postharvest fruit and vegetables.
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Effect of low-level ozone fumigations on crown rust of oats
DOE Office of Scientific and Technical Information (OSTI.GOV)
Heagle, A.S.
1970-02-01
Exposure of crown rust differential varieties of Avena spp. to 10 pphm ozone for 6 hr in the light for 10 days after infection with Puccinia coronata significantly reduced the growth of uredia. Urediospores produced on the plants exposed to ozone germinated as well, produced as many appressoria, and resulted in as much infection as spores produced on unexposed leaves. Exposure on dry leaves to 20 pphm ozone for 3 hr for 1-5 days did not affect urediospore germination, appressoria formation, or penetration. 9 references, 1 figure, 3 tables.
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INFLUENCE OF DEATH CRITERIA ON THE X-RAY SURVIVAL CURVES OF THE FUNGUS, NEUROSPORA
Uber, Fred M.; Goddard, David R.
1934-01-01
1. When ascospores of Neurospora tetrasperma were irradiated with 11 kv. X-rays, the single spore cultures obtained displayed a wide variety of mutated forms. 2. Control germinations of ascospores showed uniform behavior, ranging from 92–95 per cent germination. 3. The shape of the survival curves was found to be a function of the criterion of death. The following criteria were used: germination, growth, production of mature ascospores, and the production of normal perithecia. 4. The germination survival curve exhibited a rhythmic variation with dosage. Germination is not a significant criterion of death. 5. Half-survival dosages for growth and ascospore production were approximately 30,000 and 20,000 roentgens, respectively. 6. Multiple hit-to-kill relations were found on the basis of the quantum hit theory; no accurate analysis was possible. 7. The studies indicate that ascospore death does not result from a single well defined reaction, but rather from the integrated effects of several deleterious processes initiated by the radiation. PMID:19872801
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Evaluation of Clostridium novyi–NT spores in dogs with naturally occurring tumors
Krick, Erika L.; Sorenmo, Karin U.; Rankin, Shelley C.; Cheong, Ian; Kobrin, Barry; Thornton, Katherine; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin; Diaz, Luis A.
2015-01-01
Objective To establish the maximum tolerated dose of Clostridium novyi–NT spores in tumor-bearing dogs and evaluate spore germination within tumors and tumor response. Animals 6 client-owned dogs. Procedures A standard dose-escalation study was planned, with maximum tolerated dose defined as the highest dose at which 0 or 1 of 6 dogs had dose-limiting toxicoses (DLT). Dogs received 1 dose of C novyi–NT spores IV. Toxicoses were graded and interventions performed according to specific guidelines. Grade 3 or higher toxicosis or any toxicosis combination that substantially affected patient status was considered DLT. Clinical response was measured by use of response evaluation criteria in solid tumors at 28 days. Results The first 2 dogs had DLT. The dose was decreased. Two of the next 4 dogs had DLT; therefore, dose administration was stopped because the study endpoint had been reached. The most common toxicosis was fever (n = 6 dogs). Two dogs developed abscesses (1 within a nasal carcinoma and 1 splenic abscess) attributable to C novyi–NT infection; both required surgical intervention. Clostridium novyi–NT was cultured from 1 of 6 tumors. Five dogs were available for response assessment (4 had stable disease; 1 had progressive disease). Conclusions and Clinical Relevance Results indicated that C novyi–NT can germinate within tumors of dogs. Toxicosis, although common and sometimes severe, was manageable with treatment. Further studies in dogs with superficial tumors may allow for continued dose escalation and provide information for use in clinical trials in veterinary and human oncology. PMID:22204296
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Meiotic sex ratio variation in natural populations of Ceratodon purpureus (Ditrichaceae).
Norrell, Tatum E; Jones, Kelly S; Payton, Adam C; McDaniel, Stuart F
2014-09-01
• Sex ratio variation is a common but often unexplained phenomenon in species across the tree of life. Here we evaluate the hypothesis that meiotic sex ratio variation can contribute to the biased sex ratios found in natural populations of the moss Ceratodon purpureus.• We obtained sporophytes from several populations of C. purpureus from eastern North America. From each sporophyte, we estimated the mean spore viability by germinating replicate samples on agar plates. We estimated the meiotic sex ratio of each sporophyte by inferring the sex of a random sample of germinated spores (mean = 77) using a PCR-RFLP test. We tested for among-sporophyte variation in viability using an ANOVA and for deviations from 1:1 sex ratio using a χ(2)-test and evaluated the relationship between these quantities using a linear regression.• We found among-sporophyte variation in spore viability and meiotic sex ratio, suggesting that genetic variants that contribute to variation in both of these traits segregate within populations of this species. However, we found no relationship between these quantities, suggesting that factors other than sex ratio distorters contribute to variation in spore viability within populations.• These results demonstrate that sex ratio distortion may partially explain the population sex ratio variation seen in C. purpureus, but more generally that genetic conflict over meiotic segregation may contribute to fitness variation in this species. Overall, this study lays the groundwork for future studies on the genetic basis of meiotic sex ratio variation. © 2014 Botanical Society of America, Inc.
