Molecular diversity of Clostridium botulinum and phenotypically similar strains.

PubMed

Grenda, T; Kukier, E; Sieradzki, Z; Goldsztejn, M; Kwiatek, K

2016-12-01

This study was undertaken to examine phenotypic and genetic features of strains preliminary classified as Clostridium botulinum species. The phenotypic characteristics were assessed with different culture media and biochemical tests. The genetic characterization included detection of botulinum toxin genes by PCR and macrorestriction analysis with SmaI, XhoI and SacII by PFGE (Pulsed-field Gel Electrophoresis). Despite similar biochemical properties of all analysed strains, only 47% of them contained genes determining toxicity specific to C. botulinum species. The most valuable differentiation of C. botulinum and C. botulinum-like strains was obtained after SmaI digestion. The highest affinity was observed among C. botulinum type B profiles which was even up to 100%. It was found 100% of affinity between C. botulinum and C. botulinum-like strains, however, the similarity among C. botulinum and C. botulinum-like was generally lower than 80%.

Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum sp. nov

NASA Technical Reports Server (NTRS)

Paster, B. J.; Russell, J. B.; Yang, C. M.; Chow, J. M.; Woese, C. R.; Tanner, R.

1993-01-01

In previous studies, gram-positive bacteria which grew rapidly with peptides or an amino acid as the sole energy source were isolated from bovine rumina. Three isolates, strains C, FT (T = type strain), and SR, were considered to be ecologically important since they produced up to 20-fold more ammonia than other ammonia-producing ruminal bacteria. On the basis of phenotypic criteria, the taxonomic position of these new isolates was uncertain. In this study, the 16S rRNA sequences of these isolates and related bacteria were determined to establish the phylogenetic positions of the organisms. The sequences of strains C, FT, and SR and reference strains of Peptostreptococcus anaerobius, Clostridium sticklandii, Clostridium coccoides, Clostridium aminovalericum, Acetomaculum ruminis, Clostridium leptum, Clostridium lituseburense, Clostridium acidiurici, and Clostridium barkeri were determined by using a modified Sanger dideoxy chain termination method. Strain C, a large coccus purported to belong to the genus Peptostreptococcus, was closely related to P. anaerobius, with a level of sequence similarity of 99.6%. Strain SR, a heat-resistant, short, rod-shaped organism, was closely related to C. sticklandii, with a level of sequence similarity of 99.9%. However, strain FT, a heat-resistant, pleomorphic, rod-shaped organism, was only distantly related to some clostridial species and P. anaerobius. On the basis of the sequence data, it was clear that strain FT warranted designation as a separate species. The closest known relative of strain FT was C. coccoides (level of similarity, only 90.6%). Additional strains that are phenotypically similar to strain FT were isolated in this study.(ABSTRACT TRUNCATED AT 250 WORDS).

Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes.

PubMed

Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R

2016-06-13

In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes

PubMed Central

Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R.

2016-01-01

In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947–1961], 43 from France [1986–2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2′) A5(B3′), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements. PMID:27189984

Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, C.Y.; Crawford, D.L.

1995-12-31

A Clostridium capable of degrading 2,4,6-trinitrotoluene (TNT) cometabolically was isolated from a mixed culture obtained from a bioreactor fed TNT. This bacterium, identified as a strain of Clostridium bifermentans, and designated strain CYS-1, was able to degrade TNT via 4-amino-2,6-dinitrotoluene (4-ADNT) and 2,4-diamino-6-nitrotoluene (2,4-DANT) to aliphatic polar products which are now being identified and are assumed to be organic acids. CYS 1 cells are tolerant of TNT and capable of degrading it at starting concentrations of up to {ge}100 mg/L TNT. The number of cells inoculated and the availability of cosubstrate nutrients are significant factors influencing TNT degradation, as aremore » TNT tolerance and survival of the cells at high TNT concentrations. In liquid media, at high TNT concentrations, TNT toxicity could be overcome by increasing the amount of inoculum and supplementing the culture with appropriate rich organic cosubstrates. Under these conditions, the reduction of 4-ADNT to 2,4-DANT occurred very fast, whereas the further degradation of 2,4-DANT proceeded more slowly.« less

Strategies for improved isopropanol-butanol production by a Clostridium strain from glucose and hemicellulose through consolidated bioprocessing.

PubMed

Xin, Fengxue; Chen, Tianpeng; Jiang, Yujiang; Dong, Weiliang; Zhang, Wenming; Zhang, Min; Wu, Hao; Ma, Jiangfeng; Jiang, Min

2017-01-01

High cost of traditional substrates and formation of by-products (such as acetone and ethanol) in acetone-butanol-ethanol (ABE) fermentation hindered the large-scale production of biobutanol. Here, we comprehensively characterized a newly isolated solventogenic and xylanolytic Clostridium species, which could produce butanol at a high ratio with elimination of ethanol and conversion of acetone to more value-added product, isopropanol. Ultimately, direct butanol production from hemicellulose was achieved with efficient expression of indigenous xylanase by the novel strain via consolidated bioprocessing. A novel wild-type Clostridium sp. strain NJP7 was isolated and characterized in this study, which was capable of fermenting monosaccharides, e.g., glucose into butanol via a fermentative acetone-isopropanol-butanol pathway. With enhancement of buffering capacity and alcohol dehydrogenase activities, butanol and isopropanol titer by Clostridium sp. strain NJP7 was improved to 12.21 and 1.92 g/L, respectively, and solvent productivity could be enhanced to 0.44 g/L/h. Furthermore, with in situ extraction with biodiesel, the amount of butanol and isopropanol was finally improved to 25.58 and 5.25 g/L in the fed-batch mode. Meanwhile, Clostridium sp. strain NJP7 shows capability of direct isopropanol-butanol production from hemicelluloses with expression of indigenous xylanase. 2.06 g/L of butanol and 0.54 g/L of isopropanol were finally achieved through the temperature-shift simultaneous saccharification and fermentation, representing the highest butanol production directly from hemicellulose. The co-production of isopropanol with butanol by the newly isolated Clostridium sp. strain NJP7 would add on the economical values for butanol fermentation. Furthermore, the high isopropanol-butanol production with in situ extraction would also greatly enhance the economic feasibility for fermentative production of butanol-isopropanol in large scale. Meanwhile, its direct

Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium sp. strain RPT-4213

USDA-ARS?s Scientific Manuscript database

A novel Clostridium sp. strain RPT-4213 was found producing butyrate under strict anaerobic conditions. This strain produced 9.47 g L-1 butyric acid from MRS media (0.48 g/g glucose). RPT-4213 was also used to ferment dilute acid pretreated hydrolysates including wheat straw (WSH), corn fiber (CFH...

Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213

USDA-ARS?s Scientific Manuscript database

A newly isolated Clostridium sp. strain RPT-4213 was found to produce butyrate under anaerobic conditions. Fermentations using Lactobacilli MRS Broth produced 9.47 g L-1 butyric acid from glucose (0.48 g/g glucose). However, the strain was not capable of utilizing five carbon sugars. To assess the a...

The same clade of Clostridium botulinum strains is causing avian botulism in southern and northern Europe.

PubMed

Anza, Ibone; Skarin, Hanna; Vidal, Dolors; Lindberg, Anna; Båverud, Viveca; Mateo, Rafael

2014-04-01

Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C. botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C. botulinum type C/D through Europe and give relevant information for future epidemiological studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

The molecular-genetic analysis of Clostridium perfringens strains isolated from broilers on farms in Central Russia

USDA-ARS?s Scientific Manuscript database

The objective of the research was to perform phenotypic and molecular-genetic typing of Clostridium perfringens strains commonly spread on poultry farms in Central Russia. Samples of homogenized iliac and cecal contents from 760 broilers were assayed and 325 C. perfringens strains (42.8 %) were isol...

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production.

PubMed

Kannuchamy, Saranyah; Mukund, Nisha; Saleena, Lilly M

2016-05-11

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis. The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain. Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents.

PubMed

Carman, R J; Wilkins, T D

1991-08-30

Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-toxigenic strains of Clostridium spiroforme. Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits. Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8 micrograms/ml or more). Metronidazole was uniformly active against C. spiroforme. With MIC of 8 micrograms/ml or more, both lincomycin (11 strains) and erythromycin (9 strains) were relatively inactive against C. spiroforme, conversely, penicillin G was active (MIC for 8 strains was 0.5 micrograms/ml or less). Exposure to any one of these drugs has been implicated as a predisposing factor for C. spiroforme mediated diarrhoea of rabbits. The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold).

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-06-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed Central

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-01-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

Typing Clostridium difficile strains based on tandem repeat sequences

PubMed Central

2009-01-01

Background Genotyping of epidemic Clostridium difficile strains is necessary to track their emergence and spread. Portability of genotyping data is desirable to facilitate inter-laboratory comparisons and epidemiological studies. Results This report presents results from a systematic screen for variation in repetitive DNA in the genome of C. difficile. We describe two tandem repeat loci, designated 'TR6' and 'TR10', which display extensive sequence variation that may be useful for sequence-based strain typing. Based on an investigation of 154 C. difficile isolates comprising 75 ribotypes, tandem repeat sequencing demonstrated excellent concordance with widely used PCR ribotyping and equal discriminatory power. Moreover, tandem repeat sequences enabled the reconstruction of the isolates' largely clonal population structure and evolutionary history. Conclusion We conclude that sequence analysis of the two repetitive loci introduced here may be highly useful for routine typing of C. difficile. Tandem repeat sequence typing resolves phylogenetic diversity to a level equivalent to PCR ribotypes. DNA sequences may be stored in databases accessible over the internet, obviating the need for the exchange of reference strains. PMID:19133124

Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

DOE PAGES

Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup; ...

2016-10-28

Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup

Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

Enhanced robustness in acetone-butanol-ethanol fermentation with engineered Clostridium beijerinckii overexpressing adhE2 and ctfAB.

PubMed

Lu, Congcong; Yu, Le; Varghese, Saju; Yu, Mingrui; Yang, Shang-Tian

2017-11-01

Clostridium beijerinckii CC101 was engineered to overexpress aldehyde/alcohol dehydrogenase (adhE2) and CoA-transferase (ctfAB). Solvent production and acid assimilation were compared between the parental and engineered strains expressing only adhE2 (CC101-SV4) and expressing adhE2, ald and ctfAB (CC101-SV6). CC101-SV4 showed an early butanol production from glucose but stopped pre-maturely at a low butanol concentration of ∼6g/L. Compared to CC101, CC101-SV6 produced more butanol (∼12g/L) from glucose and was able to re-assimilate more acids, which prevented "acid crash" and increased butanol production, under all conditions studied. CC101-SV6 also showed better ability in using glucose and xylose present in sugarcane bagasse hydrolysate, and produced 9.4g/L solvents (acetone, butanol and ethanol) compared to only 2.6g/L by CC101, confirming its robustness and better tolerance to hydrolysate inhibitors. The engineered strain of C. beijerinckii overexpressing adhE2 and ctfAB should have good potential for producing butanol from lignocellulosic biomass hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dual Toxin-Producing Strain of Clostridium botulinum Type Bf Isolated from a California Patient with Infant Botulism

PubMed Central

Barash, Jason R.; Arnon, Stephen S.

2004-01-01

A retrospective study of Clostridium botulinum strains isolated from patients from California with infant botulism identified the fourth known C. botulinum strain that produces both type B and type F botulinum toxins. This unique strain represented 0.12% of the California infant botulism case isolates from 1976 to 2003. The relative concentrations of type B and F toxins produced were temperature dependent. PMID:15071029

Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials.

PubMed

Martel, A; Devriese, L A; Cauwerts, K; De Gussem, K; Decostere, A; Haesebrouck, F

2004-02-01

Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis. All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin. All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity. Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated. Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain. One strain with high-level resistance against tetracyclines carried the tet(M) gene. Sixty-three percent of the strains showed low-level resistance to lincomycin. The lnu(A) and lnu(B) genes were each only found in one strain. Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%).

Application of Lactobacillus johnsonii expressing phage endolysin for control of Clostridium perfringens.

PubMed

Gervasi, T; Lo Curto, R; Minniti, E; Narbad, A; Mayer, M J

2014-10-01

Clostridium perfringens is frequently found in food and the environment and produces potent toxins that have a negative impact on both human and animal health and particularly on the poultry industry. Lactobacillus johnsonii FI9785, isolated from the chicken gastrointestinal tract, has been demonstrated to exclude Cl. perfringens in poultry. We have investigated the interaction of wild-type Lact. johnsonii FI9785 or an engineered strain expressing a cell wall-hydrolysing endolysin with Cl. perfringens in vitro, using a batch culture designed to simulate human gastrointestinal tract conditions. Co-culture experiments indicated that acid production by Lact. johnsonii is important in pathogen control. The co-culture of the endolysin-secreting Lact. johnsonii with Cl. perfringens showed that the engineered strain had the potential to control the pathogen, but the ability to reduce Cl. perfringens numbers was not consistent. Results obtained indicate that survival of high numbers of Lact. johnsonii will be essential for effective pathogen control. Significance and impact of the study: The bacterium Lactobacillus johnsonii FI9785 reduces numbers of the pathogen Clostridium perfringens in vitro. Biocontrol was improved by engineering the strain to produce and export a cell wall-hydrolysing endolysin, but good survival of the producer strain is essential. The production of bacteriophage endolysins by commensal bacteria has the potential to improve competitive exclusion of pathogens in the gastrointestinal tract. © 2014 The Society for Applied Microbiology.

Simultaneous glucose and xylose uptake by an acetone/butanol/ethanol producing laboratory Clostridium beijerinckii strain SE-2.

PubMed

Zhang, Jie; Zhu, Wen; Xu, Haipeng; Li, Yan; Hua, Dongliang; Jin, Fuqiang; Gao, Mintian; Zhang, Xiaodong

2016-04-01

Most butanol-producing strains of Clostridium prefer glucose over xylose, leading to a slower butanol production from lignocellulose hydrolysates. It is therefore beneficial to find and use a strain that can simultaneously use both glucose and xylose. Clostridium beijerinckii SE-2 strain assimilated glucose and xylose simultaneously and produced ABE (acetone/butanol/ethanol). The classic diauxic growth behavior was not seen. Similar rates of sugar consumption (4.44 mM glucose h(-1) and 6.66 mM xylose h(-1)) were observed suggesting this strain could use either glucose or xylose as the substrate and it has a similar capability to degrade these two sugars. With different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. ABE production profiles were similar on different substrates. Transcriptional studies on the effect of glucose and xylose supplementation, however, suggests a clear glucose inhibition on xylose metabolism-related genes is still present.

Characterization of Clostridium perfringens strains isolated from clinically healthy and necrotic enteritis-afflicted broiler chickens

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens (CP) Type A strains are the main etiological factor for necrotic enteritis (NE), one of the important enteric diseases in poultry, which has gained worldwide attention during the last decade and is responsible for the annual loss of 6 billion dollars in US poultry industry. ...

Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol.

PubMed

Sedlar, Karel; Kolek, Jan; Skutkova, Helena; Branska, Barbora; Provaznik, Ivo; Patakova, Petra

2015-11-20

The strain Clostridium pasteurianum NRRL B-598 is non-type, oxygen tolerant, spore-forming, mesophilic and heterofermentative strain with high hydrogen production and ability of acetone-butanol fermentation (ethanol production being negligible). Here, we present the annotated complete genome sequence of this bacterium, replacing the previous draft genome assembly. The genome consisting of a single circular 6,186,879 bp chromosome with no plasmid was determined using PacBio RSII and Roche 454 sequencing. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D

2013-01-01

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of Llactate ( ldh) and/or acetate ( pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end products. Cellobiose-grown cultures of the ldh strain had identical biomass accumulation, fermentation end products, transcription profile and intracellular metabolite concentrations compared to its parent strain (DSM1313 hpt spo0A). The pta-deficient strain grew slower and had 30% lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol.more » A ldh pta double mutant strain evolved for faster growth had growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to pta. Free amino acids were secreted by all examined strains, with both pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for ldh pta reached 5 mM by the end of growth, or 2.7% of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to 6-fold in the pta and 16-fold in the ldh pta strain. We hypothesize that the deletions in fermentation end product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.« less

Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D

2013-01-01

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dptamore » double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Dpta and 16-fold in the Dldh Dpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP* through increased production of amino acids.« less

Acetone-butanol-ethanol production from substandard and surplus dates by Egyptian native Clostridium strains.

PubMed

Abd-Alla, Mohamed Hemida; Zohri, Abdel-Naser Ahmed; El-Enany, Abdel-Wahab Elsadek; Ali, Shimaa Mohamed

2015-04-01

One hundred and seven mesophilic isolates of Clostridium were isolated from agricultural soils cultivated with different plants in Assuit Governorate, Egypt. Eighty isolates (out of 107) showed the ability to produce ABE (Acetone, butanol and ethanol) on T6 medium ranging from 0.036 to 31.89 g/L. The highest numbers of ABE producing isolates were obtained from soil samples of potato contributing 27 isolates, followed by 18 isolates from wheat and 10 isolates from onion. On the other hand, there were three native isolates that produced ABE more than those produced by the reference isolate Clostridium acetobutylicum ATCC 824 (11.543 g/L). The three isolates were identified based on phenotypic and gene encoding 16S rRNA as Clostridium beijerinckii ASU10 (KF372577), Clostridium chauvoei ASU55 (KF372580) and Clostridium roseum ASU58 (KF372581). The highest ABE level from substandard and surplus dates was produced by C. beijerinckii ASU10 (24.07 g/L) comprising butanol 67.15% (16.16 g/L), acetone 30.73% (7.4 g/L) and ethanol 2.12% (0.51 g/L), while C. roseum ASU58 and C. chauvoei ASU55 produced ABE contributing 20.20 and 13.79 g/L, respectively. ABE production by C. acetobutylicum ATCC 824 was 15.01 g/L. This study proved that the native strains C. beijerinckii ASU10 and C. roseum ASU58 have high competitive efficacy on ABE production from economical substrate as substandard and surplus date fruits. Additionally, using this substrate without any nutritional components is considered to be a commercial substrate for desired ABE production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Relationship of compressive stress-strain response of engineering materials obtained at constant engineering and true strain rates

DOE PAGES

Song, Bo; Sanborn, Brett

2018-05-07

In this paper, a Johnson–Cook model was used as an example to analyze the relationship of compressive stress-strain response of engineering materials experimentally obtained at constant engineering and true strain rates. There was a minimal deviation between the stress-strain curves obtained at the same constant engineering and true strain rates. The stress-strain curves obtained at either constant engineering or true strain rates could be converted from one to the other, which both represented the intrinsic material response. There is no need to specify the testing requirement of constant engineering or true strain rates for material property characterization, provided that eithermore » constant engineering or constant true strain rate is attained during the experiment.« less

Relationship of compressive stress-strain response of engineering materials obtained at constant engineering and true strain rates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Bo; Sanborn, Brett

In this paper, a Johnson–Cook model was used as an example to analyze the relationship of compressive stress-strain response of engineering materials experimentally obtained at constant engineering and true strain rates. There was a minimal deviation between the stress-strain curves obtained at the same constant engineering and true strain rates. The stress-strain curves obtained at either constant engineering or true strain rates could be converted from one to the other, which both represented the intrinsic material response. There is no need to specify the testing requirement of constant engineering or true strain rates for material property characterization, provided that eithermore » constant engineering or constant true strain rate is attained during the experiment.« less

Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods.

PubMed

Rodriguez, C; Van Broeck, J; Taminiau, B; Delmée, M; Daube, G

2016-08-01

Recognised as the leading cause of nosocomial antibiotic-associated diarrhoea, the incidence of Clostridium difficile infection (CDI) remains high despite efforts to improve prevention and reduce the spread of the bacterium in healthcare settings. In the last decade, many studies have focused on the epidemiology and rapid diagnosis of CDI. In addition, different typing methods have been developed for epidemiological studies. This review explores the history of C. difficile and the current scope of the infection. The variety of available laboratory tests for CDI diagnosis and strain typing methods are also examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923).

PubMed

Wasels, François; Clément, Benjamin; Lopes Ferreira, Nicolas

2016-03-03

Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain. Copyright © 2016 Wasels et al.

Sigma Factor Regulated Cellular Response in a Non-solvent Producing Clostridium beijerinckii Degenerated Strain: A Comparative Transcriptome Analysis

PubMed Central

Zhang, Yan; Jiao, Shengyin; Lv, Jia; Du, Renjia; Yan, Xiaoni; Wan, Caixia; Zhang, Ruijuan; Han, Bei

2017-01-01

Clostridium beijerinckii DG-8052, derived from NCIMB 8052, cannot produce solvent or form spores, a phenomenon known as degeneration. To explore the mechanisms of degeneration at the gene level, transcriptomic profiles of the wild-type 8052 and DG-8052 strains were compared. Expression of 5168 genes comprising 98.6% of the genome was assessed. Interestingly, 548 and 702 genes were significantly up-regulated in the acidogenesis and solventogenesis phases of DG-8052, respectively, and mainly responsible for the phosphotransferase system, sugar metabolic pathways, and chemotaxis; meanwhile, 699 and 797 genes were significantly down-regulated, respectively, and mainly responsible for sporulation, oxidoreduction, and solventogenesis. The functions of some altered genes, including 286 and 333 at the acidogenesis and solventogenesis phases, respectively, remain unknown. Dysregulation of the fermentation machinery was accompanied by lower transcription levels of glycolysis rate-limiting enzymes (pfk and pyk), and higher transcription of cell chemotaxis genes (cheA, cheB, cheR, cheW, and cheY), controlled mainly by σ54 at acidogenesis. Meanwhile, abnormal spore formation was associated with repressed spo0A, sigE, sigF, sigG, and sigK which are positively regulated by σ70, and correspondingly inhibited expression of CoA-transferase at the solventogenesis phase. These findings indicated that morphological and physiological changes in the degenerated Clostridium strain may be related to altered expression of sigma factors, providing valuable targets for strain development of Clostridium species. PMID:28194137

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose.

PubMed

Fu, Hongxin; Yu, Le; Lin, Meng; Wang, Jufang; Xiu, Zhilong; Yang, Shang-Tian

2017-03-01

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21.