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NASA Astrophysics Data System (ADS)
Neuberger, Katja; Lux-Endrich, Astrid; Panitz, Corinna
2015-01-01
In the space experiment `Spores in artificial meteorites' (SPORES), spores of the fungus Trichoderma longibrachiatum were exposed to low-Earth orbit for nearly 2 years on board the EXPOSE-R facility outside of the International Space Station. The environmental conditions tested in space were: space vacuum at 10-7-10-4 Pa or argon atmosphere at 105 Pa as inert gas atmosphere, solar extraterrestrial ultraviolet (UV) radiation at λ > 110 nm or λ > 200 nm with fluences up to 5.8 × 108 J m-2, cosmic radiation of a total dose range from 225 to 320 mGy, and temperature fluctuations from -25 to +50°C, applied isolated or in combination. Comparable control experiments were performed on ground. After retrieval, viability of spores was analysed by two methods: (i) ethidium bromide staining and (ii) test of germination capability. About 30% of the spores in vacuum survived the space travel, if shielded against insolation. However, in most cases no significant decrease was observed for spores exposed in addition to the full spectrum of solar UV irradiation. As the spores were exposed in clusters, the outer layers of spores may have shielded the inner part. The results give some information about the likelihood of lithopanspermia, the natural transfer of micro-organisms between planets. In addition to the parameters of outer space, sojourn time in space seems to be one of the limiting parameters.
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Kheiri, A; Moosawi Jorf, S A; Malihipour, A; Saremi, H; Nikkhah, M
2016-12-01
Fusarium head blight (FHB) disease caused by Fusarium graminearum is one of the most important diseases of wheat in humid and warm areas. This disease significantly reduces yield as well as seed quality. The aim of this work was to evaluate the possibility of control of FHB by chitosan (CS) and chitosan nanoparticles (CS/NPs). In vitro, the application of various concentrations of CS and CS/NPs showed significant inhibition of both radial mycelial growth and number of colonies formed against F. graminearum. The application of 1000 and 5000ppm concentration of CS and CS/NPs produced maximum inhibition of radial mycelial growth in comparison to the control, respectively. The microscopic examination, of treated F. graminearum with the CS and CS/NPs, showed dehydration and deformation in mycelial growth and some hyphae were collapsed. The maximum percentage reduction number of colonies was observed in 5000ppm concentration of both CS and CS/NPs. To test the effect of CS and CS/NPs on spore germination, four concentrations were used for 4 and 24h incubation. The 24h incubation of F. graminearum spores with a 5000ppm solution of CS greatly reduced the number of germinating spores. In greenhouse trials, the disease severity percentage was low when CS and CS/NPs were applied before fungus inoculation on the plants and 1000ppm concentration. The spores of F. graminearum germinated on the anther, hyphae penetrated into anther and colonized the palea, lemma and glume after 24 and 72 hpi, respectively. Wherease, the spikelets treated with CS and CS/NPs were infected slowly. Light microscopy and TEM observations indicated that mycelium penetrated into the cells through stoma and transited to other cells by cell wall or plasmodesmata. Mycelial growth caused conidia into cells but CS and CS/NPs prevented of it's growth. Results showed that CS and CS/NPs could be a useful biological pesticide for controlling FHB. Copyright © 2016 Elsevier B.V. All rights reserved.
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Inhibition of Ophiognomonia clavigignenti-juglandacearum by Juglans species bark extracts
M.E. Ostry; M. Moore
2013-01-01
A rapid and reliable screening technique is needed for selecting trees with resistance to butternut canker. In a laboratory assay, reagent grade naphthoquinones and crude bark extracts of Juglans species variously inhibited spore germination and growth of Ophiognomonia clavigignenti-juglandacearum, the causal fungus of butternut...
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40 CFR 180.1315 - Natamycin; exemption from the requirement of a tolerance.
Code of Federal Regulations, 2014 CFR
2014-07-01
... from the requirement of a tolerance is established for residues of natamycin in or on mushrooms when applied as a fungistat to prevent the germination of fungal spores on mushrooms produced in enclosed mushroom production facilities. [77 FR 29548, May 18, 2012] ...
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40 CFR 180.1315 - Natamycin; exemption from the requirement of a tolerance.
Code of Federal Regulations, 2012 CFR
2012-07-01
... from the requirement of a tolerance is established for residues of natamycin in or on mushrooms when applied as a fungistat to prevent the germination of fungal spores on mushrooms produced in enclosed mushroom production facilities. [77 FR 29548, May 18, 2012] ...
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40 CFR 180.1315 - Natamycin; exemption from the requirement of a tolerance.
Code of Federal Regulations, 2013 CFR
2013-07-01
... from the requirement of a tolerance is established for residues of natamycin in or on mushrooms when applied as a fungistat to prevent the germination of fungal spores on mushrooms produced in enclosed mushroom production facilities. [77 FR 29548, May 18, 2012] ...
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Light inhibits spore germination through phytochrome in Aspergillus nidulans.
Röhrig, Julian; Kastner, Christian; Fischer, Reinhard
2013-05-01
Aspergillus nidulans responds to light in several aspects. The balance between sexual and asexual development as well as the amount of secondary metabolites produced is controlled by light. Here, we show that germination is largely delayed by blue (450 nm), red (700 nm), and far-red light (740 nm). The largest effect was observed with far-red light. Whereas 60 % of the conidia produced a germ tube after 20 h in the dark, less than 5 % of the conidia germinated under far-red light conditions. Because swelling of conidia was not affected, light appears to act at the stage of germ-tube formation. In the absence of nutrients, far-red light even inhibited swelling of conidia, whereas in the dark, conidia did swell and germinated after prolonged incubation. The blue-light signaling components, LreA (WC-1) and LreB (WC-2), and also the cryptochrome/photolyase CryA were not required for germination inhibition. However, in the phytochrome mutant, ∆fphA, the germination delay was released, but germination was delayed in the dark in comparison to wild type. This suggests a novel function of phytochrome as far-red light sensor and as activator of polarized growth in the dark.
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Epidemiology of Basil Downy Mildew.
Cohen, Yigal; Ben Naim, Yariv; Falach, Lidan; Rubin, Avia E
2017-10-01
Basil downy mildew (BDM) caused by the oomycete Peronospora belbahrii is a destructive disease of sweet basil (Ocimum basilicum) worldwide. It originated in Uganda in the 1930s and recently spread to Europe, the Middle East, Americas, and the Far East. Seed transmission may be responsible for its quick global spread. The pathogen attacks leaf blades, producing chlorotic lesions with ample dark asexual spores on the lower leaf surface. Oospores may form in the mesophyll of infected leaves. The asexual spores germinate on a wet leaf surface within 2 h and penetrate into the epidermis within 4 h. Spore germination and infection occur at a wide range of temperatures from 5 to 28.5°C. Infection intensity depends on the length of dew period, leaf temperature, and inoculum dose. The duration of latent period (from infection to sporulation) extends from 5 to 10 days, depending on temperature and light regime. The shortest is 5 days at 25°C under continuous light. Sporulation requires high humidity but not free leaf wetness. Sporulation occurs at 10 to 26°C. At the optimum temperature of 18°C, the process of sporulation requires 7.5 h at relative humidity ≥ 85%, with 3 h for sporophores emergence from stomata and 4.5 h for spore formation. Sporophores can emerge under light or darkness, but spore formation occurs in the dark only. Limited data are available on spore dispersal. Spores dispersed from sporulating plants contaminate healthy plants within 2 h of exposure. Settled spores may survive on leaf surface of healthy plants for prolonged periods, depending on temperature. Seed transmission of the disease occurs in Europe, but not in Israel or the United States. P. belbahrii in Israel also attacks species belonging to Rosemarinus, Nepeta, Agastache, Micromeria, and Salvia but not Plectranthus (coleus). A Peronospora species that infects coleus does not infect sweet basil. Control of BDM includes chemical, physical, and genetic means. The fungicide mefenoxam was highly effective in controlling the disease but resistant populations were quickly selected for in Israel and Europe rendering it ineffective. A new compound oxathiapiprolin (OSBP inhibitor) is highly effective. Nocturnal illumination of basil crops controls the disease by preventing sporulation. Daytime solar heating suppressed the disease effectively by reducing spore and mycelium viability. The most effective physical means is fanning. Nocturnal fanning prevents or limits dew deposition on leaf surfaces, and as a result, infection and sporulation diminish and epidemics are prevented. Genetic resistance occurs in wild basil and its transfer to sweet basil is under way.