PubMed

Singh, Nisha; Puri, Munish; Tuli, Deepak K; Gupta, Ravi P; Barrow, Colin J; Mathur, Anshu S

2018-06-01

To overcome the challenges associated with combined bioprocessing of lignocellulosic biomass to biofuel, finding good organisms is essential. An ethanol producing bacteria DBT-IOC-DC21 was isolated from a compost site via preliminary enrichment culture on a pure hemicellulosic substrate and identified as a Clostridium strain by 16S rRNA analysis. This strain presented broad substrate spectrum with ethanol, acetate, lactate, and hydrogen as the primary metabolic end products. The optimum conditions for ethanol production were found to be an initial pH of 7.0, a temperature of 70 °C and an L-G ratio of 0.67. Strain presented preferential hemicellulose fermentation when compared to various substrates and maximum ethanol concentration of 26.61 mM and 43.63 mM was produced from xylan and xylose, respectively. During the fermentation of varying concentration of xylan, a substantial amount of ethanol ranging from 25.27 mM to 67.29 mM was produced. An increased ethanol concentration of 40.22 mM was produced from a mixture of cellulose and xylan, with a significant effect observed on metabolic flux distribution. The optimum conditions were used to produce ethanol from 28 g L -1 rice straw biomass (RSB) (equivalent to 5.7 g L -1 of the xylose equivalents) in which 19.48 mM ethanol production was achieved. Thus, Clostridium strain DBT-IOC-DC21 has the potential to perform direct microbial conversion of untreated RSB to ethanol at a yield comparative to xylan fermentation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Clostridium species strain BOH3 tolerates and transforms inhibitors from horticulture waste hydrolysates.

PubMed

Yan, Yu; He, Jianzhong

2017-08-01

Conversion of lignocellulosic hydrolysate to biofuels is impeded by the toxic effects of inhibitors that are generated during pretreatment and hydrolysis processes. Here we describe a wild-type Clostridium sp. strain BOH3 with high tolerance to the lignocellulose-derived inhibitors and its capability to transform these inhibitors. Strain BOH3 is capable of tolerating over 60 mM furfural, 60 mM hydroxymethylfurfural, and 6.6 mM vanillin, respectively, and is able to convert 53.74 ± 0.37 mM furfural into furfuryl alcohol within 90 h. The high furfural tolerance and its biotransformation by strain BOH3, which is correlated to the high transcription levels of two short-chain dehydrogenase/reductases, enable strain BOH3 to produce 5.15 ± 0.52 g/L butanol from dilute sulfuric acid pretreated horticultural waste hydrolysate (HWH) that bypassed the detoxification step. The capability of strain BOH3 to produce butanol from un-detoxified HWH lays the foundation of cost-effective biofuel production from lignocellulosic materials.

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

PubMed Central

Robinson, Catherine D.; Auchtung, Jennifer M.; Collins, James

2014-01-01

Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota. PMID:24733099

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

PubMed

Xiong, Wei; Reyes, Luis H; Michener, William E; Maness, Pin-Ching; Chou, Katherine J

2018-03-15

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals. © 2018 Wiley Periodicals, Inc.

Presence of the epidemic North American Pulsed Field type 1 Clostridium difficile strain in hospitalized children.

PubMed

Toltzis, Philip; Kim, Jason; Dul, Michael; Zoltanski, Joan; Smathers, Sarah; Zaoutis, Theoklis

2009-04-01

A hypervirulent strain of Clostridium difficile-labeled North American Pulsed Field type 1 causes severe disease in adults. To determine the prevalence of NAP1 C. difficile in children, organisms from consecutive C. difficile toxin-positive stool samples at 2 children's hospitals microbiology laboratories were characterized. We found that 19.4% of these samples were NAP1.

Clostridium perfringens: Comparative effects of heat and osmotic stress on non-enterotoxigenic and enterotoxigenic strains.

PubMed

Abbona, Cinthia Carolina; Stagnitta, Patricia Virginia

2016-06-01

Clostridium perfringens isolates associated with food poisoning carries a chromosomal cpe gene, while non-foodborne human gastrointestinal disease isolates carry a plasmid cpe gene. The enterotoxigenic strains tested produced vegetative cells and spores with significantly higher resistance than non-enterotoxigenic strains. These results suggest that the vegetative cells and spores have a competitive advantage over non-enterotoxigenic strains. However, no explanation has been provided for the significant associations between chromosomal cpe genotypes with the high resistance, which could explain the strong relationship between chromosomal cpe isolates and C. perfringens type A food poisoning. Here, we analyse the action of physical and chemical agent on non-enterotoxigenic and enterotoxigenic regional strains. And this study tested the relationship between the sensitivities of spores and their levels SASPs (small acid soluble proteins) production in the same strains examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters.

PubMed

Raphael, Brian H; Luquez, Carolina; McCroskey, Loretta M; Joseph, Lavin A; Jacobson, Mark J; Johnson, Eric A; Maslanka, Susan E; Andreadis, Joanne D

2008-07-01

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

PubMed

Raphael, Brian H; Bradshaw, Marite; Kalb, Suzanne R; Joseph, Lavin A; Lúquez, Carolina; Barr, John R; Johnson, Eric A; Maslanka, Susan E

2014-05-01

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

PubMed

Chen, Chaoyang; Sun, Chongran; Wu, Yi-Rui

2018-03-21

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85 Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

DOE PAGES

Xiong, Wei; Reyes, Luis H.; Michener, William E.; ...

2018-04-10

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Wei; Reyes, Luis H.; Michener, William E.

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

PubMed Central

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

Molecular characterization of Clostridium perfringens strains isolated from diseased turkeys in Italy.

PubMed

Giovanardi, Davide; Drigo, Ilenia; De Vidi, Beatrice; Agnoletti, Fabrizio; Viel, Laura; Capello, Katia; Berto, Giacomo; Bano, Luca

2016-06-01

One hundred and six Clostridium perfringens field strains, isolated from diseased turkeys in Italy between 2006 and 2015, were toxinotyped by polymerase chain reaction. Strains were derived from intestines (87), livers (17) and subcutaneous tissues (2). In addition to the four major toxins, strains were also screened for NetB toxin, enterotoxin and beta2 toxin encoding genes. The intestinal gross lesions of turkeys with enteric disorders were statistically studied with respect to the presence of C. perfringens beta2 toxin encoding gene and coccidia in the gut. All the isolates belonged to the toxinotype A and were netB negative. Enterotoxin (cpe) and beta2 toxin (cpb2) encoding genes were detected in two (2.63%) and 76 (71.69%) strains, respectively. Toxinotype results agree with the few published reports concerning the genetic characterization of C. perfringens of turkey origin. On the contrary, the presence of netB and cpb2 genes differs from the results of a previous study where these genes were detected respectively in 6.6% and in 0.5% of the tested strains. Necrotic enteritis in turkeys was not statistically correlated either to the presence of cpb2 gene, or to the synergistic effect operated by coccidia, even though a high percentage of birds with these protozoa in the gut showed necrotic enteritis lesions (64.29%).

Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients.

PubMed

Reigadas, E; Muñoz-Pacheco, P; Vázquez-Cuesta, S; Alcalá, L; Marín, M; Martin, A; Bouza, E

2017-12-01

Rifaximin has been proposed as an alternative treatment for specific cases of Clostridium difficile infection (CDI) and intestinal decontamination. Rifaximin-resistant C. difficile has occasionally been reported. Antibiotic susceptibility testing relies on anaerobic agar dilution (reference method), which is cumbersome and not routinely used. There is no commercial test for detection of resistance to rifaximin. To assess resistance to rifaximin by C. difficile and to evaluate the correlation between the results of the rifampicin E-test and susceptibility to rifaximin. We compared the in vitro susceptibility of clinical CDI isolates to rifaximin over a 6-month period using the agar dilution method with susceptibility to rifampicin using the E-test. All isolates were characterized using PCR-ribotyping. Clinical data were recorded prospectively. We recovered 276 consecutive C. difficile isolates and found that 32.2% of episodes were caused by rifaximin-resistant strains. The MICs for rifaximin ranged from <0.0009-256 mg/L, with a geometric mean (GM) of 0.256 mg/L, an MIC 50/90 of 0.015/>256 mg/L. Rifaximin and rifampicin MICs were comparable, and all strains classed as resistant by agar dilution were correctly classified as resistant by E-test. The most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for rifaximin. Resistance to rifaximin was common; resistance rates were higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar dilution correlated with susceptibility to rifampicin determined using the E-test, including rifaximin-resistant strains. Our results suggest that the rifampicin E-test is a valid method for the prediction of rifaximin-resistant C. difficile. Copyright © 2017 Elsevier Ltd. All rights reserved.

Clostridium perfringens Type A Food Poisoning II. Response of the Rabbit Ileum as an Indication of Enteropathogenicity of Strains of Clostridium perfringens in Human Beings

PubMed Central

Strong, Dorothy H.; Duncan, Charles L.; Perna, Giuseppe

1971-01-01

The effect of feeding human beings individual strains of Clostridium perfringens or culture filtrates thereof was examined. The strains selected for challenge included both those which had previously been shown to produce fluid accumulation in the ligated ileum or overt diarrhea when injected into the nonligated ileum of the rabbit, or had produced both, and those which did not regularly produce these responses. Challenge doses prepared by allowing each strain to grow in beef stew for 3 hr at 46 C resulted in a 61% incidence of diarrhea when rabbit-positive cells were used. No diarrhea occurred among the subjects fed rabbit-negative strains prepared in a similar manner. The procedures employed in preparing the challenge dose appeared to influence the results obtained. When cell-free filtrates were fed, 4 of 15 persons consuming filtrates from rabbit-positive strains developed diarrhea. All subjects fed filtrates from rabbit-negative strains remained free from diarrhea. Serological tests were carried out to compare the identity of the strains of C. perfringens consumed by the subjects and those excreted in the feces. Heat resistance measured as D100 values varied greatly among the rabbit-positive strains. PMID:16557937

Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review.

PubMed

Lacey, Jake A; Johanesen, Priscilla A; Lyras, Dena; Moore, Robert J

2016-06-01

The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.

Elimination of formate production in Clostridium thermocellum.

PubMed

Rydzak, Thomas; Lynd, Lee R; Guss, Adam M

2015-09-01

The ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield is far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, genes encoding pyruvate:formate lyase (pflB) and PFL-activating enzyme (pflA) were deleted. Formate production in the resulting Δpfl strain was eliminated and acetate production decreased by 50 % on both complex and defined medium. The growth rate of the Δpfl strain decreased by 2.9-fold on defined medium and biphasic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δpfl growth rate to 80 % of the parent strain. The role of pfl in metabolic engineering strategies and C1 metabolism is discussed.

Draft genome sequences of clostridium perfringens strain LLY_N11, a pathogenic isolate of necrotic enteritis from a healthy chicken

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens strain LLY_N11 is a commensal bacterial isolate from a healthy chicken that produced a necrotic enteritis in experimental studies. Here we present the assembly and annotation of its genome, which may provide further insights into improved understanding of the molecular mechan...

Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, A; Leang, C; Ueki, T

2014-03-25

The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C.more » ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.« less

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly.

PubMed

Harkness, Justine M; Li, Jihong; McClane, Bruce A

2012-10-01

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Construction of a nontoxigenic Clostridium botulinum strain for food challenge studies.

PubMed

Bradshaw, Marite; Marshall, Kristin M; Heap, John T; Tepp, William H; Minton, Nigel P; Johnson, Eric A

2010-01-01

Clostridium botulinum produces the most poisonous natural toxin known and is a perennial concern to the food industry and to regulatory agencies due to the potential threat of food-borne botulism. To ensure the botulinal safety of foods, rigorous food challenge testing to validate food-processing conditions and food formulations has been routinely performed. Detection of the botulinum neurotoxin is performed by using a mouse bioassay and/or in vitro assays. There has been considerable interest by the food industry and regulatory agencies in minimizing or even replacing the use of animals in these challenge studies. In addition, due to stringent select-agent regulations, the testing of various foods using toxigenic C. botulinum strains requires facilities and personnel that are certified for work with this organism. For this purpose we propose to generate sets of nontoxigenic C. botulinum strains from proteolytic and nonproteolytic groups that differ from the wild-type strains only by their inability to produce botulinum neurotoxin. In this initial study we describe the generation of a nontoxigenic mutant of C. botulinum strain 62A using the ClosTron mutagenesis system by inserting a group II intron into the botulinum neurotoxin type A gene (bont/A). The mutant clones were nontoxigenic as determined by Western blots and mouse bioassays but showed physiological characteristics, including growth properties and sporulation, that were similar to those of the parent strain in laboratory media. Additional studies will be required to evaluate comparable characteristics in various food matrices. The availability of suitable nontoxigenic C. botulinum strains for food challenge studies will be beneficial for enhancing the botulinal safety of foods as well as increasing the biosafety of workers and may eliminate the use of laboratory animals.

Comparative in vitro activities of LFF571 against Clostridium difficile and 630 other intestinal strains of aerobic and anaerobic bacteria.

PubMed

Citron, Diane M; Tyrrell, Kerin L; Merriam, C Vreni; Goldstein, Ellie J C

2012-05-01

The in vitro activities of LFF571, a novel analog of GE2270A that inhibits bacterial growth by binding with high affinity for protein synthesis elongation factor Tu, fidaxomicin, and 10 other antimicrobial agents were determined against 50 strains of Clostridium difficile and 630 other anaerobic and aerobic organisms of intestinal origin. LFF571 possesses potent activity against C. difficile and most other Gram-positive anaerobes (MIC(90), ≤ 0.25 μg/ml), with the exception of bifidobacteria and lactobacilli. The MIC(90)s for aerobes, including enterococci, Staphylococcus aureus (as well as methicillin-resistant S. aureus [MRSA] isolates), Streptococcus pyogenes, and other streptococci were 0.06, 0.125, 2, and 8 μg/ml, respectively. Comparatively, fidaxomicin showed variable activity against Gram-positive organisms: MIC(90)s against C. difficile, Clostridium perfringens, and Bifidobacterium spp. were 0.5, ≤ 0.015, and 0.125 μg/ml, respectively, but >32 μg/ml against Clostridium ramosum and Clostridium innocuum. MIC(90) for S. pyogenes and other streptococci was 16 and >32 μg/ml, respectively. LFF571 and fidaxomicin were generally less active against Gram-negative anaerobes.

Characterization of Clostridium perfringens Strains Isolated from Healthy and Necrotic Enteritis-Afflicted Broiler Chickens.

PubMed

Li, Charles; Lillehoj, Hyun S; Gadde, Ujvala Deepthi; Ritter, Don; Oh, SungTaek

2017-06-01

Necrotic enteritis (NE) is an important enteric disease in poultry, and Clostridium perfringens (CP) type A strains are the primary etiology. NE is responsible for annual losses of US $6 billion to the poultry industry in the United States. An increase in the incidence of NE has been also associated with withdrawal of antibiotic growth promoters from poultry feed. In this study, CP strains isolated from healthy and NE-afflicted birds were characterized microbiologically and molecularly, and their virulence was experimentally tested in chickens. All strains were hemolytic, lecithinase positive, and identified as CP by biochemical tests. Three distinct colony morphologies were seen in brain-heart infusion media with 0.3% agarose, FeSO 4 , and ZnCl 2 . The CP strains responded differently to iron chelation with 2,2'-bidypinol. PCR toxinotyping showed that all tested strains were alpha toxin-positive, seven (N11, N10, CP1, CP5, CP13, JGS, and Del1) were beta2-toxin-positive, and only one (Del1) was necrotic enteritis toxin B-like-positive. In vivo studies indicated that most isolates, including strain N11 isolated from the normal chicken gut, were sufficiently virulent to produce NE disease in the Eimeria/CP dual infection model. The Del1 and N11 strains merit further investigation to identify their virulence factors and immune-protective antigens.

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

PubMed

Bormann, Sebastian; Baer, Zachary C; Sreekumar, Sanil; Kuchenreuther, Jon M; Dean Toste, F; Blanch, Harvey W; Clark, Douglas S

2014-09-01

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was

Mechanisms of microbial oil recovery by Clostridium acetobutylicum and Bacillus strain JF-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marsh, T.L.; Zhang, X.; Knapp, R.M.

1995-12-31

Core displacement experiments at elevated pressures were conducted to determine whether microbial processes are effective under conditions that simulate those found in an actual oil reservoir. The in-situ growth of Clostridium acetobutylicum and Bacillus strain JF-2 resulted in the recovery of residual oil. About 21 and 23% of the residual oil was recovered by C. acetobutylicum and Bacillus strain JF-2, respectively. Flooding cores with cell-free culture fluids of C. acetobutylicum with and without the addition of 50 mM acetone and 100 mM butanol did not result in the recovery of residual oil. Mathematical simulations showed that the amount of gasmore » produced by the clostridial fermentation was not showed that the amount of gas produced by the clostridial fermentation was not sufficient to recover residual oil. Oil recovery by Bacillus strain JF-2 was highly correlated to surfactant production. A biosurfactant-deficient mutant of strain JF-2 was not capable of recovering residual oil. These data show that surfactant production is an important mechanism for microbially enhanced oil recovery. The mechanism for oil recovery by C. acetobutylicum is not understood at this time, but the production of acids, solvents, or gases alone cannot explain the observed increases in oil recovery by this organism.« less

Amino acid catabolism-directed biofuel production in Clostridium sticklandii: An insight into model-driven systems engineering.

PubMed

Sangavai, C; Chellapandi, P

2017-12-01

Model-driven systems engineering has been more fascinating process for the microbial production of biofuel and bio-refineries in chemical and pharmaceutical industries. Genome-scale modeling and simulations have been guided for metabolic engineering of Clostridium species for the production of organic solvents and organic acids. Among them, Clostridium sticklandii is one of the potential organisms to be exploited as a microbial cell factory for biofuel production. It is a hyper-ammonia producing bacterium and is able to catabolize amino acids as important carbon and energy sources via Stickland reactions and the development of the specific pathways. Current genomic and metabolic aspects of this bacterium are comprehensively reviewed herein, which provided information for learning about protein catabolism-directed biofuel production. It has a metabolic potential to drive energy and direct solventogenesis as well as acidogenesis from protein catabolism. It produces by-products such as ethanol, acetate, n -butanol, n -butyrate and hydrogen from amino acid catabolism. Model-driven systems engineering of this organism would improve the performance of the industrial sectors and enhance the industrial economy by using protein-based waste in environment-friendly ways.

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese

PubMed Central

Wüthrich, Daniel; Bruggmann, Rémy; Berthoud, Hélène; Arias-Roth, Emmanuelle

2015-01-01

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively. PMID:25767226

Highly Divergent Clostridium difficile Strains Isolated from the Environment

PubMed Central

Janezic, Sandra; Potocnik, Mojca; Zidaric, Valerija; Rupnik, Maja

2016-01-01

Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile. PMID:27880843

Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline.

PubMed

Shin, Yeseul; Kang, Seok-Seong; Paek, Jayoung; Jin, Tae Eun; Song, Hong Seok; Kim, Hongik; Park, Hee-Moon; Chang, Young-Hyo

2016-06-01

Two bacterial strains, YHK0403(T) and YHK0508, isolated from soil under a corroded gas pipe line, were revealed as Gram-negative, obligately anaerobic, spore-forming and mesophilic bacteria. The cells were rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were members of the genus Clostridium and were the most closely related to Clostridium scatologenes KCTC 5588(T) (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177(T) (95.8%), Clostridium drakei KCTC 5440(T) (95.7%) and Clostridium tyrobutyricum KCTC 5387(T) (94.9%). The G + C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1γ type with meso-diaminopimelic acid. The major polar lipid was diphosphatidylglycerol (DPG), and other minor lipids were revealed as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unknown glycolipids (GL1 and GL2), an unknown aminoglycolipid (NGL), two unknown aminophospholipids (PN1 and PN2) and four unknown phospholipids (PL1 to PL4). Predominant fatty acids were C16:0 and C16:1cis9 DMA. The major end products from glucose fermentation were identified as butyrate (12.2 mmol) and acetate (9.8 mmol). Collectively, the results from a wide range of phenotypic tests, chemotaxonomic tests, and phylogenetic analysis indicated that the two isolates represent novel species of the genus Clostridium, for which the name Clostridium kogasensis sp. nov. (type strain, YHK0403(T) = KCTC 15258(T) = JCM 18719(T)) is proposed. Copyright © 2016. Published by Elsevier Ltd.

Clostridium geopurificans strain MJ1 sp. nov., a strictly anaerobic bacterium that grows via fermentation and reduces the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

PubMed

Kwon, Man Jae; Wei, Na; Millerick, Kayleigh; Popovic, Jovan; Finneran, Kevin

2014-06-01

A fermentative, non-spore forming, motile, rod-shaped bacterium, designated strain MJ1(T), was isolated from an RDX contaminated aquifer at a live-fire training site in Northwest NJ, United States. On the basis of 16S rRNA gene sequencing and DNA base composition, strain MJ1(T) was assigned to the Firmicutes. The DNA G+C content was 42.8 mol%. Fermentative growth was supported by glucose and citrate in a defined basal medium. The bacterium is a strict anaerobe that grows between at pH 6.0 and pH 8.0 and 18 and 37 °C. The culture did not grow with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as the electron acceptor or mineralize RDX under these conditions. However, MJ1(T) transformed RDX into MNX, methylenedinitramine, formaldehyde, formate, ammonium, nitrous oxide, and nitrate. The nearest phylogenetic relative with a validly published name was Desulfotomaculum guttoideum (95 % similarity). However, MJ1(T) was also related to Clostridium celerecrescens DSM 5628 (95 %), Clostridium indolis DSM 755 (94 %), and Clostridium sphenoides DSM 632 (94 %). DNA:DNA hybridization with these strains was between 6.7 and 58.7 percent. The dominant cellular fatty acids (greater than 5 % of the total, which was 99.0 % recovery) were 16:0 fatty acid methyl ester (FAME) (32.12 %), 18:1cis 11 dimethyl acetal (DMA) (16.47 %), 16:1cis 9 DMA (10.28 %), 16:1cis 9 FAME (8.10 %), and 18:1cis 9 DMA (5.36 %). On the basis of morphological, physiological, and phylogenetic data, Clostridium geopurificans is proposed as a new species in genus Clostridium, with strain MJ1(T) as the type strain.

FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

NASA Astrophysics Data System (ADS)

Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

2013-07-01

Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols

PubMed Central

2013-01-01

Background Recent progress in production of various biofuel precursors and molecules, such as fatty acids, alcohols and alka(e)nes, is a significant step forward for replacing the fossil fuels with renewable fuels. A two-step process, where fatty acids from sugars are produced in the first step and then converted to corresponding biofuel molecules in the second step, seems more viable and attractive at this stage. We have engineered an Escherichia coli strain to take care of the second step for converting short chain fatty acids into corresponding alcohols by using butyrate kinase (Buk), phosphotransbutyrylase (Ptb) and aldehyde/alcohol dehydrogenase (AdhE2) from Clostridium acetobutylicum. Results The engineered E. coli was able to convert butyric acid and other short chain fatty acids of chain length C3 to C7 into corresponding alcohols and the efficiency of conversion varied with different E. coli strain type. Glycerol proved to be a better donor of ATP and electron as compared to glucose for converting butyric acid to butanol. The engineered E. coli was able to tolerate up to 100 mM butyric acid and produced butanol with the conversion rate close to 100% under anaerobic condition. Deletion of native genes, such as fumarate reductase (frdA) and alcohol dehydrogenase (adhE), responsible for side products succinate and ethanol, which act as electron sink and could compete with butyric acid uptake, did not improve the butanol production efficiency. Indigenous acyl-CoA synthetase (fadD) was found to play no role in the conversion of butyric acid to butanol. Engineered E. coli was cultivated in a bioreactor under controlled condition where 60 mM butanol was produced within 24 h of cultivation. A continuous bioreactor with the provision of cell recycling allowed the continuous production of butanol at the average productivity of 7.6 mmol/l/h until 240 h. Conclusions E. coli engineered with the pathway from C. acetobutylicum could efficiently convert butyric acid

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

PubMed Central

Raphael, Brian H.; Joseph, Lavin A.; Meno, Sarah R.; Fernández, Rafael A.; Maslanka, Susan E.

2012-01-01

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE. PMID:23042179

Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation

DOE PAGES

Sander, Kyle B.; Wilson, Charlotte M.; M. Rodriquez, Jr.; ...

2015-12-12

Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing xylose catabolism genes for glucose and xylose co-utilization.

PubMed

Fu, Hongxin; Yang, Shang-Tian; Wang, Minqi; Wang, Jufang; Tang, I-Ching

2017-06-01

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

PubMed

Storari, Michelangelo; Wüthrich, Daniel; Bruggmann, Rémy; Berthoud, Hélène; Arias-Roth, Emmanuelle

2015-03-12

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively. Copyright © 2015 Storari et al.

Preventing clostridium difficile infection in the intensive care unit.

PubMed

Zilberberg, Marya D; Shorr, Andrew F

2013-01-01

Clostridium difficile is a formidable problem in the twenty-first century. Because of injudicious use of antibiotics, the emergence of the hypervirulent epidemic strain of this organism has been difficult to contain. The NAP1/BI/027 strain causes more-severe disease than other widely prevalent strains and affects patients who were not traditionally thought to be at risk for Clostridium difficile infection. Critically ill patients remain at high risk for this pathogen, and preventive measures, such as meticulous contact precautions, hand hygiene, environmental disinfection, and, most importantly, antibiotic stewardship, are the cornerstones of mitigation in the intensive care unit. Copyright © 2013 Elsevier Inc. All rights reserved.

Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

PubMed

Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

2016-06-01

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

2014-04-01

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

Isolation of Clostridium difficile from dogs with digestive disorders, including stable metronidazole-resistant strains.

PubMed

Orden, Cristina; Blanco, Jose L; Álvarez-Pérez, Sergio; Garcia-Sancho, Mercedes; Rodriguez-Franco, Fernando; Sainz, Angel; Villaescusa, Alejandra; Harmanus, Celine; Kuijper, Ed; Garcia, Marta E

2017-02-01

The prevalence of Clostridium difficile in 107 dogs with diverse digestive disorders attended in a Spanish veterinary teaching hospital was assessed. The microorganism was isolated from 13 dogs (12.1%) of different disease groups. Isolates belonged to PCR ribotypes 078, 106, 154 and 430 (all of them toxigenic) and 110 (non-toxigenic), and were resistant to several antimicrobial drugs. Notably, seven isolates obtained from different dogs displayed stable resistance to metronidazole. The results of this study provide further evidence that dogs can act as a reservoir of C. difficile strains of epidemic ribotypes with resistance to multiple antibiotics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum

PubMed Central

Carlson, Ellinor D.

2017-01-01

ABSTRACT With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

PubMed

Carlson, Ellinor D; Papoutsakis, Eleftherios T

2017-08-15

With recent advances in synthetic biology, CO 2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO 2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO 2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO 2 , and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO 2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum , which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO 2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO 2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO 2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum , which is natively incapable of CO 2 fixation. The expression of CODH, alone or together with the C. carboxidivorans

Clostridium neonatale sp. nov. linked to necrotizing enterocolitis in neonates and a clarification of species assignable to the genus Clostridium (Prazmowski 1880) emend. Lawson and Rainey 2016.

PubMed

Bernard, Kathryn; Burdz, Tamara; Wiebe, Deborah; Alfa, Michelle; Bernier, Anne-Marie

2018-06-11

A description of an outbreak of necrotizing enterocolitis among neonates, linked to the putative novel species Clostridium neonatale and assignable to the genus Clostridium, was previously reported in brief but that name had never been validly published (Alfa et al. Clin Inf Dis 2002;35:S101-S105). Features of this taxon group and its phylogenetic position with respect to contemporary species in the genus Clostridium were recently reviewed and still found to be unique. Therefore, we provide here a description based on biochemical, chemotaxonomic and antimicrobial susceptibility testing (AST), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, 16S rRNA gene sequencing as well as information obtained by whole genome sequencing (WGS) for strains 99A005 T and 99A006. Those two C. neonatale strains were essentially identical to each other, with genome sizes of 4 658 596-4 705 520 bp and G+C content of 28.4-28.5 mol% (WGS). AST inferred susceptibility to 14 antibiotics. MALDI-TOF spectra were unique and could potentially be used for identification. The type strain is (NML) LCDC 99A005 T [=ATCC BAA-265 T =CCUG 46077 T =St. Boniface Hospital 30686 T ]. While performing this review, we found that the names of 24 validly published species assignable to the genus Clostridium had been omitted from the emended description of the genus (Lawson and Rainey Int J Syst Evol Microbiol 2016;66 :1009-1016). Those species are listed in brief here. Lastly, based on this review, we also propose that Eubacterium budayi, Eubacterium nitritogenes and Eubacterium combesii be transferred to the emended genus Clostridium, as Clostridium budayi comb. nov., Clostridium nitritogenes comb. nov. and Clostridium combesii comb. nov., respectively.

DETECTION, RIBOTYPING AND ANTIMICROBIAL RESISTANCE PROPERTIES OF CLOSTRIDIUM DIFFICILE STRAINS ISOLATED FROM THE CASES OF DIARRHEA.

PubMed

Kouzegaran, Samaneh; Ganjifard, Mahmood; Tanha, Amir Saber

2016-10-01

Clostridium difficile is the most prevalent cause of antibiotic-associated infectious diarrhea al-around the world. Prevalence of virulent and resistant strains of Clostridium difficile is increasing now a day. The present investigation was carried out to study the prevalence, ribotyping and antibiotic resistance pattern of C. difficile isolated from diarrheic and non-diarrheic pediatrics. Four-hundred stool specimens were collected from the diarrheic and non-diarrheic pediatrics hospitalized due to the diseases other than diarrhea. Samples were cultured and their positive results were subjected to disk diffusion and PCR-based ribotyping. Thirty-five out of 400 (8.75%) samples were positive for C. difficile . Prevalence of C. difficile in diarrheic and non-diarrheic pediatrics were 11.25% and 4.16%, respectively. Male had the higher prevalence of bacteria than female ( P < 0.05). eight to twelve months old pediatrics were the most commonly infected group. R27 (14.28%), R1 (10.71%), R12 (7.14%), R13 (7.14%) and R18 (7.14%) were most commonly detected ribotypes. There were no positive results for studied ribotypes in non-diarrheic pediatrics. C. difficile strains had the highest levels of resistance against tetracycline (71.42%), erythromycin (57.14%), moxifloxacin (48.57%), metronidazole (28.57%) and clindamycin (22.85%) antibiotics. Prescription of antibiotics in diarrheic pediatrics, males and also 8-12 months old pediatrics should be done in a regular and cautious manner.

Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production.

PubMed

Zhang, Jie; Zong, Wenming; Hong, Wei; Zhang, Zhong-Tian; Wang, Yi

2018-03-09

Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30-38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel

Elimination of formate production in Clostridium thermocellum

DOE PAGES

Rydzak, Thomas; Lynd, Lee R.; Guss, Adam M.

2015-07-11

We study the ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield are far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H 2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, pyruvate:formate lyase (pfl) and respective PFL-activating enzyme were deleted. Formate production in the resulting Δpfl strain was eliminated and acetate production decreased by 50% on both complex and defined medium. Growthmore » rate of Δpfl decreased by 2.9-fold on defined medium and diauxic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δpfl growth rate to 80% of the parent strain. Finally, we discuss the role of pfl in metabolic engineering strategies and C 1 metabolism.« less

Lactic acid bacteria as protective cultures in fermented pork meat to prevent Clostridium spp. growth.

PubMed

Di Gioia, Diana; Mazzola, Giuseppe; Nikodinoska, Ivana; Aloisio, Irene; Langerholc, Tomaz; Rossi, Maddalena; Raimondi, Stefano; Melero, Beatriz; Rovira, Jordi

2016-10-17

In meat fermented foods, Clostridium spp. growth is kept under control by the addition of nitrite. The growing request of consumers for safer products has led to consider alternative bio-based approaches, the use of protective cultures being one of them. This work is aimed at checking the possibility of using two Lactobacillus spp. strains as protective cultures against Clostridium spp. in pork ground meat for fermented salami preparation. Both Lactobacillus strains displayed anti-clostridia activity in vitro using the spot agar test and after co-culturing them in liquid medium with each Clostridium strain. Only one of them, however, namely L. plantarum PCS20, was capable of effectively surviving in ground meat and of performing anti-microbial activity in carnis in a challenge test where meat was inoculated with the Clostridium strain. Therefore, this work pointed out that protective cultures can be a feasible approach for nitrite reduction in fermented meat products. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic engineering of industrial strains of Saccharomyces cerevisiae.

PubMed

Le Borgne, Sylvie

2012-01-01

Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

PubMed

Nakano, V; Ignacio, A; Llanco, L; Bueris, V; Sircili, M P; Avila-Campos, M J

2017-04-01

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid.

PubMed

Jang, Yu-Sin; Woo, Hee Moon; Im, Jung Ae; Kim, In Ho; Lee, Sang Yup

2013-11-01

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

Clostridium pabulibutyricum sp. nov., a butyric-acid-producing organism isolated from high-moisture grass silage.

PubMed

Kobayashi, Hisami; Nakasato, Takuya; Sakamoto, Mitsuo; Ohtani, Yoshihisa; Terada, Fuminori; Sakai, Ken; Ohkuma, Moriya; Tohno, Masanori

2017-12-01

A Gram-stain-variable, strictly anaerobic, rod-shaped, catalase-negative and endospore-forming bacterial strain, designated MJC39 T , was isolated from grass silage preserved in Hokkaido, Japan. Growth occurred at 20-42 °C, pH 5.0-7.0 and NaCl concentrations up to 2 % (w/v). The isolated strain MJC39 T produced butyric acid in peptone yeast extract medium with glucose. The DNA G+C content of strain MJC39 T was 34.4±0.2 mol%. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and summed feature 3 (including C16 : 1ω7c/C16 : 1ω6c). No respiratory quinones were detected. The polar lipids of strain MJC39 T were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified lipid, one unidentified aminolipid, two unidentified glycolipids, one unidentified phospholipid, one unidentified aminoglycolipid and one unidentified phosphoaminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJC39 T was a member of the genus Clostridium and is closely related to Clostridium tyrobutyricum JCM 11008 T (95.8 % similarity) and Clostridium algifaecis MB9-7 T (95.5 % similarity). Based on the genotypic, phenotypic and chemotaxonomic characteristics, strain MJC39 T represents a novel species of the genus Clostridium, for which the name Clostridium pabulibutyricum sp. nov. is proposed. The type strain is MJC39 T (=JCM 31506 T =DSM 103944 T ).

Inhibitory activity of reuterin, nisin, lysozyme and nitrite against vegetative cells and spores of dairy-related Clostridium species.

PubMed

Avila, Marta; Gómez-Torres, Natalia; Hernández, Marta; Garde, Sonia

2014-02-17

The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51-32.5 mM) and nisin (MIC values 0.05-12.5 μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values<0.20-400 μg/ml) and sodium nitrite (MIC values 18.75-150 μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Strain Types and Antimicrobial Resistance Patterns of Clostridium difficile Isolates from the United States, 2011 to 2013

PubMed Central

Goering, Richard V.; Whitmore, Joseph D.; Lynn, Ashley N. W.; Persing, David H.; Tenover, Fred C.

2014-01-01

We determined the PCR ribotypes and antimicrobial susceptibility patterns of 508 toxigenic Clostridium difficile isolates collected between 2011 and 2013 from 32 U.S. hospitals. Of the 29 PCR ribotypes identified, the 027 strain type was the most common (28.1%), although the rates varied by geographic region. Ribotype 014/020 isolates appear to be emerging. Clindamycin and moxifloxacin resistances (36.8% and 35.8%, respectively) were the most frequent resistance phenotypes observed. Reduced susceptibility to vancomycin was observed in 39.1% of 027 isolates. PMID:24752264

Comparative pathogenomics of Clostridium tetani.

PubMed

Cohen, Jonathan E; Wang, Rong; Shen, Rong-Fong; Wu, Wells W; Keller, James E

2017-01-01

Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

Clostridium difficile and Clostridium perfringens from wild carnivore species in Brazil.

PubMed

Silva, Rodrigo Otávio Silveira; D'Elia, Mirella Lauria; Tostes Teixeira, Erika Procópio; Pereira, Pedro Lúcio Lithg; de Magalhães Soares, Danielle Ferreira; Cavalcanti, Álvaro Roberto; Kocuvan, Aleksander; Rupnik, Maja; Santos, André Luiz Quagliatto; Junior, Carlos Augusto Oliveira; Lobato, Francisco Carlos Faria

2014-08-01

Despite some case reports, the importance of Clostridium perfringens and Clostridium difficile for wild carnivores remains unclear. Thus, the objective of this study was to identify C. perfringens and C. difficile strains in stool samples from wild carnivore species in Brazil. A total of 34 stool samples were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota) and the beta-2 toxin (cpb2), enterotoxin (cpe) and NetB (netb) genes. C. difficile strains were analyzed by multiplex-PCR for toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB) and also submitted to a PCR ribotyping. Unthawed aliquots of samples positive for C. difficile isolation were subjected to the detection of A/B toxins by a cytotoxicity assay (CTA). C. perfringens was isolated from 26 samples (76.5%), all of which were genotyped as type A. The netb gene was not detected, whereas the cpb2 and cpe genes were found in nine and three C. perfringens strains, respectively. C. difficile was isolated from two (5.9%) samples. A non-toxigenic strain was recovered from a non-diarrheic maned wolf (Chrysocyon brachyurus). Conversely, a toxigenic strain was found in the sample of a diarrheic ocelot (Leopardus pardallis); an unthawed stool sample was also positive for A/B toxins by CTA, indicating a diagnosis of C. difficile-associated diarrhea in this animal. The present work suggests that wild carnivore species could carry C. difficile strains and that they could be susceptible to C. difficile infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

PubMed

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M; Scholz, Holger C; Splettstoesser, Wolf D; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-12-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain

PubMed Central

Baroi, G N; Baumann, I; Westermann, P; Gavala, H N

2015-01-01

Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l−1 of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v−1) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60–80% PHWS lie between 0.37 and 0.46 g g−1 of sugar, while the selectivity for butyric acid was as high as 0.9–1.0 g g−1 of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7. PMID:26230610

Clostridium tyrobutyricum strains show wide variation in growth at different NaCl, pH, and temperature conditions.

PubMed

Ruusunen, Marjo; Surakka, Anu; Korkeala, Hannu; Lindström, Miia

2012-10-01

Outgrowth from Clostridium tyrobutyricum spores in milk can lead to butyric acid fermentation in cheeses, causing spoilage and economical loss to the dairy industry. The aim of this study was to investigate the growth of 10 C. tyrobutyricum strains at different NaCl, pH, and temperature conditions. Up to 7.5-fold differences among the maximum growth rates of different strains in the presence of 2.0% NaCl were observed. Five of 10 strains were able to grow in the presence of 3.0% NaCl, while a NaCl concentration of 3.5% was completely inhibitory to all strains. Seven of 10 strains were able to grow at pH 5.0, and up to 4- and 12.5-fold differences were observed among the maximum growth rates of different strains at pH 5.5 and 7.5, respectively. The maximum growth temperatures varied from 40.2 to 43.3°C. The temperature of 10°C inhibited the growth of all strains, while 8 of 10 strains grew at 12 and 15°C. Despite showing no growth, all strains were able to survive at 10°C. In conclusion, wide variation was observed among different C. tyrobutyricum strains in their ability to grow at different stressful conditions. Understanding the physiological diversity among the strains is important when designing food control measures and predictive models for the growth of spoilage organisms in cheese.

Metabolic Engineering of Clostridium thermocellum for Biofuel Production (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guess, Adam

2013-03-01

Adam Guss of Oak Ridge National Lab presents on Metabolic engineering of Clostridium thermocellum for biofuel production at the 8th Annual Genomics of Energy & Environment Meeting on March 28, 2013 in Walnut Creek, CA.

Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children

NASA Astrophysics Data System (ADS)

Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie

2014-12-01

Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.

Engineering Clostridium beijerinckii with the Cbei_4693 gene knockout for enhanced ferulic acid tolerance.

PubMed

Liu, Jun; Guo, Ting; Shen, Xiaoning; Xu, Jiahui; Wang, Junzhi; Wang, Yanyan; Liu, Dong; Niu, Huanqing; Liang, Lei; Ying, Hanjie

2016-07-10

A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.

PubMed

Liew, Fungmin; Henstra, Anne M; Kӧpke, Michael; Winzer, Klaus; Simpson, Sean D; Minton, Nigel P

2017-03-01

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.

PubMed

Wang, Shaohua; Dong, Sheng; Wang, Pixiang; Tao, Yong; Wang, Yi

2017-05-15

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk , two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum.

PubMed

Thompson, R Adam; Layton, Donovan S; Guss, Adam M; Olson, Daniel G; Lynd, Lee R; Trinh, Cong T

2015-11-01

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model's capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. The model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced

Lumbar Discitis Caused by Clostridium perfringens

PubMed Central

Popoff, M. R.; Degand, Nicolas; Lotte, Laurene; Bouvet, Philippe; Baudin, Guillaume; Cua, Eric; Roger, Pierre-Marie; Ruimy, Raymond

2014-01-01

We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of β-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern. PMID:25056327

Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain.

PubMed

Baroi, G N; Baumann, I; Westermann, P; Gavala, H N

2015-09-01

Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l(-1) of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v(-1) ) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60-80% PHWS lie between 0.37 and 0.46 g g(-1) of sugar, while the selectivity for butyric acid was as high as 0.9-1.0 g g(-1) of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II

PubMed Central

Woudstra, Cedric; Lambert, Dominic; Anniballi, Fabrizio; De Medici, Dario; Austin, John

2013-01-01

Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum. PMID:23603687

In silico metabolic engineering of Clostridium ljungdahlii for synthesis gas fermentation.

PubMed

Chen, Jin; Henson, Michael A

2016-11-01

Synthesis gas fermentation is one of the most promising routes to convert synthesis gas (syngas; mainly comprised of H 2 and CO) to renewable liquid fuels and chemicals by specialized bacteria. The most commonly studied syngas fermenting bacterium is Clostridium ljungdahlii, which produces acetate and ethanol as its primary metabolic byproducts. Engineering of C. ljungdahlii metabolism to overproduce ethanol, enhance the synthesize of the native byproducts lactate and 2,3-butanediol, and introduce the synthesis of non-native products such as butanol and butyrate has substantial commercial value. We performed in silico metabolic engineering studies using a genome-scale reconstruction of C. ljungdahlii metabolism and the OptKnock computational framework to identify gene knockouts that were predicted to enhance the synthesis of these native products and non-native products, introduced through insertion of the necessary heterologous pathways. The OptKnock derived strategies were often difficult to assess because increase product synthesis was invariably accompanied by decreased growth. Therefore, the OptKnock strategies were further evaluated using a spatiotemporal metabolic model of a syngas bubble column reactor, a popular technology for large-scale gas fermentation. Unlike flux balance analysis, the bubble column model accounted for the complex tradeoffs between increased product synthesis and reduced growth rates of engineered mutants within the spatially varying column environment. The two-stage methodology for deriving and evaluating metabolic engineering strategies was shown to yield new C. ljungdahlii gene targets that offer the potential for increased product synthesis under realistic syngas fermentation conditions. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

PubMed

Kim, Young Jin; Kim, Si Hyun; Park, Hyun-Jung; Park, Hae-Geun; Park, Dongchul; Song, Sae Am; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kook, Joong-Ki; Kim, Hye Ran; Shin, Jeong Hwan

2016-08-01

All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome characterization of a novel binary toxin-positive strain of Clostridium difficile and comparison with the epidemic 027 and 078 strains.

PubMed

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

Functional analysis of an feoB mutant in Clostridium perfringens strain 13.

PubMed

Awad, Milena M; Cheung, Jackie K; Tan, Joanne E; McEwan, Alastair G; Lyras, Dena; Rood, Julian I

2016-10-01

Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile▿†

PubMed Central

Forgetta, Vincenzo; Oughton, Matthew T.; Marquis, Pascale; Brukner, Ivan; Blanchette, Ruth; Haub, Kevin; Magrini, Vince; Mardis, Elaine R.; Gerding, Dale N.; Loo, Vivian G.; Miller, Mark A.; Mulvey, Michael R.; Rupnik, Maja; Dascal, Andre; Dewar, Ken

2011-01-01

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains. PMID:21508155

Metabolically engineered glucose-utilizing Shewanella strains under anaerobic conditions.

PubMed

Choi, Donggeon; Lee, Sae Bom; Kim, Sohyun; Min, Byoungnam; Choi, In-Geol; Chang, In Seop

2014-02-01

Comparative genome analysis of Shewanella strains predicted that the strains metabolize preferably two- and three-carbon carbohydrates as carbon/electron source because many Shewanella genomes are deficient of the key enzymes in glycolysis (e.g., glucokinase). In addition, all Shewanella genomes are known to have only one set of genes associated with the phosphotransferase system required to uptake sugars. To engineer Shewanella strains that can utilize five- and six-carbon carbohydrates, we constructed glucose-utilizing Shewanella oneidensis MR-1 by introducing the glucose facilitator (glf; ZMO0366) and glucokinase (glk; ZMO0369) genes of Zymomonas mobilis. The engineered MR-1 strain was able to grow on glucose as a sole carbon/electron source under anaerobic conditions. The glucose affinity (Ks) and glucokinase activity in the engineered MR-1 strain were 299.46 mM and 0.259 ± 0.034 U/g proteins. The engineered strain was successfully applied to a microbial fuel cell system and exhibited current generation using glucose as the electron source. Copyright © 2013 Elsevier Ltd. All rights reserved.

Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

PubMed

Timbermont, Leen; De Smet, Lina; Van Nieuwerburgh, Filip; Parreira, Valeria R; Van Driessche, Gonzalez; Haesebrouck, Freddy; Ducatelle, Richard; Prescott, John; Deforce, Dieter; Devreese, Bart; Van Immerseel, Filip

2014-04-05

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

PubMed Central

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M.; Scholz, Holger C.; Splettstoesser, Wolf D.; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-01-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse. PMID:22012011

Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

PubMed

Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

2013-10-01

The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

PubMed

Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

2017-03-01

Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

PubMed Central

Li, Jihong; Freedman, John C.; Evans, Daniel R.

2017-01-01

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. PMID:28052992

Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitham, Jason M.; Moon, Ji Won; Rodriguez Jr, Miguel

Background: Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C.more » thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.Results: Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h -1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6

Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

DOE PAGES

Whitham, Jason M.; Moon, Ji Won; Rodriguez Jr, Miguel; ...

2018-04-05

Background: Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C.more » thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.Results: Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h -1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6

Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

NASA Technical Reports Server (NTRS)

Shing, Y. W.; Akagi, J. M.; Himes, R. H.

1972-01-01

It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.

PubMed

Hon, Shuen; Olson, Daniel G; Holwerda, Evert K; Lanahan, Anthony A; Murphy, Sean J L; Maloney, Marybeth I; Zheng, Tianyong; Papanek, Beth; Guss, Adam M; Lynd, Lee R

2017-07-01

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Capturing the response of Clostridium acetobutylicum to chemical stressors using a regulated genome-scale metabolic model

DOE PAGES

Dash, Satyakam; Mueller, Thomas J.; Venkataramanan, Keerthi P.; ...

2014-10-14

Clostridia are anaerobic Gram-positive Firmicutes containing broad and flexible systems for substrate utilization, which have been used successfully to produce a range of industrial compounds. Clostridium acetobutylicum has been used to produce butanol on an industrial scale through acetone-butanol-ethanol (ABE) fermentation. A genome-scale metabolic (GSM) model is a powerful tool for understanding the metabolic capacities of an organism and developing metabolic engineering strategies for strain development. The integration of stress related specific transcriptomics information with the GSM model provides opportunities for elucidating the focal points of regulation.

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum

DOE PAGES

Thompson, R. Adam; Layton, Donovan S.; Guss, Adam M.; ...

2015-10-21

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model smore » capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. Furthermore, the model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol

Kinetic modeling of batch fermentation for Populus hydrolysate tolerant mutant and wild type strains of Clostridium thermocellum.

PubMed

Linville, Jessica L; Rodriguez, Miguel; Mielenz, Jonathan R; Cox, Chris D

2013-11-01

The extent of inhibition of two strains of Clostridium thermocellum by a Populus hydrolysate was investigated. A Monod-based model of wild type (WT) and Populus hydrolysate tolerant mutant (PM) strains of the cellulolytic bacterium C. thermocellum was developed to quantify growth kinetics in standard media and the extent of inhibition to a Populus hydrolysate. The PM was characterized by a higher growth rate (μmax=1.223 vs. 0.571 h(-1)) and less inhibition (KI,gen=0.991 vs. 0.757) in 10% v/v Populus hydrolysate compared to the WT. In 17.5% v/v Populus hydrolysate inhibition of PM increased slightly (KI,gen=0.888), whereas the WT was strongly inhibited and did not grow in a reproducible manner. Of the individual inhibitors tested, 4-hydroxybenzoic acid was the most inhibitory, followed by galacturonic acid. The PM did not have a greater ability to detoxify the hydrolysate than the WT. Copyright © 2013 Elsevier Ltd. All rights reserved.

Strain engineering of van der Waals heterostructures.

PubMed

Vermeulen, Paul A; Mulder, Jefta; Momand, Jamo; Kooi, Bart J

2018-01-18

Modifying the strain state of solids allows control over a plethora of functional properties. The weak interlayer bonding in van der Waals (vdWaals) materials such as graphene, hBN, MoS 2 , and Bi 2 Te 3 might seem to exclude strain engineering, since strain would immediately relax at the vdWaals interfaces. Here we present direct observations of the contrary by showing growth of vdWaals heterostructures with persistent in-plane strains up to 5% and we show that strain relaxation follows a not yet reported process distinctly different from strain relaxation in three-dimensionally bonded (3D) materials. For this, 2D bonded Bi 2 Te 3 -Sb 2 Te 3 and 2D/3D bonded Bi 2 Te 3 -GeTe multilayered films are grown using Pulsed Laser Deposition (PLD) and their structure is monitored in situ using Reflective High Energy Electron Diffraction (RHEED) and post situ analysis is performed using Transmission Electron Microscopy (TEM). Strain relaxation is modeled and found to solely depend on the layer being grown and its initial strain. This insight demonstrates that strain engineering of 2D bonded heterostructures obeys different rules than hold for epitaxial 3D materials and opens the door to precise tuning of the strain state of the individual layers to optimize functional performance of vdWaals heterostructures.

In vitro and in vivo antagonistic activity of new probiotic culture against Clostridium difficile and Clostridium perfringens.

PubMed

Golić, Nataša; Veljović, Katarina; Popović, Nikola; Djokić, Jelena; Strahinić, Ivana; Mrvaljević, Igor; Terzić-Vidojević, Amarela

2017-05-06

Genus Clostridium accompanies more than 200 known species and at least 30 among them are associated with human and animal diseases. At the moment, the treatment of clostridial infections is based on use of antibiotics. However, due to the European ban on the use of antibiotics in livestock production, novel therapeutic strategies for treatment of these hardly curable infections have been evaluated. Hence, in this study the antimicrobial effect of newly designed probiotic culture consisted of natural isolates Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44 against Clostridium difficile and Clostridium perfringens was analyzed. The probiotic culture showed strong in vitro antimicrobial effect on C. difficile (human clinical isolate). In addition, individual strains and the probiotic combination exhibited immunomodulatory activity. The probiotic combination significantly increased the proliferation of GALT lymphocytes. At the other hand, none of the bacterial treatments (individual strains and the combination) induced the production of proinflammatory cytokines IL-6 and IL-1β by intestinal epithelial cells, Caco-2. Interestingly, Caco-2 cells exposed to the probiotic combination produced significantly elevated amount of TGFβ pointing to potential protecting effect of the probiotic. In addition, the results of field trial on spontaneously infected goats revealed reduction of C. perfringens in goats (below the detection threshold) after the probiotic treatment. The results of this study indicated that the novel probiotic deserves to be further investigated as a promising antimicrobial agent against C. difficile and C. perfringens.

Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum.

PubMed

Tian, Liang; Papanek, Beth; Olson, Daniel G; Rydzak, Thomas; Holwerda, Evert K; Zheng, Tianyong; Zhou, Jilai; Maloney, Marybeth; Jiang, Nannan; Giannone, Richard J; Hettich, Robert L; Guss, Adam M; Lynd, Lee R

2016-01-01

Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Here, we started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. The resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. A two step selection method successfully improved the ethanol yield and the titer. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism.

PubMed

Biswas, Ranjita; Wilson, Charlotte M; Giannone, Richard J; Klingeman, Dawn M; Rydzak, Thomas; Shah, Manesh B; Hettich, Robert L; Brown, Steven D; Guss, Adam M

2017-01-01

Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H 2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech , were deleted from the chromosome of C. thermocellum . While ethanol yield increased, the growth rate of Δ hydG decreased substantially compared to wild type. Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ∆hydG , whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG , 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ΔhydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG . Clostridium thermocellum ΔhydGΔech had a higher growth rate than ΔhydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate. Sulfur metabolism is perturbed in C. thermocellum ΔhydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

PubMed

Paul, Catherine J; Twine, Susan M; Tam, Kevin J; Mullen, James A; Kelly, John F; Austin, John W; Logan, Susan M

2007-05-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification▿

PubMed Central

Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J.; Mullen, James A.; Kelly, John F.; Austin, John W.; Logan, Susan M.

2007-01-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region. PMID:17351097

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

DOE PAGES

Hon, Shuen; Olson, Daniel G.; Holwerda, Evert K.; ...

2017-06-27

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields andmore » titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. Here, this suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.« less

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hon, Shuen; Olson, Daniel G.; Holwerda, Evert K.

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields andmore » titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. Here, this suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.« less

Identification of Novel Linear Megaplasmids Carrying a ß-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains

PubMed Central

Franciosa, Giovanna; Scalfaro, Concetta; Di Bonito, Paola; Vitale, Marco; Aureli, Paolo

2011-01-01

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen. PMID

The Draft Genome Sequence of Clostridium sp. Strain NJ4, a Bacterium Capable of Producing Butanol from Inulin Through Consolidated Bioprocessing.

PubMed

Jiang, Yujia; Lu, Jiasheng; Chen, Tianpeng; Yan, Wei; Dong, Weiliang; Zhou, Jie; Zhang, Wenming; Ma, Jiangfeng; Jiang, Min; Xin, Fengxue

2018-05-23

A novel butanogenic Clostridium sp. NJ4 was successfully isolated and characterized, which could directly produce relatively high titer of butanol from inulin through consolidated bioprocessing (CBP). The assembled draft genome of strain NJ4 is 4.09 Mp, containing 3891 encoded protein sequences with G+C content of 30.73%. Among these annotated genes, a levanase, a hypothetical inulinase, and two bifunctional alcohol/aldehyde dehydrogenases (AdhE) were found to play key roles in the achievement of ABE production from inulin through CBP.

Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

PubMed

Chukwu, Emelda E; Nwaokorie, Francisca O; Coker, Akitoye O; Avila-Campos, Mario J; Solis, Rosa L; Llanco, Luis A; Ogunsola, Folasade T

2016-12-01

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Clostridium scatologenes strain SL1 isolated as an acetogenic bacterium from acidic sediments.

PubMed

Küsel, K; Dorsch, T; Acker, G; Stackebrandt, E; Drake, H L

2000-03-01

A strictly anaerobic, H2-utilizing bacterium, strain SL1, was isolated from the sediment of an acidic coal mine pond. Cells of strain SL1 were sporulating, motile, long rods with a multilayer cell wall. Growth was observed at 5-35 degrees C and pH 3.9-7.0. Acetate was the sole end product of H2 utilization and was produced in stoichiometries indicative of an acetyl-CoA-pathway-dependent metabolism. Growth and substrate utilization also occurred with CO/CO2, vanillate, syringate, ferulate, ethanol, propanol, 1-butanol, glycerine, cellobiose, glucose, fructose, mannose, xylose, formate, lactate, pyruvate and gluconate. With most substrates, acetate was the main or sole product formed. Growth in the presence of H2/CO2 or CO/CO2 was difficult to maintain in laboratory cultures. Methoxyl, carboxyl and acrylate groups of various aromatic compounds were O-demethylated, decarboxylated and reduced, respectively. Small amounts of butyrate were produced during the fermentation of sugars. The acrylate group of ferulate was reduced. Nitrate, sulfate, thiosulfate, dimethylsulfoxide and Fe(III) were not utilized as electron acceptors. Analysis of the 16S rRNA gene sequence of strain SL1 demonstrated that it is closely related to Clostridium scatologenes (99.6% sequence similarity), an organism characterized as a fermentative anaerobe but not previously shown to be capable of acetogenic growth. Comparative experiments with C. scatologenes DSM 757T demonstrated that it utilized H2/CO2 (negligible growth), CO/CO2 (negligible growth), formate, ethanol and aromatic compounds according to stoichiometries indicative of the acetyl-CoA pathway. CO dehydrogenase, formate dehydrogenase and hydrogenase activities were present in both strain SL1 and C. scatologenes DSM 757T. These results indicate that (i) sediments of acidic coal mine ponds harbour acetogens and (ii) C. scatologenes is an acetogen that tends to lose its capacity to grow acetogenically under H2/CO2 or CO/CO2 after prolonged

Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.

PubMed

Vidunas, Eugene; Mathews, Antony; Weaver, Michele; Cai, Ping; Koh, Eun Hee; Patel-Brown, Sujata; Yuan, Hailey; Zheng, Zi-Rong; Carriere, Marjolaine; Johnson, J Erik; Lotvin, Jason; Moran, Justin

2016-07-01

A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and β-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis.

PubMed

Wade, Ben; Keyburn, Anthony L; Haring, Volker; Ford, Mark; Rood, Julian I; Moore, Robert J

2016-12-25

Necrotic enteritis of poultry is an emerging disease of substantial economic importance, but aspects of the pathogenesis of this multi-factorial disease are still unclear. We recently demonstrated that the ability of avian strains of the causative bacterium, Clostridium perfringens, to bind to specific collagen types correlated strongly with their virulence and we postulated that binding of the pathogen to collagen types IV and V and gelatin may involve the putative adhesin-encoding gene cnaA, which is found in the VR-10B locus. In this study we have used site-directed mutagenesis to demonstrate that disruption of the cnaA gene leads to a reduction in the expression of the three genes immediately downstream of cnaA and reduced adherence to collagen types IV and V and gelatin. In addition, a cnaA mutant of strain EHE-NE18 was no longer capable of causing necrotic enteritis in a chicken disease induction model and had a significantly reduced ability to colonise the chicken intestinal mucosa. These results were confirmed by generating and analysing a similar mutant in an independent necrotic enteritis causing C. perfringens strain. This study expands our understanding of the mechanisms involved in necrotic enteritis pathogenesis by demonstrating the importance of C. perfringens adherence to extracellular matrix proteins. Copyright © 2016. Published by Elsevier B.V.

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

DOE PAGES

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; ...

2018-02-20

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

PubMed

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

2018-06-01

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.

Genome Sequence of Clostridium paraputrificum 373-A1 Isolated in Chile from a Patient Infected with Clostridium difficile

PubMed Central

Guerrero-Araya, Enzo; Plaza-Garrido, Angela; Díaz-Yañez, Fernando; Pizaro-Guajardo, Marjorie; Valenzuela, Sandro L.; Meneses, Claudio; Gil, Fernando

2016-01-01

Clostridium paraputrificum is a gut microbiota member reported in several cases of bacteremia and coinfections. So far, only one genome sequence of a C. paraputrificum (AGR2156) isolate is available. Here, we present the draft genome of C. paraputrificum strain 373-A1, isolated from stools from a patient with C. difficile infection. PMID:27811092

Pulsotype Diversity of Clostridium botulinum Strains Containing Serotypes A and/or B Genes

PubMed Central

Halpin, Jessica L.; Joseph, Lavin; Dykes, Janet K.; McCroskey, Loretta; Smith, Elise; Toney, Denise; Stroika, Steven; Hise, Kelley; Maslanka, Susan; Lúquez, Carolina

2017-01-01

Clostridium botulinum strains are prevalent in the environment and produce a potent neurotoxin that causes botulism, a rare but serious paralytic disease. In 2010, a national PulseNet database was established to curate C. botulinum pulsotypes and facilitate epidemiological investigations, particularly for serotypes A and B strains frequently associated with botulism cases in the United States. Between 2010 and 2014 we performed pulsed-field gel electrophoresis (PFGE) using a PulseNet protocol, uploaded the resulting PFGE patterns into a national database, and analyzed data according to PulseNet criteria (UPGMA clustering, Dice coefficient, 1.5% position tolerance, and 1.5% optimization). A retrospective data analysis was undertaken on 349 entries comprised of type A and B strains isolated from foodborne and infant cases to determine epidemiological relevance, resolution of the method, and the diversity of the database. Most studies to date on the pulsotype diversity of C. botulinum have encompassed very small sets of isolates; this study, with over 300 isolates, is more comprehensive than any published to date. Epidemiologically linked isolates had indistinguishable patterns, except in four instances and there were no obvious geographic trends noted. Simpson’s Index of Diversity (D) has historically been used to demonstrate species diversity and abundance within a group, and is considered a standard descriptor for PFGE databases. Simpson’s Index was calculated for each restriction endonuclease (SmaI, XhoI), the pattern combination SmaI-XhoI, as well as for each toxin serotype. The D values indicate that both enzymes provided better resolution for serotype B isolates than serotype A. XhoI as the secondary enzyme provided little additional discrimination for C. botulinum. SmaI patterns can be used to exclude unrelated isolates during a foodborne outbreak, but pulsotypes should always be considered concurrently with available epidemiological data. PMID:28692343

Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species-specific genomic properties and numerous putative antibiotic resistance determinants.

PubMed

Dehoux, Pierre; Marvaud, Jean Christophe; Abouelleil, Amr; Earl, Ashlee M; Lambert, Thierry; Dauga, Catherine

2016-10-21

Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. Genomic comparison of C. bolteae and C. clostridiofrome revealed

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: genetic markers and characterization of cellulolytic potential.

PubMed

Koeck, Daniela E; Zverlov, Vladimir V; Liebl, Wolfgang; Schwarz, Wolfgang H

2014-07-01

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters. Copyright © 2014 Elsevier GmbH. All rights reserved.

Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum

DOE PAGES

Tian, Liang; Papanek, Beth; Olson, Daniel G.; ...

2016-06-02

Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Results We started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yieldmore » and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. Moreover, the resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. Conclusions A two step selection method successfully improved the ethanol yield and the titer. Finaly, this evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.« less

Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light.

PubMed

Kusakabe, Tamami; Tatsuke, Tsuneyuki; Tsuruno, Keigo; Hirokawa, Yasutaka; Atsumi, Shota; Liao, James C; Hanai, Taizo

2013-11-01

Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by adc) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days. © 2013 Published by Elsevier Inc.

Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst.

PubMed

Gak, Eugene; Tyurin, Michael; Kiriukhin, Michael

2014-05-01

The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min (p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min (p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO2/H2 gas blend. Biocatalyst Clostridium sp. MT871RG- 11IBR6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min (p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO2/H2 blend thus contributing to the reversal of global warming.

Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy

PubMed Central

Iacobino, Angelo; Scalfaro, Concetta; Franciosa, Giovanna

2013-01-01

We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the

Clostridium difficile infection

PubMed Central

Vedantam, Gayatri; Clark, Andrew; Chu, Michele; McQuade, Rebecca; Mallozzi, Michael; Viswanathan, V. K.

2012-01-01

Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets. PMID:22555464

Is there a relationship between the presence of the binary toxin genes in Clostridium difficile strains and the severity of C. difficile infection (CDI)?

PubMed

Berry, C E; Davies, K A; Owens, D W; Wilcox, M H

2017-12-01

Some strains of Clostridium difficile produce a binary toxin, in addition to the main C. difficile virulence factors (toxins A and B). There have been conflicting reports regarding the role of binary toxin and its relationship to the severity of C. difficile infection (CDI). Samples, isolates and clinical data were collected as part of a prospective multicentre diagnostic study. Clostridium difficile isolates (n = 1259) were tested by polymerase chain reaction (PCR) assay to detect binary toxin genes cdtA and cdtB. The PCR binary toxin gene results were compared with clinical severity and outcome data, including 30-day all-cause mortality. The 1259 isolates corresponded to 1083 different patients (October 2010 to September 2011). The prevalence of binary toxin positive strains was significantly higher in faecal samples with detectable toxin A/B than in those without toxin but that were positive by cytotoxigenic culture (26.3% vs. 10.3%, p < 0.001). The presence of binary toxin correlated moderately with markers of CDI severity (white cell count, serum albumin concentration and serum creatinine concentration). However, the risk ratio for all-cause mortality was 1.68 for binary toxin positive patients and patients were significantly less likely to survive if they had CDI caused by a binary toxin gene positive strain, even after adjusting for age (p < 0.001). The presence of binary toxin genes does not predict the clinical severity of CDI, but it is significantly associated with the risk of all-cause mortality.

EDITORIAL: Excelling under strain: band engineering in nanomaterials Excelling under strain: band engineering in nanomaterials

NASA Astrophysics Data System (ADS)

Demming, Anna

2013-08-01

A little stress or strain has been known to improve the performance of athletes, actors and of course nanomaterials alike. In fact strain in silicon is now a major engineering tool for improving the performance of devices, and is ubiquitously used in device design and fabrication. Strain engineering alters a material's band structure, a model of electron behaviour that describes how as atoms come together in a solid, their discrete electron orbitals overlap to ultimately give rise to bands of allowed energy levels. In a strained crystal lattice of silicon or silicon germanium the distance between atoms in the lattice is greater than usual and the bands of allowed energy levels change. This July marks 100 years since Bohr submitted his paper 'On the constitution of atoms and molecules' [1] where he describes the structure of the atom in terms of discrete allowed energy levels. The paper was a seminal contribution to the development of quantum mechanics and laid the initial theoretical precepts for band gap engineering in devices. In this issue Nrauda and a collaboration of researchers in Europe and Australia study the growth of defect-free SiGe islands on pre-patterned silicon [2]. They analyse the strain in the islands and determine at what point lattice dislocations set in with a view to informing implementation of strain engineering in devices. The effects of strain on band structure in silicon and germanium were already studied and reported in the 1950s [3, 4]. Since then the increasing focus on nanoscale materials and the hunger for control of electronic properties has prompted further study of strain effects. The increased surface area to volume ratio in nanostructures changes the strain behaviour with respect to bulk materials, and this can also be exploited for handling and fine tuning strain to manipulate material properties. It is perhaps no surprise that graphene, one of the most high-profile materials in current nanotechnology research, has attracted

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

PubMed

Bradshaw, J G; Peeler, J T; Twedt, R M

1977-09-01

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

PubMed Central

Bradshaw, J G; Peeler, J T; Twedt, R M

1977-01-01

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators. PMID:199113

Genetic manipulation of clostridium acetobutylicum for enhanced butanol production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blaschek, H.P.; Holt, S.

Recent developments in the genetic manipulation of the acetone-butanol-ethanol fermentation microorganism, Clostridium acetobutylicum will be discussed. This specifically involves the characterization of an M13-like genetic system for C. acetobutylicum based on the pCAK1 phagemid, as well as the development of a plasmid-based vector based on the indigenous pDM11 plasmid recovered from C. acetobutylicum NCIB 6443. In addition, a macrorestriction map of the C. acetobutylicum ATCC 824 genome was constructed by utilizing two-dimensional transverse alternating field electrophoresis combined with reciprocal enzyme digestions and hybridization with previously cloned genes. We also describe the genetic engineering of a C. acetobutylicum strain with amplifiedmore » encloglucanase activity and to development and characterization of C. acetobutylicum hyper-amylolytic mutants with enhanced potential for commercial processes and evaluate their ability to produce butanol under batch and continuous culture conditions.« less

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291.