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Wurster, Sebastian; Thielen, Vanessa; Weis, Philipp; Walther, Paul; Elias, Johannes; Waaga-Gasser, Ana Maria; Dragan, Mariola; Dandekar, Thomas; Einsele, Hermann; Löffler, Jürgen; Ullmann, Andrew J
2017-11-17
Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.
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Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis.
Zhang, Jie; Huang, Wuren; Yuan, Chuanfei; Lu, Yuzhen; Yang, Bing; Wang, Cheng-Yuan; Zhang, Peng; Dobens, Leonard; Zou, Zhen; Wang, Chengshu; Ling, Erjun
2017-01-01
Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and β-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.
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SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis.
Eijlander, Robyn T; de Jong, Anne; Krawczyk, Antonina O; Holsappel, Siger; Kuipers, Oscar P
2014-01-01
Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information. To facilitate obtaining a complete overview as well as new insights concerning this complex and tightly regulated process, we have developed a database-driven knowledge platform called SporeWeb (http://sporeweb.molgenrug.nl) that focuses on gene regulatory networks during sporulation in the Gram-positive bacterium Bacillus subtilis. Dynamic features allow the user to navigate through all stages of sporulation with review-like descriptions, schematic overviews on transcriptional regulation and detailed information on all regulators and the genes under their control. The Web site supports data acquisition on sporulation genes and their expression, regulon network interactions and direct links to other knowledge platforms or relevant literature. The information found on SporeWeb (including figures and tables) can and will be updated as new information becomes available in the literature. In this way, SporeWeb offers a novel, convenient and timely reference, an information source and a data acquisition tool that will aid in the general understanding of the dynamics of the complete sporulation cycle.
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Mucor circinelloides induces platelet aggregation through integrin αIIbβ3 and FcγRIIA.
Ghuman, Harlene; Shepherd-Roberts, Alicia; Watson, Stephanie; Zuidscherwoude, Malou; Watson, Steve P; Voelz, Kerstin
2018-01-03
Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbβ3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA 2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbβ3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.
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Management of Clostridium difficile Infection
Al-Jashaami, Layth S.
2016-01-01
Since the discovery of Clostridium difficile infection (CDI) in the 1970s, there has been an increase in the incidence, severity, and recurrence rate of the disease. We reviewed the recent CDI literature in PubMed published before February 28, 2016 that focused on advances in therapy. Despite a large number of studies describing methods for diagnosing the disease, there is currently no definitive test that identifies this infection with certainty, which complicates therapy. Recommended therapy for CDI includes oral metronidazole for mild cases and oral vancomycin or fidaxomicin for moderate to severe cases, each given for 10 to 14 days. For infection with spore-forming C difficile, this length of treatment may be insufficient to lead to cure; however, continuing antibiotics for longer periods of time may unfavorably alter the microbiome, preventing recovery. Treatment with metronidazole has been associated with an increasing failure rate, and the only clear recommended form of metronidazole for treatment of CDI is the intravenous formulation for patients unable to take oral medications. For vancomycin or fidaxomicin treatment of first CDI recurrences, the drug used in the initial bout can be repeated. For second or future recurrences, vancomycin can be given in pulsed or tapered doses. New modalities of treatment, such as bacteriotherapy and immunotherapy, show promise for the treatment of recurrent CDI. PMID:27917075
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Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi
2015-06-01
Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Exogenous superoxide dismutase may lose its antidotal ability on rice leaves
USDA-ARS?s Scientific Manuscript database
Leaf diffusates of the resistant rice cultivars suppressed spore germination of blast fungus (Magnaporthe grisea). Bovine Cu-Zn superoxide dismutase (SOD) added to the diffusate abolished its toxicity. However, the enzyme added to the inoculum did not affect the toxicity of the diffusate. Even the s...