PubMed

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

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses

PubMed Central

Rusk, Anthony W.; Tung, David; Miller, Maria; Roix, Jeffrey; Khanna, Kristen V.; Murthy, Ravi; Benjamin, Robert S.; Helgason, Thorunn; Szvalb, Ariel D.; Bird, Justin E.; Roy-Chowdhuri, Sinchita; Zhang, Halle H.; Qiao, Yuan; Karim, Baktiar; McDaniel, Jennifer; Elpiner, Amanda; Sahora, Alexandra; Lachowicz, Joshua; Phillips, Brenda; Turner, Avenelle; Klein, Mary K.; Post, Gerald; Diaz, Luis A.; Riggins, Gregory J.; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Bettegowda, Chetan; Huso, David L.; Varterasian, Mary

2015-01-01

Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were observed in 6 of 16 dogs (37.5%), with three complete and three partial responses. On the basis of these encouraging results, we treated a human patient who had an advanced leiomyosarcoma with an intratumoral injection of C. novyi-NT spores. This treatment reduced the tumor within and surrounding the bone. Together, these results show that C. novyi-NT can precisely eradicate neoplastic tissues and suggest that further clinical trials of this agent in selected patients are warranted. PMID:25122639

Multihospital Outbreak of Clostridium difficile Infection, Cleveland, Ohio, USA

PubMed Central

Jump, Robin L.P.; Riggs, Michelle M.; Sethi, Ajay K.; Pultz, Michael J.; Ellis-Reid, Tracie; Riebel, William; Gerding, Dale N.; Salata, Robert A.

2010-01-01

To determine whether a multihospital Clostridium difficile outbreak was associated with epidemic strains and whether use of particular fluoroquinolones was associated with increased infection rates, we cultured feces from C. difficile–infected patients. Use of fluoroquionolones with enhanced antianaerobic activity was not associated with increased infection rates. PMID:20409374

Cellular Uptake and Mode-of-Action of Clostridium difficile Toxins.

PubMed

Papatheodorou, Panagiotis; Barth, Holger; Minton, Nigel; Aktories, Klaus

2018-01-01

Research on the human gut pathogen Clostridium difficile and its toxins has gained much attention, particularly as a consequence of the increasing threat to human health presented by emerging hypervirulent strains. Toxin A (TcdA) and B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (Clostridium difficile transferase). As C. difficile toxins are the causative agents of C. difficile-associated diseases (CDAD), such as antibiotics-associated diarrhea and pseudomembranous colitis, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Notably, a high proportion of studies on C. difficile toxins were performed in European laboratories. In this chapter we will highlight important recent advances in C. difficile toxins research.

Field performance of a genetically engineered strain of pink bollworm.

PubMed

Simmons, Gregory S; McKemey, Andrew R; Morrison, Neil I; O'Connell, Sinead; Tabashnik, Bruce E; Claus, John; Fu, Guoliang; Tang, Guolei; Sledge, Mickey; Walker, Adam S; Phillips, Caroline E; Miller, Ernie D; Rose, Robert I; Staten, Robert T; Donnelly, Christl A; Alphey, Luke

2011-01-01

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.

Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

PubMed Central

Parreira, Valeria R.; Marri, Pradeep R.; Rosey, Everett L.; Gong, Joshua; Songer, J. Glenn; Vedantam, Gayatri; Prescott, John F.

2010-01-01

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne. PMID:20532244

Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis.

PubMed

Lepp, Dion; Roxas, Bryan; Parreira, Valeria R; Marri, Pradeep R; Rosey, Everett L; Gong, Joshua; Songer, J Glenn; Vedantam, Gayatri; Prescott, John F

2010-05-24

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes approximately 85 and approximately 70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.

A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

PubMed

Mazuet, C; Yoon, E-J; Boyer, S; Pignier, S; Blanc, T; Doehring, I; Meziane-Cherif, D; Dumant-Forest, C; Sautereau, J; Legeay, C; Bouvet, P; Bouchier, C; Quijano-Roy, S; Pestel-Caron, M; Courvalin, P; Popoff, M R

2016-07-01

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to β-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the β-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Increase in ethanol yield via elimination of lactate production in an ethanol-tolerant mutant of Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biswas, Ranjita; Prabhu, Sandeep; Lynd, Lee R

2014-01-01

Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previouslymore » developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) ldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) ldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.« less

Clostridium perfringens, necrotic enteritis and its vaccination in broiler chickens

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens type A strains are the main etiological factors for necrotic enteritis (NE), one of the economically important gastrointestinal diseases in poultry responsible for the annual loss of 2 billion dollars in US poultry industry. NE has gained worldwide importance during the last...

Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes

PubMed Central

2014-01-01

Background Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species. Results Strains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases. Conclusions Bovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro. PMID:24479821

Vaginal and Rectal Clostridium sordellii and Clostridium perfringens Presence Among Women in the United States.

PubMed

Chong, Erica; Winikoff, Beverly; Charles, Dyanna; Agnew, Kathy; Prentice, Jennifer L; Limbago, Brandi M; Platais, Ingrida; Louie, Karmen; Jones, Heidi E; Shannon, Caitlin

2016-02-01

To characterize the presence of Clostridium sordellii and Clostridium perfringens in the vagina and rectum, identify correlates of presence, and describe strain diversity and presence of key toxins. We conducted an observational cohort study in which we screened a diverse cohort of reproductive-aged women in the United States up to three times using vaginal and rectal swabs analyzed by molecular and culture methods. We used multivariate regression models to explore predictors of presence. Strains were characterized by pulsed-field gel electrophoresis and tested for known virulence factors by polymerase chain reaction assays. Of 4,152 participants enrolled between 2010 and 2013, 3.4% (95% confidence interval [CI] 2.9-4.0) were positive for C sordellii and 10.4% (95% CI 9.5-11.3) were positive for C perfringens at baseline. Among the 66% with follow-up data, 94.7% (95% CI 88.0-98.3) of those positive for C sordellii and 74.4% (95% CI 69.0-79.3) of those positive for C perfringens at baseline were negative at follow-up. At baseline, recent gynecologic surgery was associated with C sordellii presence, whereas a high body mass index was associated with C perfringens presence in adjusted models. Two of 238 C sordellii isolates contained the lethal toxin gene, and none contained the hemorrhagic toxin gene. Substantial strain diversity was observed in both species with few clusters and no dominant clones identified. The relatively rare and transient nature of C sordellii and C perfringens presence in the vagina and rectum makes it inadvisable to use any screening or prophylactic approach to try to prevent clostridial infection. ClinicalTrials.gov, www.clinicaltrials.gov, NCT01283828.

Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lynd, Lee R.; Guss, Adam M.; Himmel, Mike

2016-11-01

Recent advances are addressed pertaining to consolidated bioprocessing (CBP) of plant cell walls to ethanol using two thermophilic, saccharolytic bacteria: the cellulose-fermenting Clostridium thermocellum and the hemicellulose- fermenting ermoanaerobacterium saccharolyticum. On the basis of the largest comparative dataset assembled to date, it appears that C. thermocellum is substantially more effective at solubilizing unpretreated plant cell walls than industry-standard fungal cellulase, and that this is particularly the case for more recalcitrant feedstocks. e distinctive central metabolism of C. thermocellum appears to involve more extensive energy coupling (e.g., on the order of 5 ATP per glucosyl moiety) than most fermentative anaerobes. Ethanolmore » yields and titers realized by engineered strains of T. saccharolyticum meet standards for industrial feasibility and provide an important proof of concept as well as a model that may be emulated in other organisms. Progress has also been made with C. thermocellum, although not yet to this extent. e current state of strain development is summarized and outstanding challenges for commercial application are discussed. We speculate that CBP organism development is more promising starting with naturally occurring cellulolytic microbes as compared to starting with noncellulolytic hosts.« less

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing Class I heat shock protein GroESL.

PubMed

Suo, Yukai; Fu, Hongxin; Ren, Mengmeng; Yang, Xitong; Liao, Zhengping; Wang, Jufang

2018-02-01

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum, were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20-0.21 g/L·h for the wild-type strain) due to the shortened lag phase. Copyright © 2017 Elsevier Ltd. All rights reserved.

SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

PubMed Central

Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

2007-01-01

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

PubMed

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.

Incorporating comparative genomics into the design-test-learn cycle of microbial strain engineering.

PubMed

Sardi, Maria; Gasch, Audrey P

2017-08-01

Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/acetate ratio.

PubMed

Suo, Yukai; Ren, Mengmeng; Yang, Xitong; Liao, Zhengping; Fu, Hongxin; Wang, Jufang

2018-05-01

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio.

Artificial symbiosis for acetone-butanol-ethanol (ABE) fermentation from alkali extracted deshelled corn cobs by co-culture of Clostridium beijerinckii and Clostridium cellulovorans.

PubMed

Wen, Zhiqiang; Wu, Mianbin; Lin, Yijun; Yang, Lirong; Lin, Jianping; Cen, Peilin

2014-07-15

Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to solvents (acetone, butanol and ethanol) production from the renewable and inexpensive substrates, for example, lignocellulose, on account of the depletion of oil resources, increasing gasoline prices and deteriorating environment. Limited to current tools for genetic manipulation, it is difficult to develop a genetically engineered microorganism with combined ability of lignocellulose utilization and solvents production. Mixed culture of cellulolytic microorganisms and solventogenic bacteria provides a more convenient and feasible approach for ABE fermentation due to the potential for synergistic utilization of the metabolic pathways of two organisms. But few bacteria pairs succeeded in producing biobutanol of high titer or high productivity without adding butyrate. The aim of this work was to use Clostridium cellulovorans 743B to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be subsequently utilized by Clostridium beijerinckii NCIMB 8052 to produce butanol in one pot reaction. A stable artificial symbiotic system was constructed by co-culturing a celluloytic, anaerobic, butyrate-producing mesophile (C. cellulovorans 743B) and a non-celluloytic, solventogenic bacterium (C. beijerinckii NCIMB 8052) to produce solvents by consolidated bioprocessing (CBP) with alkali extracted deshelled corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. Under optimized conditions, the co-culture degraded 68.6 g/L AECC and produced 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and 0.87 g/L ethanol) in less than 80 h. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain during the co

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

PubMed

Lacey, Jake A; Allnutt, Theodore R; Vezina, Ben; Van, Thi Thu Hao; Stent, Thomas; Han, Xiaoyan; Rood, Julian I; Wade, Ben; Keyburn, Anthony L; Seemann, Torsten; Chen, Honglei; Haring, Volker; Johanesen, Priscilla A; Lyras, Dena; Moore, Robert J

2018-05-22

Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

PubMed Central

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

Survey of Clostridium difficile in retail seafood in College Station, Texas

USDA-ARS?s Scientific Manuscript database

The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America with the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer t...

Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

PubMed Central

Ismaiel, A A; Zhu, C X; Colby, G D; Chen, J S

1993-01-01

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Images PMID:8349550

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion.

PubMed

Hector, Ronald E; Dien, Bruce S; Cotta, Michael A; Qureshi, Nasib

2011-09-01

Saccharomyces' physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04-0.17 h(-1). Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17-0.31 g/l/h, with ethanol yields 0.18-0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13-17% more ethanol than the parental control strains because of their ability to ferment xylose.

Hungatella effluvii gen. nov., sp. nov., an obligately anaerobic bacterium isolated from an effluent treatment plant, and reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov.

PubMed

Kaur, Sukhpreet; Yawar, Mir; Kumar, P Anil; Suresh, K

2014-03-01

A Gram-stain-positive, rod-shaped, spore-forming and strictly anaerobic bacterium, designated UB-B.2(T), was isolated from an industrial effluent anaerobic digester sample. It grew optimally at 30 °C and pH 7.0. Comparative analysis of the 16S rRNA gene sequence confirmed that strain UB-B.2(T) was closely related to Clostridium hathewayi DSM 13479(T) (97.84% similarity), a member of rRNA gene cluster XIVa of the genus Clostridium, and formed a coherent cluster with other related members of the Blautia (Clostridium) coccoides rRNA group in phylogenetic analyses. The end products of glucose fermentation by strain UB-B.2(T) were acetate and propionate. The G+C content of the DNA was 51.4 mol%. Although strain UB-B.2(T) showed 97.8% 16S rRNA gene sequence identity to the type strain of C. hathewayi, it exhibited only 38.4% relatedness at the whole-genome level. It also showed differences from its closest phylogenetic relative, C. hathewayi DSM 13479(T), in phenotypic characteristics such as hydrolysis of aesculin, starch and urea and fermentation end products. Both strains showed phenotypic differences from the members of rRNA gene cluster XIVa of the genus Clostridium. Based on these differences, C. hathewayi DSM 13479(T) and strain UB-B.2(T) were identified as representatives of a new genus of the family Clostridiaceae. Thus, we propose the reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov., the type species of the new genus (type strain DSM 13479(T) = CCUG 43506(T) = MTCC 10951(T)). Strain UB-B.2(T) ( = MTCC 11101(T) = DSM 24995(T)) is assigned to the novel species Hungatella effluvii gen. nov., sp. nov as the type strain.

Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate.

PubMed

Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

2016-08-01

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

Strain engineered pyrochlore at high pressure

DOE PAGES

Rittman, Dylan R.; Turner, Katlyn M.; Park, Sulgiye; ...

2017-05-22

Strain engineering is a promising method for next-generation materials processing techniques. Here, we use mechanical milling and annealing followed by compression in diamond anvil cell to tailor the intrinsic and extrinsic strain in pyrochlore, Dy 2Ti 2O 7 and Dy 2Zr 2O 7. Raman spectroscopy, X-ray pair distribution function analysis, and X-ray diffraction were used to characterize atomic order over short-, medium-, and long-range spatial scales, respectively, under ambient conditions. Raman spectroscopy and X-ray diffraction were further employed to interrogate the material in situ at high pressure. High-pressure behavior is found to depend on the species and concentration of defectsmore » in the sample at ambient conditions. Overall, we show that defects can be engineered to lower the phase transformation onset pressure by ~50% in the ordered pyrochlore Dy 2Ti 2O 7, and lower the phase transformation completion pressure by ~20% in the disordered pyrochlore Dy 2Zr 2O 7. Lastly, these improvements are achieved without significantly sacrificing mechanical integrity, as characterized by bulk modulus.« less

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

PubMed

Martirosian, G; Kuipers, S; Verbrugh, H; van Belkum, A; Meisel-Mikolajczyk, F

1995-08-01

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and

Protein design in systems metabolic engineering for industrial strain development.

PubMed

Chen, Zhen; Zeng, An-Ping

2013-05-01

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

PubMed

Kim, Sun-Ki; Jo, Jung-Hyun; Jin, Yong-Su; Seo, Jin-Ho

2017-05-01

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

USDA-ARS?s Scientific Manuscript database

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in C. perfringens isolates ...

Acetone production with metabolically engineered strains of Acetobacterium woodii.

PubMed

Hoffmeister, Sabrina; Gerdom, Marzena; Bengelsdorf, Frank R; Linder, Sonja; Flüchter, Sebastian; Öztürk, Hatice; Blümke, Wilfried; May, Antje; Fischer, Ralf-Jörg; Bahl, Hubert; Dürre, Peter

2016-07-01

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL(-1)h(-1) in bottle fermentation to 26.4mgL(-1)h(-1) in continuous gas fermentation. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

PubMed

Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

2013-02-01

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Anisotropic-Strain-Induced Band Gap Engineering in Nanowire-Based Quantum Dots.

PubMed

Francaviglia, Luca; Giunto, Andrea; Kim, Wonjong; Romero-Gomez, Pablo; Vukajlovic-Plestina, Jelena; Friedl, Martin; Potts, Heidi; Güniat, Lucas; Tütüncüoglu, Gözde; Fontcuberta I Morral, Anna

2018-04-11

Tuning light emission in bulk and quantum structures by strain constitutes a complementary method to engineer functional properties of semiconductors. Here, we demonstrate the tuning of light emission of GaAs nanowires and their quantum dots up to 115 meV by applying strain through an oxide envelope. We prove that the strain is highly anisotropic and clearly results in a component along the NW longitudinal axis, showing good agreement with the equations of uniaxial stress. We further demonstrate that the strain strongly depends on the oxide thickness, the oxide intrinsic strain, and the oxide microstructure. We also show that ensemble measurements are fully consistent with characterizations at the single-NW level, further elucidating the general character of the findings. This work provides the basic elements for strain-induced band gap engineering and opens new avenues in applications where a band-edge shift is necessary.

Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

PubMed Central

Linville, Jessica L.; Rodriguez, Miguel; Land, Miriam; Syed, Mustafa H.; Engle, Nancy L.; Tschaplinski, Timothy J.; Mielenz, Jonathan R.; Cox, Chris D.

2013-01-01

Background An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. Methodology/Principal Findings In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v). Conclusion/Significance The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

PubMed

Secore, Susan; Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S; Bodmer, Jean-Luc; Heinrichs, Jon H

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains

PubMed Central

Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R.; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S.; Bodmer, Jean-Luc; Heinrichs, Jon H.

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile. PMID:28125650

Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation

PubMed Central

Kurka, Hedwig; Ehrenreich, Armin; Ludwig, Wolfgang; Monot, Marc; Rupnik, Maja; Barbut, Frederic; Indra, Alexander; Dupuy, Bruno; Liebl, Wolfgang

2014-01-01

PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S–23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation. PMID:24482682

In-Plane Heterostructures Enable Internal Stress Assisted Strain Engineering in 2D Materials.

PubMed

Liu, Feng; Wang, Tzu-Chiang; Tang, Qiheng

2018-04-01

Conventional methods to induce strain in 2D materials can hardly catch up with the sharp increase in requirements to design specific strain forms, such as the pseudomagnetic field proposed in graphene, funnel effect of excitons in MoS 2 , and also the inverse funnel effect reported in black phosphorus. Therefore, a long-standing challenge in 2D materials strain engineering is to find a feasible scheme that can be used to design given strain forms. In this article, combining the ability of experimentally synthetizing in-plane heterostructures and elegant Eshelby inclusion theory, the possibility of designing strain fields in 2D materials to manipulate physical properties, which is called internal stress assisted strain engineering, is theoretically demonstrated. Particularly, through changing the inclusion's size, the stress or strain gradient can be controlled precisely, which is never achieved. By taking advantage of it, the pseudomagnetic field as well as the funnel effect can be accurately designed, which opens an avenue to practical applications for strain engineering in 2D materials. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Draft Genome Sequence of Clostridium pasteurianum NRRL B-598, a Potential Butanol or Hydrogen Producer.

PubMed

Kolek, Jan; Sedlár, Karel; Provazník, Ivo; Patáková, Petra

2014-03-20

We present a draft genome sequence of Clostridium pasteurianum NRRL B-598. This strain ferments saccharides by two-stage acetone-butanol (AB) fermentation, is oxygen tolerant, and has high hydrogen yields.

Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining tra...

In vitro antimicrobial activities of metabolites from vaginal Lactobacillus strains against Clostridium perfringens isolated from a woman's vagina.

PubMed

Amin, Mansour; Moradi Choghakabodi, Parastoo; Alhassan Hamidi, Mohammad; Najafian, Mahin; Farajzadeh Sheikh, Ahmad

2017-01-01

More than 50 different species of bacteria may live in a woman's vagina, with lactobacilli being the predominant microorganism found in healthy adult females. Lactobacilli are relevant as a barrier to infection and are important in the impairment of colonization by pathogens, owing to competitive adherence to adhesion sites in the vaginal epithelium and their capacity to produce antimicrobial compounds. The aim of the present study was to demonstrate the inhibitory capability of Lactobacillus metabolites against Clostridium perfringens, an anaerobic Gram-positive bacterium. These bacteria were isolated from vaginal swabs by using culture-dependent approaches, and the bacteriostatic effect of Lactobacillus metabolites, extracted from different isolates, was assessed using a modified E test. Among the 100 vaginal swabs, 59 (59%) samples showed the presence of Lactobacillus strains and only one sample contained C. perfringens. Lactobacillus metabolites demonstrated the significant potency of in vitro activity against C. perfringens, with minimal inhibitory concentration values ranging from 15.6 μg/mL to 31.2 μg/mL. This study suggests that women without vaginal Lactobacillus strains may be susceptible to nonindigenous and potentially harmful microorganisms. Copyright © 2016. Published by Elsevier Taiwan LLC.

Genome engineering and gene expression control for bacterial strain development.

PubMed

Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

2015-01-01

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Risk factors for Clostridium difficile infection in a hepatology ward.

PubMed

Vanjak, Dominique; Girault, Guillaume; Branger, Catherine; Rufat, Pierre; Valla, Dominique-Charles; Fantin, Bruno

2007-02-01

During 2001, Clostridium difficile infection was observed in 23 patients hospitalized in a hepatology ward (attack rate, 0.9%). Since strain typing ruled out a clonal dissemination, we performed a case-control study. In addition to antibiotic use as a risk factor, the C. difficile infection rate was higher among patients with autoimmune hepatitis (P<.01).

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-04-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed Central

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-01-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor. PMID:9119480

Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

PubMed

Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

2017-01-01

A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

Stable coexistence of five bacterial strains as a cellulose-degrading community.

PubMed

Kato, Souichiro; Haruta, Shin; Cui, Zong Jun; Ishii, Masaharu; Igarashi, Yasuo

2005-11-01

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

Strain-engineered growth of two-dimensional materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Geun Ho; Amani, Matin; Rasool, Haider

The application of strain to semiconductors allows for controlled modification of their band structure. This principle is employed for the manufacturing of devices ranging from high-performance transistors to solid-state lasers. Traditionally, strain is typically achieved via growth on lattice-mismatched substrates. For two-dimensional (2D) semiconductors, this is not feasible as they typically do not interact epitaxially with the substrate. Here in this paper, we demonstrate controlled strain engineering of 2D semiconductors during synthesis by utilizing the thermal coefficient of expansion mismatch between the substrate and semiconductor. Using WSe 2 as a model system, we demonstrate stable built-in strains ranging from 1%more » tensile to 0.2% compressive on substrates with different thermal coefficient of expansion. Consequently, we observe a dramatic modulation of the band structure, manifested by a strain-driven indirect-to-direct bandgap transition and brightening of the dark exciton in bilayer and monolayer WSe 2, respectively. The growth method developed here should enable flexibility in design of more sophisticated devices based on 2D materials.« less

Strain-engineered growth of two-dimensional materials

DOE PAGES

Ahn, Geun Ho; Amani, Matin; Rasool, Haider; ...