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Aldars-García, Laila; Sanchis, Vicente; Ramos, Antonio J; Marín, Sonia
2017-06-01
The aim of this work was to assess the temporal relationship among quantified germination, mycelial growth and aflatoxin B 1 (AFB1) production from colonies coming from single spores, in order to find the best way to predict as accurately as possible the presence of AFB1 at the early stages of contamination. Germination, mycelial growth, probability of growth and probability of AFB1 production of an isolate of Aspergillus flavus were determined at 25 °C and two water activities (0.85 and 0.87) on 3% Pistachio Extract Agar (PEA). The percentage of germinated spores versus time was fitted to the modified Gompertz equation for the estimation of the germination parameters (geometrical germination time and germination rate). The radial growth curve for each colony was fitted to a linear model for the estimation of the apparent lag time for growth and the growth rate, and besides the time to visible growth was estimated. Binary data obtained from growth and AFB1 studies were modeled using logistic regression analysis. Both water activities led to a similar fungal growth and AFB1 production. In this study, given the suboptimal set conditions, it has been observed that germination is a stage far from the AFB1 production process. Once the probability of growth started to increase it took 6 days to produce AFB1, and when probability of growth was 100%, only a 40-57% probability of detection of AFB1 production was predicted. Moreover, colony sizes with a radius of 1-2 mm could be a helpful indicator of the possible AFB1 contamination in the commodity. Despite growth models may overestimate the presence of AFB1, their use would be a helpful tool for producers and manufacturers; from our data 5% probability of AFB1 production (initiation of production) would occur when a minimum of 60% probability of growth is observed. Legal restrictions are quite severe for these toxins, thus their control from the early stages of contamination throughout the food chain is of paramount importance. Copyright © 2016 Elsevier Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Addae, Ebenezer
Bacillus anthracis is a gram positive, rod shaped and spore forming bacteria. It causes anthrax, a deadly human and animal disease that can kill its victims in three days. The spores of B. anthracis can survive extreme environmental conditions for decades and germinate when exposed to proper conditions. Due to its potential as a bio-weapon, effective disinfectants that pose less harm to the environment and animals are urgently needed. Metal nanoparticles have the potential of killing microbial cells and spores. We present here the effect of Gold/Copper Sulphide core/shell (Au/CuS) nanoparticles on B. anthracis cells and spores. The results indicated that the continuous presence of 0.83 microM during the spore growth in nutrient medium completely inhibited spore outgrowth. Au/CuS nanoparticles at concentration of 4.15 μM completely inactivated B. anthracis cells (x 107) after 30 min of pre-treatment in any of the three buffers including water, PBS, and nutrient broth. However, the same and even higher concentrations of nanoparticles produce no significant spore (x 105) killing after 24 h of pre-treatment. SEM imaging, EDS analysis, and DNA extrusion experiments revealed that nanoparticles damaged the cell membrane causing DNA and cytosolic content efflux and eventually cell death. The study demonstrated the strong antimicrobial activity of Au/CuS nanoparticles to B. anthracis cells and revealed that Au/CuS NPs showed more effective inactivation effect against the cells than they did against the spores.
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Riley, Eammon P; Trinquier, Aude; Reilly, Madeline L; Durchon, Marine; Perera, Varahenage R; Pogliano, Kit; Lopez-Garrido, Javier
2018-04-01
Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation-specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell- and developmental stage-specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ H and σ A , during sporulation. The results suggest that σ H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth. © 2018 John Wiley & Sons Ltd.
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ORIENTATION AND LOCUS OF TROPIC PHOTORECEPTOR MOLECULES IN SPORES OF BOTRYTIS AND OSMUNDA
Jaffe, Lionel; Etzold, Helmut
1962-01-01
Study of the tropic responses of Botrytis cinerea and Osmunda cinnamomea spores to blue light shows the photoreceptor molecules to be highly dichroic and oriented: in Botrytis their axes of maximum absorption lie perpendicular to the nearby cell surface; in Osmunda, parallel. The chief evidence lies in a comparison of their responses to plane polarized light—both germinate parallel to the vibration planes (defined by the axis of vibration of the electric vector and the axis of light propagation)—with those to partial illumination with unpolarized light: Botrytis grows from its brighter part; Osmunda, from its darker. The degree of orientation produced by polarized light corresponds, at high intensities, to that produced by the imposition of such large (about 100 per cent) intensity differences across a cell as to preclude all alternatives to oriented dichroic receptors. The photoreceptors of the Botrytis spore lie within the cell wall's inner half. The chief evidence lies in the component of its tropic responses to polarized light within the vibration plane: germination peaks about 10° off the vibration axis. This deviation arises from focusing which is effective only in the wall's inner half. At high intensities, anomalies appear in Botrytis which are interpreted as "centering," i.e., a tendency toward growth from the center of two or more equally illuminated points of a cell rather than from one of them. PMID:14450869
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Sella, Sandra R B R; Gouvea, Patricia Milla; Gomes, Vanessa F; Vandenberghe, Luciana P S; Minozzo, João Carlos; Soccol, Carlos Ricardo
2013-02-01
The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.
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Oomes, S J C M; van Zuijlen, A C M; Hehenkamp, J O; Witsenboer, H; van der Vossen, J M B M; Brul, S
2007-11-30
Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.
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Deschênes, Philippe; Chano, Frédéric; Dionne, Léa-Laurence; Pittet, Didier; Longtin, Yves
2017-08-01
The efficacy of the World Health Organization (WHO)-recommended handwashing technique against Clostridium difficile is uncertain, and whether it could be improved remains unknown. Also, the benefit of using a structured technique instead of an unstructured technique remains unclear. This study was a prospective comparison of 3 techniques (unstructured, WHO, and a novel technique dubbed WHO shortened repeated [WHO-SR] technique) to remove C difficile. Ten participants were enrolled and performed each technique. Hands were contaminated with 3 × 10 6 colony forming units (CFU) of a nontoxigenic strain containing 90% spores. Efficacy was assessed using the whole-hand method. The relative efficacy of each technique and of a structured (either WHO or WHO-SR) vs an unstructured technique were assessed by Mann-Whitney U test and Wilcoxon signed-rank test. The median effectiveness of the unstructured, WHO, and WHO-SR techniques in log 10 CFU reduction was 1.30 (interquartile range [IQR], 1.27-1.43), 1.71 (IQR, 1.34-1.91), and 1.70 (IQR, 1.54-2.42), respectively. The WHO-SR technique was significantly more efficacious than the unstructured technique (P = .01). Washing hands with a structured technique was more effective than washing with an unstructured technique (median, 1.70 vs 1.30 log 10 CFU reduction, respectively; P = .007). A structured washing technique is more effective than an unstructured technique against C difficile. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Moeller, Ralf; Berger, Thomas; Matthiä, Daniel; Okayasu, Ryuichi; Kitamura, H.; Reitz, Guenther
Based on their unique resistance to various space parameters, bacterial spores (mainly spores of Bacillus subtilis) are one of the model systems used for astrobiological studies. More re-cently, spores of B. subtilis have been applied for experimental research on the likelihood of interplanetary transfer of life. Since its first postulation by Arrhenius in 1903, the pansper-mia hypothesis has been revisited many-times, e.g. after the discovery of several lunar and Martian meteorites on Earth [1,2]. These information provided intriguing evidence that rocks may naturally be transferred between the terrestrial planets. The scenario of panspermia, now termed "lithopanspermia" involves three basic hypothetical steps: (i) the escape process, i.e. removal to space of biological material, which has survived being lifted from the surface to high altitudes; (ii) interim state in space, i.e., survival of the biological material over time scales comparable with interplanetary or interstellar passage; (iii) the entry process, i.e. nondestruc-tive deposition of the biological material on another planet [2]. In our research, spores of B. subtilis were used to study the effects of galactic cosmic radiation on spore survival and induced mutations. On an interplanetary journey, outside a protective magnetic field, spore-containing rocks would be exposed to bombardment by high-energy charged particle radiation from galac-tic sources and from the sun. Air-dried spore layers on three different host materials (i.e., non-porous igneous rocks (gabbro), quartz, and spacecraft analog material (aluminum)) were irradiated with accelerated heavy ions (Helium and Iron) with a LET (linear energy transfer) ˆ of 2 and 200 keV/Am, at the Heavy Ion Medical Accelerator (HIMAC) at the National In-stitute of Radiological Sciences, (NIRS), Chiba, Japan in the frame of the HIMAC research project 20B463 "Characterization of heavy ion-induced damage in Bacillus subtilis spores and their global transcriptional response during spore germination" (Moeller et al., 2008 [3]). To simulate the interplanetary journey of a meteorite, stacks of spore-samples on gabbro slides in different depths were exposed. Spore survival and the rate of the induced mutations (i.e., sporulation-deficiency (Spo-)) depended on the LET of the applied species of ions as well as on the location (and depth) of the irradiated spores in the artificial meteorite. The exposure to high LET iron ions led to a low level of spore survival and increased frequency of mutation to Spo-compared to low-energy charged particles compared to the low LET helium ions. In order to obtain insights on the role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination (HR) and apurinic/apyrimidinic (AP) endonucleases in B. subtilis spore resistance to high-energy charged particles has been studied in parallel. Spores deficient in NHEJ and AP endonucleases were significantly more sensitive to HZE particle bombardment than were the HR-mutant and wild-type spores, indicating that NHEJ and AP endonucleases provide DNA break repair pathways during spore germination. ((References: [1] Arrhenius, S. 1903. Die Verbreitung des Lebens im Weltenraum. Umschau 7:481-485.; [2] Nicholson, W. L. 2009. Ancient micronauts: interplanetary transport of microbes by cosmic impacts. Trends Mi-crobiol. 17:243-250.; [3] Moeller, R., P. Setlow, G. Horneck, T. Berger, G. Reitz, P. Rettberg, A. J. Doherty, R. Okayasu, and W. L. Nicholson. 2008. Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X-rays and high-energy charged-particle bombardment. J. Bacteriol. 190:1134-1140.))