2017-09-20

The application of strain to semiconductors allows for controlled modification of their band structure. This principle is employed for the manufacturing of devices ranging from high-performance transistors to solid-state lasers. Traditionally, strain is typically achieved via growth on lattice-mismatched substrates. For two-dimensional (2D) semiconductors, this is not feasible as they typically do not interact epitaxially with the substrate. Here in this paper, we demonstrate controlled strain engineering of 2D semiconductors during synthesis by utilizing the thermal coefficient of expansion mismatch between the substrate and semiconductor. Using WSe 2 as a model system, we demonstrate stable built-in strains ranging from 1%more » tensile to 0.2% compressive on substrates with different thermal coefficient of expansion. Consequently, we observe a dramatic modulation of the band structure, manifested by a strain-driven indirect-to-direct bandgap transition and brightening of the dark exciton in bilayer and monolayer WSe 2, respectively. The growth method developed here should enable flexibility in design of more sophisticated devices based on 2D materials.« less

Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen.

PubMed

Hargreaves, Katherine R; Flores, Cesar O; Lawley, Trevor D; Clokie, Martha R J

2014-08-26

Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an

Strain measurements in a rotary engine housing

NASA Technical Reports Server (NTRS)

Lee, C. M.; Bond, T. H.; Addy, H. E.; Chun, K. S.; Lu, C. Y.

1989-01-01

The development of structural design tools for Rotary Combustion Engines (RCE) using Finite Element Modeling (FEM) requires knowledge about the response of engine materials to various service conditions. This paper describes experimental work that studied housing deformation as a result of thermal, pressure and mechanical loads. The measurement of thermal loads, clamping pressure, and deformation was accomplished by use of high-temperature strain gauges, thermocouples, and a high speed data acquisition system. FEM models for heat transfer stress analysis of the rotor housing will be verified and refined based on these experimental results.

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

PubMed

Silva, Rodrigo Otávio Silveira; Dorella, Fernanda Alves; Figueiredo, Henrique Cesar Pereira; Costa, Érica Azevedo; Pelicia, Vanessa; Ribeiro, Bruna Letícia Devidé; Ribeiro, Marcio Garcia; Paes, Antonio Carlos; Megid, Jane; Lobato, Francisco Carlos Faria

2017-12-01

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa. Copyright © 2017 Elsevier Ltd. All rights reserved.

MESSI: metabolic engineering target selection and best strain identification tool.

PubMed

Kang, Kang; Li, Jun; Lim, Boon Leong; Panagiotou, Gianni

2015-01-01

Metabolic engineering and synthetic biology are synergistically related fields for manipulating target pathways and designing microorganisms that can act as chemical factories. Saccharomyces cerevisiae's ideal bioprocessing traits make yeast a very attractive chemical factory for production of fuels, pharmaceuticals, nutraceuticals as well as a wide range of chemicals. However, future attempts of engineering S. cerevisiae's metabolism using synthetic biology need to move towards more integrative models that incorporate the high connectivity of metabolic pathways and regulatory processes and the interactions in genetic elements across those pathways and processes. To contribute in this direction, we have developed Metabolic Engineering target Selection and best Strain Identification tool (MESSI), a web server for predicting efficient chassis and regulatory components for yeast bio-based production. The server provides an integrative platform for users to analyse ready-to-use public high-throughput metabolomic data, which are transformed to metabolic pathway activities for identifying the most efficient S. cerevisiae strain for the production of a compound of interest. As input MESSI accepts metabolite KEGG IDs or pathway names. MESSI outputs a ranked list of S. cerevisiae strains based on aggregation algorithms. Furthermore, through a genome-wide association study of the metabolic pathway activities with the strains' natural variation, MESSI prioritizes genes and small variants as potential regulatory points and promising metabolic engineering targets. Users can choose various parameters in the whole process such as (i) weight and expectation of each metabolic pathway activity in the final ranking of the strains, (ii) Weighted AddScore Fuse or Weighted Borda Fuse aggregation algorithm, (iii) type of variants to be included, (iv) variant sets in different biological levels.Database URL: http://sbb.hku.hk/MESSI/. © The Author(s) 2015. Published by Oxford University

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V

2011-01-01

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less

Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose.

PubMed

Gaida, Stefan Marcus; Liedtke, Andrea; Jentges, Andreas Heinz Wilhelm; Engels, Benedikt; Jennewein, Stefan

2016-01-13

Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such as Clostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced. We therefore developed a metabolic engineering approach in C. cellulolyticum to expand its natural product spectrum and to fine tune the engineered metabolic pathways. Here we report the metabolic engineering of C. cellulolyticum to produce n-butanol, a next-generation biofuel and important chemical feedstock, directly from crystalline cellulose. We introduced the CoA-dependent pathway for n-butanol synthesis from C. acetobutylicum and measured the expression of functional enzymes (using targeted proteomics) and the abundance of metabolic intermediates (by LC-MS/MS) to identify potential bottlenecks in the n-butanol biosynthesis pathway. We achieved yields of 40 and 120 mg/L n-butanol from cellobiose and crystalline cellulose, respectively, after cultivating the bacteria for 6 and 20 days. The analysis of enzyme activities and key intracellular metabolites provides a robust framework to determine the metabolic flux through heterologous pathways in C. cellulolyticum, allowing further improvements by fine tuning individual steps to improve the yields of n-butanol.

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

PubMed

Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

2014-12-01

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239.

PubMed

Yasugi, Mayo; Otsuka, Keisuke; Miyake, Masami

2016-10-01

Clostridium perfringens type A is a common source of food-borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food-borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co-cultured with Caco-2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co-cultured in Roswell Park Memorial Institute-1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO 3 ] 2 ) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO 3 ) 2 -deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO 3 ) 2 down-regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down-regulating Spo0A-regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

PubMed

Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

2004-05-20

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

Clostridium difficile-associated disease: impact of the updated SHEA/IDSA guidelines.

PubMed

Kincaid, Scott E

2010-12-01

Clostridium difficile-associated disease (CDAD) is an increasingly difficult condition to treat because of the emergence of antibiotic resistance and highly pathogenic strains of bacteria. These newly identified strains affect patients in every facet of health care, from individuals in the community to those in intensive care units and all points in between. Appropriate management regarding diagnosis, infection control, pharmacotherapy, and prevention is the key to good outcomes in all patient populations. Geriatric patients are particularly at risk of acquiring CDAD as a result of their gradually declining immune systems and increased exposure to health care facilities. Therefore, they merit a higher level of attention when CDAD is suspected. In an effort to identify the best practices, the Society for Healthcare Epidemiology of America in conjunction with the Infectious Diseases Society of America developed guidelines regarding the diagnosis and management of Clostridium-associated disease. By utilizing these guidelines to educate other health care practitioners and by considering the recommendations in their own practice, pharmacists can have a positive impact on every facet of CDAD prevention and management.

Strain-Engineered Nanomembrane Substrates for Si/SiGe Heterostructures

NASA Astrophysics Data System (ADS)

Sookchoo, Pornsatit

For Group IV materials, including silicon, germanium, and their alloys, although they are most widely used in the electronics industry, the development of photonic devices is hindered by indirect band gaps and large lattice mismatches. Thus, any heterostructures involving Si and Ge (4.17% lattice mismatch) are subject to plastic relaxation by dislocation formation in the heterolayers. These defects make many devices impossible and at minimum degrade the performance of those that are possible. Fabrication using elastic strain engineering in Si/SiGe nanomembranes (NMs) is an approach that is showing promise to overcome this limitation. A key advantage of such NM substrates over conventional bulk substrates is that they are relaxed elastically and therefore free of dislocations that occur in the conventional fabrication of SiGe substrates, which are transferred to the epilayers and roughen film interfaces. In this thesis, I use the strain engineering of NMs or NM stacks to fabricate substrates for the epitaxial growth of many repeating units of Si/SiGe heterostructure, known as a 'superlattice', by the elastic strain sharing of a few periods of the repeating unit of Si/SiGe heterolayers or a Si/SiGe/Si tri-layer structure. In both cases, the process begins with the epitaxial growth of Si/SiGe heterolayers on silicon-on-insulator (SOI), where each layer thickness is designed to stay below its kinetic critical thickness for the formation of dislocations. The heterostructure NMs are then released by etching of the SiO2 sacrificial layer in hydrofluoric acid. The resulting freestanding NMs are elastically relaxed by the sharing of strain between the heterolayers. The NMs can be bonded in-place to their host substrate or transferred to another host substrate for the subsequent growth of many periods of superlattice film. The magnitude of strain sharing in these freestanding NMs is influenced by their layer thicknesses and layer compositions. As illustrated in this

Clostridium difficile infection among immunocompromised patients in Rio de Janeiro, Brazil and detection of moxifloxacin resistance in a ribotype 014 strain.

PubMed

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.

Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Guanhui; University of Chinese Academy of Sciences, Beijing; Dong, Hongjun

Highlights: • Genomes of a butanol tolerant strain and its parent strain were deciphered. • Comparative genomic and proteomic was applied to understand butanol tolerance. • None differentially expressed proteins have mutations in its corresponding genes. • Mutations in ribosome might be responsible for the global difference of proteomics. - Abstract: Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19 g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work,more » we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.« less

[Characteristics of Clostridium difficile infection in a high complexity hospital and report of the circulation of the NAP1/027 hypervirulent strain in Colombia].

PubMed

Gualtero, Sandra Milena; Abril, Lina Alejandra; Camelo, Nathalia; Sanchez, Susi Daniela; Davila, Fabián Antonio; Arias, Gerson; Silva, Edwin; Bustos, Ingrid Gissel; Josa, Diego Fernando; Torres, Isabel Cristina; Zambrano, Luis Carlos; Pareja, María José

2017-12-01

Clostridium difficile is the main pathogen related to healthcare-associated diarrhea and it is the cause of 20 to 30% of diarrhea cases caused by antibiotics. In Colombia and Latin America, the knowledge about the epidemiological behavior of this infection is limited. To describe the characteristics of a series of patients with C. difficile infection. We performed a descriptive case series study of patients with C. difficile infection hospitalized in the Fundación Clínica Shaio from January, 2012, to November, 2015. We analyzed 36 patients. The average age was 65 years. The risk factors associated with the infection were: previous use of antibiotics (94.4%), prior hospitalization in the last three months (66.7%) and use of proton pump inhibitors (50%). The most common comorbidities were chronic kidney disease (41.7%) and diabetes mellitus (30.6%). The most frequent symptoms were more than three loose stools per day (97.1%) and abdominal pain (42.9%). According to the severity of the disease, 44.4% of cases were classified as mild to moderate, 38.9% as severe, and 11.1% as complicated or severe. The detection of the toxin by PCR (GeneXpert) was the most common diagnostic procedure (63.8%). Global mortality during hospitalization was 8%. We identified four strains with serotype NAP1/027 and nine samples positive for binary toxin. Clostridium difficile infection should be suspected in patients with diarrhea and traditional risk factors associated with this disease. We report the circulation of the hypervirulent strain serotype NAP1/027 in Colombia, which should be countered with epidemiological surveillance and a prompt diagnosis.

Clostridium difficile infection

PubMed Central

Smits, Wiep Klaas; Lyras, Dena; Lacy, D. Borden; Wilcox, Mark H.; Kuijper, Ed J.

2017-01-01

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis — the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota. PMID:27158839

NanR Regulates nanI Sialidase Expression by Clostridium perfringens F4969, a Human Enteropathogenic Strain.

PubMed

Li, Jihong; Evans, Daniel R; Freedman, John C; McClane, Bruce A

2017-09-01

Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N -acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI , are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo . Copyright © 2017 American Society for Microbiology.

Occurrence and prevalence of Clostridium perfringens in polar bears from Svalbard, Norway.

PubMed

Jores, Joerg; Derocher, Andrew E; Staubach, Christoph; Aschfalk, Ansgar

2008-01-01

To obtain insight into the occurrence and prevalence of Clostridium perfringens and its major toxins in polar bears (Ursus maritimus), we took fecal samples for bacteriologic analysis from live-captured bears in the Svalbard Archipelago, Norway, in 2001. Clostridium perfringens was isolated from 40 of 92 samples (44%). Thirty strains were further characterized by determining toxin type and were classified to be type A, while one was also positive for the gene encoding beta2-toxin. Despite the fact that C. perfringens type A has been associated with fatal diseases in several animal species as well as in humans, our data indicate that C. perfringens type A is an normal inhabitant of the gastrointestinal tract of polar bears.

Delay time and Hartman effect in strain engineered graphene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xi, E-mail: xchen@shu.edu.cn; Deng, Zhi-Yong; Ban, Yue, E-mail: yban@shu.edu.cn

2014-05-07

Tunneling times, including group delay and dwell time, are studied for massless Dirac electrons transmitting through a one-dimensional barrier in strain-engineered graphene. The Hartman effect, the independence of group delay on barrier length, is induced by the strain effect, and associated with the transmission gap and the evanescent mode. The influence of barrier height/length and strain modulus/direction on the group delay is also discussed, which provides the flexibility to control the group delay with applications in graphene-based devices. The relationship between group delay and dwell time is finally derived to clarify the nature of the Hartman effect.

Expression of adhA from different organisms in Clostridium thermocellum.

PubMed

Zheng, Tianyong; Cui, Jingxuan; Bae, Hye Ri; Lynd, Lee R; Olson, Daniel G

2017-01-01

Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum . Based on sequence identity to T. saccharolyticum adhA , we chose adhA genes from 10 other organisms: Clostridium botulinum , Methanocaldococcus bathoardescens , Thermoanaerobacterium ethanolicus , Thermoanaerobacter mathranii , Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum , Caldicellulosiruptor saccharolyticus , Fervidobacterium nodosum , Marinitoga piezophila , and Thermotoga petrophila . All 11 adhA genes (including T. saccharolyticum adhA ) were expressed in C. thermocellum and fermentation end products were analyzed. All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA . Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum , and that this appears to be a general property of adhA genes.

Clostridium difficile binary toxin CDT

PubMed Central

Gerding, Dale N; Johnson, Stuart; Rupnik, Maja; Aktories, Klaus

2014-01-01

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed. PMID:24253566

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

PubMed

Kim, Soo Rin; Skerker, Jeffrey M; Kong, In Iok; Kim, Heejin; Maurer, Matthew J; Zhang, Guo-Chang; Peng, Dairong; Wei, Na; Arkin, Adam P; Jin, Yong-Su

2017-03-01

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

PubMed

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M; Fawley, Warren N; Paredes-Sabja, Daniel; Wilcox, Mark; Gonzalez, Angel

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients' data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia

PubMed Central

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M.; Fawley, Warren N.; Paredes-Sabja, Daniel; Wilcox, Mark

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia. PMID:29649308

First Report Worldwide of an Infant Botulism Case Due to Clostridium botulinum Type E▿

PubMed Central

Lúquez, Carolina; Dykes, Janet K.; Yu, Patricia A.; Raphael, Brian H.; Maslanka, Susan E.

2010-01-01

Clostridium botulinum type E has been associated with botulism in adults but never in infants. Infant botulism type E cases have been associated with neurotoxigenic strains of C. butyricum. We report the first infant botulism case due to C. botulinum type E worldwide. PMID:19906896

Secretion of clostridium cellulase by E. coli

DOEpatents

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

High temperature strain gage technology for gas turbine engines

NASA Technical Reports Server (NTRS)

Fichtel, Edward J.; Mcdaniel, Amos D.

1994-01-01

This report summarizes the results of a six month study that addressed specific issues to transfer the Pd-13Cr static strain sensor to a gas turbine engine environment. The application issues that were addressed include: (1) evaluation of a miniature, variable potentiometer for use as the ballast resistor, in conjunction with a conventional strain gage signal conditioning unit; (2) evaluation of a metal sheathed, platinum conductor leadwire assembly for use with the three-wire sensor; and (3) subjecting the sensor to dynamic strain cyclic testing to determine fatigue characteristics. Results indicate a useful static strain gage system at all temperature levels up to 1350 F. The fatigue characteristics also appear to be very promising, indicating a potential use in dynamic strain measurement applications. The procedure, set-up, and data for all tests are presented in this report. This report also discusses the specific strain gage installation technique for the Pd-13Cr gage because of its potential impact on the quality of the output data.

The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

PubMed

Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G; Lynd, Lee R

2015-04-01

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be

A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a Complete tcdA Gene

PubMed Central

Marín, Mercedes; Martín, Adoración; Rupnik, Maja

2014-01-01

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A− B+ CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site. PMID:25428159

The incidence of Clostridioides difficile and Clostridium perfringens netF-positive strains in diarrheic dogs.

PubMed

Diniz, Amanda Nadia; Coura, Fernanda Morcatti; Rupnik, Maja; Adams, Vicki; Stent, Thomas L; Rood, Julian I; de Oliveira, Carlos Augusto; Lobato, Francisco Carlos Faria; Silva, Rodrigo Otávio Silveira

2018-02-01

The aim of this study was to examine the incidence of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic dogs. Also, the presence of other common canine enteropathogens was examined. Toxigenic C. difficile and C. perfringens positive for the NetF-encoding gene (netF) were detected in 11 (11.9%) and seven (7.6%) diarrheic dogs, respectively. Three dogs were diagnosed simultaneously with toxigenic C. difficile and netF-positive C. perfringens. Among other enteropathogens, Giardia sp. was the most common agent detected in dogs positive for toxigenic C. difficile or netF-positive C. perfringens. The results suggest that C. difficile and C. perfringens occur more frequently as a primary cause of diarrhea. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct selection of Clostridium acetobutylicum fermentation mutants by a proton suicide method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cueto, P.H.; Mendez, B.S.

Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

Draft Genome Sequence of Clostridium mangenotii TR, Isolated from the Fecal Material of a Timber Rattlesnake

PubMed Central

Cochran, Philip A.; Dowd, Scot E.; Andersen, Kylie; Anderson, Nichole; Brennan, Rachel; Brook, Nicole; Callaway, Tracie; Diamante, Kimberly; Duberstine, Annie; Fitch, Karla; Freiheit, Heidi; Godlewski, Chantel; Gorman, Kelly; Haubrich, Mark; Hernandez, Mercedes; Hirtreiter, Amber; Ivanoski, Beth; Jaminet, Xochitl; Kirkpatrick, Travis; Kratowicz, Jennifer; Latus, Casey; Leable, Tiegen; Lingafelt, Nicole; Lowe, DeAnna; Lowrance, Holly; Malsack, Latiffa; Mazurkiewicz, Julie; Merlos, Persida; Messley, Jamie; Montemurro, Dawn; Nakitare, Samora; Nelson, Christine; Nye, Amber; Pazera, Valerie; Pierangeli, Gina; Rellora, Ashley; Reyes, Angelica; Roberts, Jennifer; Robins, Shadara; Robinson, Jeshannah; Schultz, Alissa; Seifert, Sara; Sigler, Elona; Spangler, Julie; Swift, Ebony; TenCate, Rebecca; Thurber, Jessica; Vallee, Kristin; Wamboldt, Jennifer; Whitten, Shannon; Woods, De’andrea; Wright, Amanda; Yankunas, Darin

2014-01-01

Here, we report the draft genome sequence of Clostridium mangenotii strain TR, which was isolated from the fecal material of a timber rattlesnake. This bacterium is nonpathogenic but contains 68 genes involved in virulence, disease, and defense. PMID:24407632

First Report of an Infant Botulism Case Due to Clostridium botulinum Type Af

PubMed Central

de Jong, Laura I. T.; Fernández, Rafael A.; Pareja, Virtudes; Giaroli, Gabriel; Guidarelli, Sergio R.; Dykes, Janet K.

2014-01-01

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide. PMID:25502535

Quantitative analysis and predictive engineering of self-rolling of nanomembranes under anisotropic mismatch strain

NASA Astrophysics Data System (ADS)

Chen, Cheng; Song, Pengfei; Meng, Fanchao; Li, Xiao; Liu, Xinyu; Song, Jun

2017-12-01

The present work presents a quantitative modeling framework for investigating the self-rolling of nanomembranes under different lattice mismatch strain anisotropy. The effect of transverse mismatch strain on the roll-up direction and curvature has been systematically studied employing both analytical modeling and numerical simulations. The bidirectional nature of the self-rolling of nanomembranes and the critical role of transverse strain in affecting the rolling behaviors have been demonstrated. Two fabrication strategies, i.e., third-layer deposition and corner geometry engineering, have been proposed to predictively manipulate the bidirectional rolling competition of strained nanomembranes, so as to achieve controlled, unidirectional roll-up. In particular for the strategy of corner engineering, microfabrication experiments have been performed to showcase its practical application and effectiveness. Our study offers new mechanistic knowledge towards understanding and predictive engineering of self-rolling of nanomembranes with improved roll-up yield.

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

PubMed

Lo, Yung-Chung; Huang, Chi-Yu; Cheng, Chieh-Lun; Lin, Chiu-Yue; Chang, Jo-Shu

2011-09-01

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Clostridium thiosulfatireducens sp. nov., a proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor.

PubMed

Hernández-Eugenio, Guadalupe; Fardeau, Marie-Laure; Cayol, Jean-Luc; Patel, Bharat K C; Thomas, Pierre; Macarie, Hervé; Garcia, Jean-Louis; Ollivier, Bernard

2002-09-01

A strictly anaerobic, gram-positive, sporulating rod (0.5-0.6 x 2.0-4.0 microm), designated strain Lup 21T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating cheese-factory wastewater. Strain Lup 21T was motile by means of peritrichous flagella, had a G+C content of 31.4 mol% and grew optimally at 37 degrees C, pH 7.4, in the absence of NaCl. It is a heterotrophic micro-organism, utilizing proteinaceous compounds (gelatin, peptides, Casamino acids and various single amino acids) but unable to use any of the carbohydrates tested as a carbon and energy source. It reduced thiosulfate and elemental sulfur to sulfide in the presence of Casamino acids as carbon and energy sources. Acetate, butyrate, isobutyrate, isovalerate, CO2 and sulfide were end products from oxidation of gelatin and Casamino acids in the presence of thiosulfate as an electron acceptor. In the absence of thiosulfate, serine, lysine, methionine and histidine were fermented. On the basis of 16S rRNA similarity, strain Lup 21T was related to members of the low-G+C Clostridiales group, Clostridium subterminale DSM 6970T being the closest relative (with a sequence similarity of 99.4%). DNA-DNA hybridization was 56% with this species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the isolate was designated as a novel species of the genus Clostridium, Clostridium thiosulfatireducens sp. nov. The type strain is strain Lup 21T (= DSM 13105T = CIP 106908T).

Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children.

PubMed

Finegold, Sydney M; Summanen, Paula H; Downes, Julia; Corbett, Karen; Komoriya, Tomoe

2017-06-01

We studied stool specimens from 33 autistic children aged 2-9 years with gastrointestinal (GI) abnormalities and 13 control children without autism and without GI symptoms. We performed quantitative comparison of all Clostridium species and Clostridium perfringens strains from the fecal microbiota by conventional, selective anaerobic culture methods. We isolated C. perfringens strains and performed PCR analysis for the main C. perfringens toxin genes, alpha, beta, beta2, epsilon, iota and C. perfringens enterotoxin gene. Our results indicate that autistic subjects with gastrointestinal disease harbor statistically significantly (p = 0.031) higher counts of C. perfringens in their gut compared to control children. Autistic subjects also harbor statistically significantly (p = 0.015) higher counts of beta2-toxin gene-producing C. perfringens in their gut compared to control children, and the incidence of beta2-toxin gene-producing C. perfringens is significantly higher in autistic subjects compared to control children (p = 0.014). Alpha toxin gene was detected in all C. perfringens strains studied. C. perfringens enterotoxin gene was detected from three autistic and one control subject. Beta, epsilon, and iota toxin genes were not detected from autistic or control subjects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Motility and Flagellar Glycosylation in Clostridium difficile▿ †

PubMed Central

Twine, Susan M.; Reid, Christopher W.; Aubry, Annie; McMullin, David R.; Fulton, Kelly M.; Austin, John; Logan, Susan M.

2009-01-01

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein. PMID:19749038

Phase-Change Memory Materials by Design: A Strain Engineering Approach.

PubMed

Zhou, Xilin; Kalikka, Janne; Ji, Xinglong; Wu, Liangcai; Song, Zhitang; Simpson, Robert E

2016-04-20

Van der Waals heterostructure superlattices of Sb2 Te1 and GeTe are strain-engineered to promote switchable atomic disordering, which is confined to the GeTe layer. Careful control of the strain in the structures presents a new degree of freedom to design the properties of functional superlattice structures for data storage and photonics applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlation between fecal calprotectin levels, disease severity and the hypervirulent ribotype 027 strain in patients with Clostridium difficile infection.

PubMed

Peretz, Avi; Tkhawkho, Linda; Pastukh, Nina; Brodsky, Diana; Halevi, Chen Namimi; Nitzan, Orna

2016-06-22

Clostridium difficile is the most common infectious etiology of nosocomial diarrhea. Fecal calprotectin (fc) is a sensitive marker of intestinal inflammation, found to be associated with enteric bacterial infections and inflammatory bowel disease. We evaluated fc levels using a Chemiluminescent immunoassay method, in hospitalized patients with C. difficile infection (CDI) diagnosed by molecular stool examination and assessed correlation with virulent ribotype 027 strain infection, antibiotic susceptibility by gradient Etest strip performed on C. difficile colonies and clinical and laboratory measures of disease severity. Statistical analysis was performed for correlation of fc levels with clinical and laboratory parameters, disease severity and patient outcomes. Overall 29 patients with CDI were admitted at the Poria medical center in northern Israel, during June 2014-May 2015. Resistance to metronidazole was found in 3 (10.3 %) isolates and to vancomycin in 5 (17.2 %) isolates. Regarding patient outcomes, within 30 days of CDI diagnosis, recurrence of disease occurred in 10 (34.5 %) patients and 2 patients (6.9 %) died. Seven (24.1 %) isolates were C. difficile ribotype 027. Mean fc level was 331.4 μg/g (21-932). Higher fc levels were found in patients with C. difficile ribotype 027 (p < 0.0005). Fc levels were also correlated with elevated peripheral blood white cell count (p = 0.0007). A trend for higher fc levels was found in patients with a higher clostridium severity score index (p = 0.0633). No correlation was found between fecal calprotectin levels and age, sex, functional status, community versus hospital acquired CDI, antibiotic susceptibility, fever, and creatinine levels. Our study highlights the fact that fc has a potential role as a biomarker of disease severity and binary toxin producing ribotype associated disease.

Impact of the NAP-1 strain on disease severity, mortality, and recurrence of healthcare-associated Clostridium difficile infection.

PubMed

Bauer, Karri A; Johnston, Jessica E W; Wenzler, Eric; Goff, Debra A; Cook, Charles H; Balada-Llasat, Joan-Miquel; Pancholi, Preeti; Mangino, Julie E

2017-12-01

Studies are conflicting regarding the association of the North American pulsed-field gel electrophoresis type 1 (NAP1) strain in Clostridium difficile infection (CDI) and outcomes. We evaluated the association of NAP1 with healthcare-associated CDI disease severity, mortality, and recurrence at our academic medical center. Healthcare-associated CDI cases were identified from November 1, 2011 through January 31, 2013. Multivariable regression models were used to evaluate the associations of NAP1 with severe disease (based on the Hines VA severity score index), mortality, and recurrence. Among 5424 stool specimens submitted to the Clinical Microbiology Laboratory, 292 (5.4%) were positive for C. difficile by polymerase chain reaction (PCR) on or after hospital day 4; 70 (24%) of these specimens also tested positive for NAP1. During the study period, 247 (85%) patients had non-severe disease and 45 (15%) patients had severe disease. Among patients with non-severe disease, 65 (26%) had NAP1 and among patients with severe disease, 5 (11%) had NAP1. After controlling for potential confounders, NAP1 was not associated with an increased likelihood of severe disease (adjusted odds ratio [aOR] = 0.35; 95% confidence interval [CI], 0.13-0.93), in-hospital mortality (aOR = 1.02; 95% CI, 0.53-1.96), or recurrence (aOR = 1.16, 95% CI, 0.36-3.77). The NAP1 strain did not increase disease severity, mortality, or recurrence in this study, although the incidence of NAP1-positive healthcare associated-CDI was low. The role of strain typing in outcomes and treatment selection in patients with healthcare-associated CDI remains uncertain. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and Characterization of a Toxic Moiety of Low Molecular Weight from Clostridium botulinum Type A

PubMed Central

Gerwing, Julia; Dolman, Claude E.; Bains, Hardial S.

1965-01-01

Gerwing, Julia (The University of British Columbia, Vancouver, B.C., Canada), Claude E. Dolman, and Hardial S. Bains. Isolation and characterization of a toxic moiety of low molecular weight from Clostridium botulinum type A. J. Bacteriol. 89:1383–1386. 1965.—A toxic moiety of low molecular weight has been isolated from a type A strain of Clostridium botulinum, by a method involving ammonium sulfate precipitation and elution through diethylaminoethyl cellulose at pH 5.6. By means of electrophoresis and ultracentrifugation, the toxic substance was shown to be homogeneous; a molecular weight of 12,200 was calculated. Images PMID:14293025

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718.

PubMed

Li, Jihong; Freedman, John C; McClane, Bruce A

2015-10-01

Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators

Fermentative hydrogen production from Jerusalem artichoke by Clostridium tyrobutyricum expressing exo-inulinase gene.

PubMed

Jiang, Ling; Wu, Qian; Xu, Qing; Zhu, Liying; Huang, He

2017-08-11

Clostridium tyrobutyricum ATCC25755 has been reported as being able to produce significant quantities of hydrogen. In this study, the exo-inulinase encoding gene cloned from Paenibacillus polymyxa SC-2 was into the expression plasmid pSY6 and expressed in the cells of C. tyrobutyricum. The engineered C. tyrobutyricum strain efficiently fermented the inulin-type carbohydrates from Jerusalem artichoke, without any pretreatment being necessary for the production of hydrogen. A comparatively high hydrogen yield (3.7 mol/mol inulin-type sugar) was achieved after 96 h in a batch process with simultaneous saccharification and fermentation (SSF), with an overall volumetric productivity rate of 620 ± 60 mL/h/L when the initial total sugar concentration of the inulin extract was increased to 100 g/L. Synthesis of inulinase in the batch SSF culture was closely associated with strain growth until the end of the exponential phase, reaching a maximum activity of 28.4 ± 0.26 U/mL. The overall results show that the highly productive and abundant biomass crop Jerusalem artichoke can be a good substrate for hydrogen production, and that the application of batch SSF for its conversion has the potential to become a cost-effective process in the near future.

Dietary trehalose enhances virulence of epidemic Clostridium difficile.

PubMed

Collins, J; Robinson, C; Danhof, H; Knetsch, C W; van Leeuwen, H C; Lawley, T D; Auchtung, J M; Britton, R A

2018-01-18

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Strain Engineering to Modify the Electrochemistry of Energy Storage Electrodes

PubMed Central

Muralidharan, Nitin; Carter, Rachel; Oakes, Landon; Cohn, Adam P.; Pint, Cary L.

2016-01-01

Strain engineering has been a critical aspect of device design in semiconductor manufacturing for the past decade, but remains relatively unexplored for other applications, such as energy storage. Using mechanical strain as an input parameter to modulate electrochemical potentials of metal oxides opens new opportunities intersecting fields of electrochemistry and mechanics. Here we demonstrate that less than 0.1% strain on a Ni-Ti-O based metal-oxide formed on superelastic shape memory NiTi alloys leads to anodic and cathodic peak potential shifts by up to ~30 mV in an electrochemical cell. Moreover, using the superelastic properties of NiTi to enable strain recovery also recovers the electrochemical potential of the metal oxide, providing mechanistic evidence of strain-modified electrochemistry. These results indicate that mechanical energy can be coupled with electrochemical systems to efficiently design and optimize a new class of strain-modulated energy storage materials. PMID:27283872

Clostridium perfringens type A–E toxin plasmids

PubMed Central

Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

2014-01-01

Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

Strain engineered barium strontium titanate for tunable thin film resonators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khassaf, H.; Khakpash, N.; Sun, F.

2014-05-19

Piezoelectric properties of epitaxial (001) barium strontium titanate (BST) films are computed as functions of composition, misfit strain, and temperature using a non-linear thermodynamic model. Results show that through adjusting in-plane strains, a highly adaptive rhombohedral ferroelectric phase can be stabilized at room temperature with outstanding piezoelectric response exceeding those of lead based piezoceramics. Furthermore, by adjusting the composition and the in-plane misfit, an electrically tunable piezoelectric response can be obtained in the paraelectric state. These findings indicate that strain engineered BST films can be utilized in the development of electrically tunable and switchable surface and bulk acoustic wave resonators.

The impact of horizontal gene transfer on the biology of Clostridium difficile.

PubMed

Roberts, Adam P; Allan, Elaine; Mullany, Peter

2014-01-01

Clostridium difficile infection (CDI) is now recognised as the main cause of healthcare associated diarrhoea. Over the recent years there has been a change in the epidemiology of CDI with certain related strains dominating infection. These strains have been termed hyper-virulent and have successfully spread across the globe. Many C. difficile strains have had their genomes completely sequenced allowing researchers to build up a very detailed picture of the contribution of horizontal gene transfer to the adaptive potential, through the acquisition of mobile DNA, of this organism. Here, we review and discuss the contribution of mobile genetic elements to the biology of this clinically important pathogen. © 2014 Elsevier Ltd All rights reserved.

[Culture conditions of engineered strain of L-asparaginase and the recombinant plasmid stability].

PubMed

Wang, Y; Qian, S; Ye, J; Meng, G; Zhang, S

1999-12-01

The growth curves of engineered strain JM105(pASN) were different in LB and M-3 media. The expression level and activity of L-asparaginase were affected apparently by both biomass and induction time. Glucose repression of production of L-asparaginase was found. The stability of the recombinant plasmid pASN in different host strains and in LB and M-3 media was determined. After cultivation inLB broth and M-3 media at 30 degrees C for more than 50 generations without antibiotic selection, then induced at 42 degrees C for up to 5 h, the engineered strains were proved to be stable, except for DHA alpha (pASN).

Carriage of Clostridium difficile by Wild Urban Norway Rats (Rattus norvegicus) and Black Rats (Rattus rattus)

PubMed Central

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

Carriage of Clostridium difficile by wild urban Norway rats (Rattus norvegicus) and black rats (Rattus rattus).

PubMed

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.

Dihydrodaidzein-producing Clostridium-like intestinal bacterium, strain TM-40, affects in vitro metabolism of daidzein by fecal microbiota of human male equol producer and non-producers.

PubMed

Tamura, Motoi; Hori, Sachiko; Nakagawa, Hiroyuki

2011-01-01

Much attention has been focused on the biological effects of equol, a metabolite of daidzein produced by intestinal microbiota. However, little is known about the role of isoflavone metabolizing bacteria in the intestinal microbiota. Recently, we isolated a dihydrodaidzein (DHD)-producing Clostridium-like bacterium, strain TM-40, from human feces. We investigated the effects of strain TM-40 on in vitro daidzein metabolism by human fecal microbiota from a male equol producer and two male equol non-producers. In the fecal suspension from the male equol non-producer and DHD producer, DHD was detected in the in vitro fecal incubation of daidzein after addition of TM-40. The DHD concentration increased as the concentration of strain TM-40 increased. In the fecal suspension from the equol producer, the fecal equol production was increased by the addition of strain TM-40. The occupation ratios of Bifidobacterium and Lactobacillales were higher in the equol non-producers than in the equol producer. Adding isoflavone-metabolizing bacteria to the fecal microbiota should facilitate the estimation of the metabolism of isoflavonoids by fecal microbiota. Studies on the interactions among equol-producing microbiota and DHD-producing bacteria might lead to clarification of some of the mechanisms regulating the production of equol by fecal microbiota.

Dihydrodaidzein-producing Clostridium-like intestinal bacterium, strain TM-40, affects in vitro metabolism of daidzein by fecal microbiota of human male equol producer and non-producers

PubMed Central

TAMURA, Motoi; HORI, Sachiko; NAKAGAWA, Hiroyuki

2011-01-01

Much attention has been focused on the biological effects of equol, a metabolite of daidzein produced by intestinal microbiota. However, little is known about the role of isoflavone metabolizing bacteria in the intestinal microbiota. Recently, we isolated a dihydrodaidzein (DHD)-producing Clostridium-like bacterium, strain TM-40, from human feces. We investigated the effects of strain TM-40 on in vitro daidzein metabolism by human fecal microbiota from a male equol producer and two male equol non-producers. In the fecal suspension from the male equol non-producer and DHD producer, DHD was detected in the in vitro fecal incubation of daidzein after addition of TM-40. The DHD concentration increased as the concentration of strain TM-40 increased. In the fecal suspension from the equol producer, the fecal equol production was increased by the addition of strain TM-40. The occupation ratios of Bifidobacterium and Lactobacillales were higher in the equol non-producers than in the equol producer. Adding isoflavone-metabolizing bacteria to the fecal microbiota should facilitate the estimation of the metabolism of isoflavonoids by fecal microbiota. Studies on the interactions among equol-producing microbiota and DHD-producing bacteria might lead to clarification of some of the mechanisms regulating the production of equol by fecal microbiota. PMID:25045313

Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus.

PubMed

O'Flaherty, Sarah; Klaenhammer, Todd R

2016-10-15

Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid

Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus

PubMed Central

Klaenhammer, Todd R.

2016-01-01

ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is

Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

DOE PAGES

Papanek, Beth A.; Biswas, Ranjita; Rydzak, Thomas; ...

2015-09-12

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulosemore » Avicel, effectively eliminating formate, acetate, and lactate production and reducing H 2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4 mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO 2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, with the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.« less

Characterizing Strain Variation in Engineered E. coli Using a Multi-Omics-Based Workflow

DOE PAGES

Brunk, Elizabeth; George, Kevin W.; Alonso-Gutierrez, Jorge; ...

2016-05-19

Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proofmore » of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks.« less

Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production

PubMed Central

Varman, Arul M.; Xiao, Yi; Pakrasi, Himadri B.

2013-01-01

Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions. PMID:23183979

Production of L-lactic acid from metabolically engineered strain of Enterobacter aerogenes ATCC 29007.

PubMed

Thapa, Laxmi Prasad; Lee, Sang Jun; Park, Chulhwan; Kim, Seung Wook

2017-07-01

In this study, L-lactic acid production was investigated from metabolically engineered strain of E. aerogenes ATCC 29007. The engineered strain E. aerogenes SUMI01 (Δpta) was generated by the deletion of phosphate acetyltransferase (pta) gene from the chromosome of E. aerogenes ATCC 29007 and deletion was confirmed by colony PCR. Under the optimized fermentation conditions, at 37°C and pH 6 for 84h, the L-lactic acid produced by engineered strain E. aerogenes SUMI01 (Δpta) in flask fermentation using 100g/L mannitol as the carbon source was 40.05g/L as compared to that of the wild type counterpart 20.70g/L. At the end of the batch fermentation in bioreactor the production of L-lactic acid reached to 46.02g/L and yield was 0.41g/g by utilizing 112.32g/L mannitol. This is the first report regarding the production of L-lactic acid from Enterobacter species. We believe that this result may provide valuable guidelines for further engineering Enterobacter strain for the improvement of L-lactic acid production. Copyright © 2017 Elsevier Inc. All rights reserved.

Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

PubMed

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.

Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

PubMed Central

2011-01-01

Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

Association of healthcare exposure with acquisition of different Clostridium difficile strain types in patients with recurrent infection or colonization after clinical resolution of initial infection.

PubMed

Thabit, A K; Housman, S T; Burnham, C D; Nicolau, D P

2016-02-01

Following the resolution of an episode of Clostridium difficile infection (CDI), the factors associated with acquisition of different C. difficile strain types in patients with recurrent infection or persistent colonization have not been evaluated. To explore factors with potential correlation with acquisition of different C. difficile strain types in patients clinically cured of CDI through long-term follow-up across the continuum of care. Polymerase chain reaction ribotyping was performed on C. difficile isolates recovered at baseline and follow-up (days 19-38) from stool samples of patients successfully treated for CDI, and those who had recurrence and/or colonization following symptom resolution. Chart review was conducted to determine factors associated with acquisition of a different C. difficile ribotype. Of 25 patients initially cured of CDI, five had a recurrence and eight were colonized at follow-up. Patients did not differ with regard to age, sex, and whether the initial infection was with the BI/NAP1/027 strain. Ribotyping revealed that two out of five patients had recurrence attributed to a different strain type. Three of the colonized patients demonstrated strain switching compared with five patients who carried the same baseline strain. All patients (both infected and colonized) with different C. difficile ribotypes were exposed to the healthcare system. Exposure to antibiotics and proton pump inhibitors were not related to strain switching. Exposure to healthcare, but not to antibiotics or proton pump inhibitors, was consistently associated with recurrence or colonization with a different C. difficile ribotype. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

PubMed

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Strain-engineering stabilization of BaTi O3 -based polar metals

NASA Astrophysics Data System (ADS)

Ma, Chao; Jin, Kui-juan; Ge, Chen; Yang, Guo-zhen

2018-03-01

Polar metals, which possess ferroelectriclike polar structure and conductivity simultaneously, have attracted wide interest since the first solid example, LiOs O3 (below 140 K), was discovered. However, the lack of room-temperature polar metals hinders further research and applications. Thus abundant properties of polar metals are unexplored. Here, with first-principles calculations, we report that the polar metal phase can be stabilized in the strain-engineered BaTi O3 with electron doping. The mechanism relates to the competition between the shifting of the t2 g energy levels and the narrowing of their bandwidth. Surprisingly, it is predicted that the ferroelectric-to-paraelectric transition temperature can be increased by electron doping when the strain is large enough, which holds potential for room-temperature polar metals. Our results indicate that strain engineering is a promising way to achieve BaTi O3 -based polar metals, and they should have practical significance for obtaining easily accessible, ecofriendly, and potential room-temperature polar metals.

Co-culturing a novel Bacillus strain with Clostridium tyrobutyricum ATCC 25755 to produce butyric acid from sucrose.

PubMed

Dwidar, Mohammed; Kim, Seil; Jeon, Byoung Seung; Um, Youngsoon; Mitchell, Robert J; Sang, Byoung-In

2013-03-04

Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755T; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755T to ferment sucrose to butyric acid. B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755T to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755T cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h. Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated.

First Case of Infant Botulism Caused by Clostridium baratii Type F in California

PubMed Central

Barash, Jason R.; Tang, Tania W. H.; Arnon, Stephen S.

2005-01-01

In late 2003 a severely hypotonic neonate, just 38 h old at onset of illness, was found to have infant botulism caused by neurotoxigenic Clostridium baratii type F. Environmental investigations failed to identify a source of this strain. This is the youngest patient reported to have infant botulism and the fifth instance of infant botulism caused by C. baratii type F. PMID:16082001

First report of an infant botulism case due to Clostridium botulinum type Af.

PubMed

de Jong, Laura I T; Fernández, Rafael A; Pareja, Virtudes; Giaroli, Gabriel; Guidarelli, Sergio R; Dykes, Janet K; Lúquez, Carolina

2015-02-01

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Strain-engineered diffusive atomic switching in two-dimensional crystals

PubMed Central

Kalikka, Janne; Zhou, Xilin; Dilcher, Eric; Wall, Simon; Li, Ju; Simpson, Robert E.

2016-01-01

Strain engineering is an emerging route for tuning the bandgap, carrier mobility, chemical reactivity and diffusivity of materials. Here we show how strain can be used to control atomic diffusion in van der Waals heterostructures of two-dimensional (2D) crystals. We use strain to increase the diffusivity of Ge and Te atoms that are confined to 5 Å thick 2D planes within an Sb2Te3–GeTe van der Waals superlattice. The number of quintuple Sb2Te3 2D crystal layers dictates the strain in the GeTe layers and consequently its diffusive atomic disordering. By identifying four critical rules for the superlattice configuration we lay the foundation for a generalizable approach to the design of switchable van der Waals heterostructures. As Sb2Te3–GeTe is a topological insulator, we envision these rules enabling methods to control spin and topological properties of materials in reversible and energy efficient ways. PMID:27329563

Putative function of hypothetical proteins expressed by Clostridium perfringens type A strains and their protective efficacy in mouse model.