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Gravity-directed calcium current in germinating spores of Ceratopteris richardii
NASA Technical Reports Server (NTRS)
Chatterjee, A.; Porterfield, D. M.; Smith, P. S.; Roux, S. J.
2000-01-01
Gravity directs the early polar development in single cells of Ceratopteris richardii Brogn. It acts over a limited period of time during which it irreversibly determines the axis of the spore cell's development. A self-referencing calcium selective electrode was utilized to record the net movement of calcium across the cell membrane at different positions around the periphery of the spore during the period in which gravity orients the polarity of the spore. A movement of calcium into the cell along the bottom and out of the cell along the top was detected. This movement was specific, polarized, and strongest in a direction that opposed the vector of gravity. Treatment with nifedipine, a calcium-channel blocker, diminished the calcium current and caused the cell to lose its responsiveness to the orienting influence of gravity. Results shown suggest that calcium plays a crucial role in the ability of a single cell to respond to gravity and in the subsequent establishment of its polarity.
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Bressuire-Isoard, Christelle; Bornard, Isabelle; Henriques, Adriano O; Carlin, Frédéric; Broussolle, Véronique
2016-01-01
The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Tian, Jing-Hui; Glenn, Gregory; Flyer, David; Zhou, Bin; Liu, Ye; Sullivan, Eddie; Wu, Hua; Cummings, James F; Elllingsworth, Larry; Smith, Gale
2017-07-24
Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB (027) ). TcdB (027) is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB (003) ). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB (003) , TcdB (027) and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB (003) /TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB (003) /TcdA/TcdB (027) ) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Mold inhibition on unseasoned southern pine
Carol A. Clausen; Vina W. Yang
2003-01-01
Concerns about indoor air quality due to mold growth have increased dramatically in the United States. In the absence of moisture management, fungicides need to be developed for indoor use to control mold establishment. An ideal fungicide for prevention of indoor mold growth on wood-based materials needs to specifically prevent spore germination and provide long-term...
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USDA-ARS?s Scientific Manuscript database
Chitinases function in the digestion of chitin molecules, which are present principally in insects and fungi. In plants, chitinase genes play important roles in defense, and their expression can be triggered in response to both biotic and abiotic stresses. In this study, we cloned and characterized ...
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A review on acidifying treatments for vegetable canned food.
Derossi, A; Fiore, A G; De Pilli, T; Severini, C
2011-12-01
As is well known, pasteurization treatments are not sufficient for destroying heat resistance of spore forming microorganisms, which are prevented from germination and growing by pH reducing. So, the acidification process becomes one of the most important pre-treatments for the canning industry. It is commonly applied before pasteurization treatment with the purpose of inhibiting spore germination and for reducing heat resistance of the microorganism, thereby allowing to reduce the time or temperature values of the heat treatment. With the aim to reduce the pH of vegetables several techniques are available but their application is not easy to plan. Often, industries define operative conditions only on the basis of empirical experience, thus increasing the risk of microbial growth or imparting an unpleasant sour taste. With the aim of highlighting the correct plan and management of acidification treatments to reach safety without degrading quality of canned fruit and vegetables, the topics that are reviewed and discussed are the effects of low pH on heat resistance of the most important microorganisms, acidification techniques and significant process variables, the effect of low pH on sensorial properties, and future trends.
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Alam, Mansoor; Dharni, Seema; Abdul-Khaliq; Srivastava, Santosh Kumar; Samad, Abdul; Gupta, Mahesh Kumar
2012-08-01
A bacterial strain, Streptomyces sp. CIMAP- A1 was isolated from Geranium rhizosphere and identified by morphological, physiological, biochemical and molecular characters (16S rDNA gene sequence). Phylogenetically, it was found most closely related to S. vinacendrappus, strain NRRL-2363 with 99% sequence similarity. The strain had potential antagonistic activity (in vitro) against wide range of phytopathogenic fungi like Stemphylium sp., Botrytis cinerea, Sclerotinia sclerotiorum, Colletotrichum spp., Curvularia spp., Corynespora cassicola and Thielavia basicola. The extracellular secondary metabolites produced by the strain in the culture filtrates significantly inhibited the spore germination, growth of germ tube of the germinated spores and radial growth of Alternaria alternata, Colletotrichum acutatum, Curvularia andropogonis and Fusarium moniliforme. The extraction of culture filtrate with solvents and purification by following VLC and PTLC methods always yielded a 10th fraction antifungal compound showing activity against wide range of phytopathogenic fungi. The strain was able to produce siderophores and indole-3-acetic acid. The strain was found to enhance the growth and biomass production of Geranium. It increased 11.3% fresh shoot biomass of Geranium and 21.7% essential oil yield.
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Kollmann, Albert; Brault, Agathe; Touton, Isabelle; Dubroca, Jacqueline; Chaplain, Véronique; Mougin, Christian
2003-01-01
The effect of nonylphenol on fungi following the application of contaminated sewage sludge on agricultural soil was studied in laboratory experiments. Nonylphenol bioavailability and adsorption were determined in the soil alone and soil-sludge mixtures. Mixing the soil with sludge made it possible to measure the nonylphenol concentration in the soil solution, which comprised between 6.6 x 10(-6) and 3.8 x 10(-7) M, according to the sludge. We then examined the dose-response relationship between nonylphenol concentration in the culture medium and both biomass production and germination rate of the spores from several strains of filamentous fungi. When applied in this range of concentration, nonylphenol was without noticeable short-term effect on these endpoints. Long-term exposure of fungi to nonylphenol was also assessed. The most intensive effect was a strong stimulation of spore production and germination in Fusarium oxysporum Schlechtendahl. Biomass production by the Fusarium strains also increased. Finally, nonylphenol was shown to induce laccase production in Trametes versicolor. We conclude that the potential of nonylphenol to adversely affect several soil fungi remains low.
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Clostridium difficile Diarrhea in the Elderly: Current Issues and Management Options.
Mizusawa, Masako; Doron, Shira; Gorbach, Sherwood
2015-08-01
Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in healthcare settings. Along with antimicrobial exposure, advanced age has been shown to be a significant risk factor for the development and recurrence of, and mortality from, CDI. The substantial burden of CDI in the elderly may be related to frequent healthcare exposure, the necessity for more medications, altered intestinal microbiota, and complicated comorbidities. A diagnosis of CDI is based on evidence of toxin, or the C. difficile organism itself, in a stool sample in the presence of clinical signs and symptoms. Only symptomatic patients should be tested for CDI, and routine surveillance or repeat testing on asymptomatic patients as a test of cure is discouraged. Antibiotic discontinuation alone can improve or resolve CDI in some patients, and concomitant use of antibiotics is associated with decreased response to CDI treatment. Metronidazole, vancomycin, and fidaxomicin are the therapeutic agents currently available for CDI, with the selection of these agents being based on disease severity, history of recurrence, and cost. The recurrence rate after initial treatment is 20-30%. The first recurrence can be treated with the same therapeutic agent and, for subsequent recurrences, vancomycin in a tapered and/or pulsed regimen is recommended. Fecal microbiota transplantation has shown remarkable effectiveness for recurrent anti-refractory CDI, although caution is advised in treating immunocompromised hosts and those with toxic megacolon. C. difficile can be transmitted directly and indirectly via contact with patients or their environment; therefore, isolation precautions should be initiated at the first suspicion of CDI. C. difficile spores can survive for a long time on environmental surfaces, and the patient's room and all equipment used in the room should be disinfected. In order to manage CDI in the elderly, timely diagnosis, appropriate treatment based on severity of illness, and effective infection control are essential.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Konstantin,J.; Gough, K.; Julian, R.
2008-01-01
Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids atmore » about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.« less
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Mtimet, Narjes; Guégan, Stéphanie; Durand, Lucile; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier
2016-05-01
Thermophilic spore-forming bacteria are potential contaminants in several industrial sectors involving high temperatures (40-65 °C) in the manufacturing process. Among those thermophilic spore-forming bacteria, Thermoanaerobacterium thermosaccharolyticum, called "the swelling canned food spoiler", has generated interest over the last decade in the food sector. The aim of this study was to investigate and to model pH effect on growth, heat resistance and recovery abilities after a heat-treatment of T. thermosaccharolyticum DSM 571. Growth and sporulation were conducted on reinforced clostridium media and liver broth respectively. The highest spore heat resistances and the greatest recovery ability after a heat-treatment were obtained at pH condition allowing maximal growth rate. Growth and sporulation boundaries were estimated, then models using growth limits as main parameters were extended to describe and quantify the effect of pH on recovery of injured spores after a heat-treatment. So, cardinal values were used as a single set of parameters to describe growth, sporulation and recovery abilities. Besides, this work suggests that T. thermosaccharolyticum preserve its ability for germination and outgrowth after a heat-treatment at a low pH where other high resistant spore-forming bacteria like Geobacillus stearothermophilus are unable to grow. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Water Behavior in Bacterial Spores by Deuterium NMR Spectroscopy
2015-01-01
Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium–hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water. PMID:24950158
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Hayer, Kimran; Stratford, Malcolm
2013-01-01
The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia. PMID:23995938
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Hayer, Kimran; Stratford, Malcolm; Archer, David B
2013-11-01
The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.