PubMed

Alam, Syed Imteyaz; Dwivedi, Pratistha

2016-10-01

The whole genome sequencing and annotation of Clostridium perfringens strains revealed several genes coding for proteins of unknown function with no significant similarities to genes in other organisms. Our previous studies clearly demonstrated that hypothetical proteins CPF_2500, CPF_1441, CPF_0876, CPF_0093, CPF_2002, CPF_2314, CPF_1179, CPF_1132, CPF_2853, CPF_0552, CPF_2032, CPF_0438, CPF_1440, CPF_2918, CPF_0656, and CPF_2364 are genuine proteins of C. perfringens expressed in high abundance. This study explored the putative role of these hypothetical proteins using bioinformatic tools and evaluated their potential as putative candidates for prophylaxis. Apart from a group of eight hypothetical proteins (HPs), a putative function was predicted for the rest of the hypothetical proteins using one or more of the algorithms used. The phylogenetic analysis did not suggest an evidence of a horizontal gene transfer event except for HP CPF_0876. HP CPF_2918 is an abundant extracellular protein, unique to C. perfringens species with maximum strain coverage and did not show any significant match in the database. CPF_2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF_2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF_2918 protein induced antibody production, a mixed Th1 and Th2 kind of response, and provided partial protection to immunized mice in direct C. perfringens challenge. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular characteristics of Clostridium difficile strains from patients with a first recurrence more than 8 weeks after the primary infection.

PubMed

Chen, Yijian; Rashid, Mamun Ur; Huang, Haihui; Fang, Hong; Nord, Carl Erik; Wang, Minggui; Weintraub, Andrej

2017-08-01

Nearly all published studies of recurrent Clostridium difficile infections (CDI) report recurrent CDI within 8 weeks after the primary infection. This study explored the molecular characteristics of C. difficile isolates from the first recurrent CDI more than 8 weeks after the primary infection. Consecutive hospitalized patients with a recurrent CDI more than 8 weeks after a primary infection were enrolled prospectively from January 2008 to February 2011. All C. difficile isolates of the primary and recurrent infections were collected and subjected to polymerase chain reaction ribotyping and antimicrobial susceptibility testing. There were 62 cases of CDI in this study, which included 32 cases (51.6%) of recurrence due to the same ribotype of C. difficile, 26 (41.9%) cases due to a different ribotype, and four (6.5%) cases with 2-4 recurrences due to the same or different strains. One hundred and forty C. difficile isolates were obtained, which included 62 primary CDI isolates and 78 recurrent isolates. Ribotype 020 was the most common C. difficile strain in primary and recurrent infections. Ribotype 001 accounted for 15.4% (10/78) of recurrent infections and 3.2% (2/62) of primary infections (p = 0.0447). The minimum inhibitory concentration at 90% (MIC 90 ) values of linezolid, moxifloxacin, and clindamycin against type 001 strains were much higher, compared to the three other common ribotypes. Recurrent CDI more than 8 weeks after a primary infection can be caused by the same or different C. difficile ribotype at similar percentages. Ribotype 001 C. difficile strains, which have a lower susceptibility to antimicrobials, were isolated more frequently in patients with a recurrent CDI. Copyright © 2015. Published by Elsevier B.V.

Characterization of Clostridium Baratii Type F Strains Responsible for an Outbreak of Botulism Linked to Beef Meat Consumption in France.

PubMed

Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Bouchier, Christiane; Criscuolo, Alexis; Bouvet, Philippe; Trehard, Hélène; Jourdan Da Silva, Nathalie; Popoff, Michel

2017-02-01

A second botulism outbreak due to Clostridium baratii occurred in France in August 2015 and included three patients who had their meal in a restaurant the same day. We report the characterization of C. baratii isolates including whole genome sequencing (WGS). Four C. baratii isolates collected in August 2015 from the outbreak 2 were analysed for toxin production and typing as well as for genetic characterization. WGS was done using using the NEBNext Ultra DNA Library Prep kit for Illumina (New England Biolabs) and sequenced on MiSeq machine (Illumina) in paired-end reads of 250 bases. The phylogenetic tree was generated based on the UPGMA method with genetic distances computed by using the Kimura two-parameter model. Evolutionary analyses were conducted in Bionumerics (V.6.6 Applied Maths). Three C. baratii isolates for patient's stools and one isolate from meat produced botulinum neurotoxin (BoNT) type F and retained a bont/F7 gene in OrfX cluster. All isolates were identical according to the WGS. However, phylogeny of the core genome showed that the four C. baratii strains were distantly related to that of the previous C. baratii outbreak in France in 2014 and from the other C. baratii strains reported in databanks. The fact that the strains isolated from the patients and meat samples were genetically identical supports that the meat used for the Bolognese sauce was responsible for this second botulism outbreak in France. These isolates were unrelated to that from the first C. baratii outbreak in France in 2014 indicating a distinct source of contamination. WGS provided robust determination of genetic relatedness and information regarding BoNT typing and toxin gene locus genomic localization.

Surface-layer (S-layer) of human and animal Clostridium difficile strains and their behaviour in adherence to epithelial cells and intestinal colonization.

PubMed

Spigaglia, Patrizia; Barketi-Klai, Amira; Collignon, Anne; Mastrantonio, Paola; Barbanti, Fabrizio; Rupnik, Maja; Janezic, Sandra; Kansau, Imad

2013-09-01

Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen-host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SlpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SlpA variants. Since each SlpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99-100 % was found among the SlpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SlpA of a poultry isolate, PR 014/020, which showed 99 % identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SlpA variants (v014/020a-e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human IT1106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SlpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/020b) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was

Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production

PubMed Central

Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R.; McClane, Bruce A.

2015-01-01

The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involves their production of a variant small acid soluble protein-4 that binds more tightly to spore DNA compared to the small acid soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the USA. During this foodborne disease, C. perfringens is ingested with food and then, using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

Comparative genomic analysis of single-molecule sequencing and hybrid approaches for finishing the Clostridium autoethanogenum JA1-1 strain DSM 10061 genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Steven D; Nagaraju, Shilpa; Utturkar, Sagar M

Background Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published. Results A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G +more » C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

PubMed

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.

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

USDA-ARS?s Scientific Manuscript database

Saccharomyces physiology and fermentation related properties vary broadly among industrial strains. In this study, six industrial strains of varied genetic background were engineered to ferment xylose. Aerobic growth rates on xylose were 0.040 h**-1 to 0.167 h**-1. Fermentation of xylose, glucose/xy...

The pangenome of the genus Clostridium.

PubMed

Udaondo, Zulema; Duque, Estrella; Ramos, Juan-Luis

2017-07-01

The pangenome for the genus Clostridium sensu stricto, which was obtained using highly curated and annotated genomes from 16 species is presented; some of these cause disease, while others are used for the production of added-value chemicals. Multilocus sequencing analysis revealed that species of this genus group into at least two clades that include non-pathogenic and pathogenic strains, suggesting that pathogenicity is dispersed across the phylogenetic tree. The core genome of the genus includes 546 protein families, which mainly comprise those involved in protein translation and DNA repair. The GS-GOGAT may represent the central pathway for generating organic nitrogen from inorganic nitrogen sources. Glycerol and glucose metabolism genes are well represented in the core genome together with a set of energy conservation systems. A metabolic network comprising proteins/enzymes, RNAs and metabolites, whose topological structure is a non-random and scale-free network with hierarchically structured modules was built. These modules shed light on the interactions between RNAs, proteins and metabolites, revealing biological features of transcription and translation, cell wall biosynthesis, C1 metabolism and N metabolism. Network analysis identified four nodes that function as hubs and bottlenecks, namely, coenzyme A, HPr kinases, S-adenosylmethionine and the ribonuclease P-protein, suggesting pivotal roles for them in Clostridium. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Isolation of Clostridium absonum and its cultural and biochemical properties.

PubMed

Hayase, M; Mitsui, N; Tamai, K; Nakamura, S; Nishida, S

1974-01-01

A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

Determining the cause of recurrent Clostridium difficile infection using whole genome sequencing.

PubMed

Sim, James Heng Chiak; Truong, Cynthia; Minot, Samuel S; Greenfield, Nick; Budvytiene, Indre; Lohith, Akshar; Anikst, Victoria; Pourmand, Nader; Banaei, Niaz

2017-01-01

Understanding the contribution of relapse and reinfection to recurrent Clostridium difficile infection (CDI) has implications for therapy and infection prevention, respectively. We used whole genome sequencing to determine the relation of C. difficile strains isolated from patients with recurrent CDI at an academic medical center in the United States. Thirty-five toxigenic C. difficile isolates from 16 patients with 19 recurrent CDI episodes with median time of 53.5days (range, 13-362) between episodes were whole genome sequenced on the Illumina MiSeq platform. In 84% (16) of recurrences, the cause of recurrence was relapse with prior strain of C. difficile. In 16% (3) of recurrent episodes, reinfection with a new strain of C. difficile was the cause. In conclusion, the majority of CDI recurrences at our institution were due to infection with the same strain rather than infection with a new strain. Copyright © 2016 Elsevier Inc. All rights reserved.

The identification of four histidine kinases that influence sporulation in Clostridium thermocellum.

PubMed

Mearls, Elizabeth B; Lynd, Lee R

2014-08-01

In this study, we sought to identify genes involved in the onset of spore formation in Clostridium thermocellum via targeted gene deletions, gene over-expression, and transcriptional analysis. We determined that three putative histidine kinases, clo1313_0286, clo1313_2735 and clo1313_1942 were positive regulators of sporulation, while a fourth kinase, clo1313_1973, acted as a negative regulator. Unlike Bacillus or other Clostridium species, the deletion of a single positively regulating kinase was sufficient to abolish sporulation in this organism. Sporulation could be restored in these asporogenous strains via overexpression of any one of the positive regulators, indicating a high level of redundancy between these kinases. In addition to having a sporulation defect, deletion of clo1313_2735 produced L-forms. Thus, this kinase may play an additional role in repressing L-form formation. This work suggests that C. thermocellum enters non-growth states based on the sensory input from multiple histidine kinases. The ability to control the development of non-growth states at the genetic level has the potential to inform strategies for improved strain development, as well as provide valuable insight into C. thermocellum biology. Copyright © 2014 Elsevier Ltd. All rights reserved.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

PubMed

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

CRISPR Diversity and Microevolution in Clostridium difficile

PubMed Central

Andersen, Joakim M.; Shoup, Madelyn; Robinson, Cathy; Britton, Robert; Olsen, Katharina E.P.; Barrangou, Rodolphe

2016-01-01

Abstract Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes. PMID:27576538

Toxin Plasmids of Clostridium perfringens

PubMed Central

Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

2013-01-01

SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

Environmental and High-Strain Rate effects on composites for engine applications

NASA Technical Reports Server (NTRS)

Chamis, C. C.; Smith, G. T.

1982-01-01

The Lewis Research Center is conducting a series of programs intended to investigate and develop the application of composite materials to structural components for turbojet engines. A significant part of that effort is directed to establishing resistance, defect growth, and strain rate characteristics of composite materials over the wide range of environmental and load conditions found in commercial turbojet engine operations. Both analytical and experimental efforts are involved.

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

PubMed Central

Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

2013-01-01

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

Characterization of clostridium perfringens netB+tpel+ type A atrains isolated from necrotic enteritis-afflicted broiler chickens

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens (CP) Type A strains are the key etiological factor in induction of necrotic enteritis (NE), one of the important enteric diseases in poultry, responsible for the annual loss of $ 6 billions to worldwide poultry industry. Several CP toxin genes were found to be critical in the...

Co-culturing a novel Bacillus strain with Clostridium tyrobutyricum ATCC 25755 to produce butyric acid from sucrose

PubMed Central

2013-01-01

Background Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755T; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755T to ferment sucrose to butyric acid. Results B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755T to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755T cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h. Conclusions Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated. PMID:23452443

Fully printable, strain-engineered electronic wrap for customizable soft electronics.

PubMed

Byun, Junghwan; Lee, Byeongmoon; Oh, Eunho; Kim, Hyunjong; Kim, Sangwoo; Lee, Seunghwan; Hong, Yongtaek

2017-03-24

Rapid growth of stretchable electronics stimulates broad uses in multidisciplinary fields as well as industrial applications. However, existing technologies are unsuitable for implementing versatile applications involving adaptable system design and functions in a cost/time-effective way because of vacuum-conditioned, lithographically-predefined processes. Here, we present a methodology for a fully printable, strain-engineered electronic wrap as a universal strategy which makes it more feasible to implement various stretchable electronic systems with customizable layouts and functions. The key aspects involve inkjet-printed rigid island (PRI)-based stretchable platform technology and corresponding printing-based automated electronic functionalization methodology, the combination of which provides fully printed, customized layouts of stretchable electronic systems with simplified process. Specifically, well-controlled contact line pinning effect of printed polymer solution enables the formation of PRIs with tunable thickness; and surface strain analysis on those PRIs leads to the optimized stability and device-to-island fill factor of strain-engineered electronic wraps. Moreover, core techniques of image-based automated pinpointing, surface-mountable device based electronic functionalizing, and one-step interconnection networking of PRIs enable customized circuit design and adaptable functionalities. To exhibit the universality of our approach, multiple types of practical applications ranging from self-computable digital logics to display and sensor system are demonstrated on skin in a customized form.

Fully printable, strain-engineered electronic wrap for customizable soft electronics

NASA Astrophysics Data System (ADS)

Byun, Junghwan; Lee, Byeongmoon; Oh, Eunho; Kim, Hyunjong; Kim, Sangwoo; Lee, Seunghwan; Hong, Yongtaek

2017-03-01

Rapid growth of stretchable electronics stimulates broad uses in multidisciplinary fields as well as industrial applications. However, existing technologies are unsuitable for implementing versatile applications involving adaptable system design and functions in a cost/time-effective way because of vacuum-conditioned, lithographically-predefined processes. Here, we present a methodology for a fully printable, strain-engineered electronic wrap as a universal strategy which makes it more feasible to implement various stretchable electronic systems with customizable layouts and functions. The key aspects involve inkjet-printed rigid island (PRI)-based stretchable platform technology and corresponding printing-based automated electronic functionalization methodology, the combination of which provides fully printed, customized layouts of stretchable electronic systems with simplified process. Specifically, well-controlled contact line pinning effect of printed polymer solution enables the formation of PRIs with tunable thickness; and surface strain analysis on those PRIs leads to the optimized stability and device-to-island fill factor of strain-engineered electronic wraps. Moreover, core techniques of image-based automated pinpointing, surface-mountable device based electronic functionalizing, and one-step interconnection networking of PRIs enable customized circuit design and adaptable functionalities. To exhibit the universality of our approach, multiple types of practical applications ranging from self-computable digital logics to display and sensor system are demonstrated on skin in a customized form.

Bending strain engineering in quantum spin hall system for controlling spin currents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Bing; Jin, Kyung-Hwan; Cui, Bin

Quantum spin Hall system can exhibit exotic spin transport phenomena, mediated by its topological edge states. The concept of bending strain engineering to tune the spin transport properties of a quantum spin Hall system is demonstrated. Here, we show that bending strain can be used to control the spin orientation of counter-propagating edge states of a quantum spin system to generate a non-zero spin current. This physics mechanism can be applied to effectively tune the spin current and pure spin current decoupled from charge current in a quantum spin Hall system by control of its bending curvature. Moreover, the curvedmore » quantum spin Hall system can be achieved by the concept of topological nanomechanical architecture in a controllable way, as demonstrated by the material example of Bi/Cl/Si(111) nanofilm. This concept of bending strain engineering of spins via topological nanomechanical architecture affords a promising route towards the realization of topological nano-mechanospintronics.« less

Bending strain engineering in quantum spin hall system for controlling spin currents

DOE PAGES

Huang, Bing; Jin, Kyung-Hwan; Cui, Bin; ...

2017-06-16

Quantum spin Hall system can exhibit exotic spin transport phenomena, mediated by its topological edge states. The concept of bending strain engineering to tune the spin transport properties of a quantum spin Hall system is demonstrated. Here, we show that bending strain can be used to control the spin orientation of counter-propagating edge states of a quantum spin system to generate a non-zero spin current. This physics mechanism can be applied to effectively tune the spin current and pure spin current decoupled from charge current in a quantum spin Hall system by control of its bending curvature. Moreover, the curvedmore » quantum spin Hall system can be achieved by the concept of topological nanomechanical architecture in a controllable way, as demonstrated by the material example of Bi/Cl/Si(111) nanofilm. This concept of bending strain engineering of spins via topological nanomechanical architecture affords a promising route towards the realization of topological nano-mechanospintronics.« less

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

PubMed Central

Pyne, Michael E.; Sokolenko, Stanislav; Liu, Xuejia; Srirangan, Kajan; Bruder, Mark R.; Aucoin, Marc G.; Moo-Young, Murray

2016-01-01

ABSTRACT Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter−1). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter−1), yielding a high n-butanol selectivity of 0.83 g n-butanol g−1 of solvents compared to 0.51 g n-butanol g−1 of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCE Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing

Genomic analyses of Clostridium perfringens isolates from five toxinotypes.

PubMed

Hassan, Karl A; Elbourne, Liam D H; Tetu, Sasha G; Melville, Stephen B; Rood, Julian I; Paulsen, Ian T

2015-05-01

Clostridium perfringens can be isolated from a range of environments, including soil, marine and fresh water sediments, and the gastrointestinal tracts of animals and humans. Some C. perfringens strains have attractive industrial applications, e.g., in the degradation of waste products or the production of useful chemicals. However, C. perfringens has been most studied as the causative agent of a range of enteric and soft tissue infections of varying severities in humans and animals. Host preference and disease type in C. perfringens are intimately linked to the production of key extracellular toxins and on this basis toxigenic C. perfringens strains have been classified into five toxinotypes (A-E). To date, twelve genome sequences have been generated for a diverse collection of C. perfringens isolates, including strains associated with human and animal infections, a human commensal strain, and a strain with potential industrial utility. Most of the sequenced strains are classified as toxinotype A. However, genome sequences of representative strains from each of the other four toxinotypes have also been determined. Analysis of this collection of sequences has highlighted a lack of features differentiating toxinotype A strains from the other isolates, indicating that the primary defining characteristic of toxinotype A strains is their lack of key plasmid-encoded extracellular toxin genes associated with toxinotype B to E strains. The representative B-E strains sequenced to date each harbour many unique genes. Additional genome sequences are needed to determine if these genes are characteristic of their respective toxinotypes. Copyright © 2014. Published by Elsevier Masson SAS.

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.

PubMed

Demeke, Mekonnen M; Dietz, Heiko; Li, Yingying; Foulquié-Moreno, María R; Mutturi, Sarma; Deprez, Sylvie; Den Abt, Tom; Bonini, Beatriz M; Liden, Gunnar; Dumortier, Françoise; Verplaetse, Alex; Boles, Eckhard; Thevelein, Johan M

2013-06-21

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. An industrial yeast strain for bioethanol production with

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

PubMed Central

2013-01-01

Background The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions An industrial yeast strain for

Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition

USDA-ARS?s Scientific Manuscript database

We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5'ace-1'cre-1'ndvB) was shown to produce cellobionate directly from cellulose ...

Clostridium difficile: Investigating Transmission Patterns between Infected and Colonized Patients using whole Genome Sequencing.

PubMed

Kong, L Y; Eyre, D W; Corbeil, J; Raymond, F; Walker, A S; Wilcox, M H; Crook, D W; Michaud, S; Toye, B; Frost, E; Dendukuri, N; Schiller, I; Bourgault, A M; Dascal, A; Oughton, M; Longtin, Y; Poirier, L; Brassard, P; Turgeon, N; Gilca, R; Loo, V G

2018-05-28

Whole genome sequencing (WGS) studies can enhance our understanding of the role of patients with asymptomatic Clostridium difficile colonization in transmission. Isolates obtained from patients with Clostridium difficile infection (CDI) and colonization identified in a study conducted during 2006 - 2007 at six Canadian hospitals underwent typing by pulsed-field gel electrophoresis, multilocus sequence typing, and WGS. Isolates from incident CDI cases not in the initial study were also sequenced where possible. Ward movement and typing data were combined to identify plausible donors for each CDI case, as defined by shared time and space within predefined limits. Proportions of plausible donors for CDI cases that were colonized, infected, or both were examined. Five hundred and fifty-four isolates were sequenced successfully, 353 from colonized and 201 from CDI cases. The NAP1/027/ST1 strain was the most common strain, found in 124 (62%) of infected and 92 (26%) of colonized patients. A donor with a plausible ward link was found for 81 CDI cases (40%) using WGS with a threshold of ≤2 single nucleotide variants to determine relatedness. Sixty-five (32%) CDI cases could be linked to both infected and colonized donors. Exclusive linkages to infected and colonized donors were found for 28 (14%) and 12 (6%) CDI cases, respectively. Colonized patients contribute to transmission, but CDI cases are more likely linked to other infected patients than colonized patients in this cohort with high rates of NAP1/027/ST1 strain, highlighting the importance of local prevalence of virulent strains in determining transmission dynamics.

High prevalence of Clostridium difficile on retail root vegetables, Western Australia.

PubMed

Lim, S C; Foster, N F; Elliott, B; Riley, T V

2018-02-01

The incidence of community-associated Clostridium difficile infection (CA-CDI) in Australia has increased since mid-2011. With reports of clinically important C. difficile strains being isolated from retail foods in Europe and North America, a foodborne source of C. difficile in cases of CA-CDI is a possibility. This study represents the first to investigate the prevalence and genotypes of C. difficile in Australian retail vegetables. A total of 300 root vegetables grown in Western Australia (WA) were collected from retail stores and farmers' markets. Three vegetables of the same kind bought from the same store/market were treated as one sample. Selective enrichment culture, toxin profiling and PCR ribotyping were performed. Clostridium difficile was isolated from 30% (30/100) of pooled vegetable samples, 55·6% of organic potatoes, 50% of nonorganic potatoes, 22·2% of organic beetroots, 5·6% of organic onions and 5·3% of organic carrots. Over half (51·2%, 22/43) the isolates were toxigenic. Many of the ribotypes of C. difficile isolated were common among human and Australian animals. Clostridium difficile could be found commonly on retail root vegetables of WA. This may be potential sources for CA-CDI. This study enhances knowledge of possible sources of C. difficile in the Australian community, outside the hospital setting. © 2017 The Society for Applied Microbiology.

Clostridium difficile Infection and Fecal Microbiota Transplant

PubMed Central

Liubakka, Alyssa; Vaughn, Byron P.

2017-01-01

Clostridium difficile infection (CDI) is a major source of morbidity and mortality for hospitalized patients. Although most patients have a clinical response to existing antimicrobial therapies, recurrent infection develops in up to 30% of patients. Fecal microbiota transplant is a novel approach to this complex problem, with an efficacy rate of nearly 90% in the setting of multiple recurrent CDI. This review covers the current epidemiology of CDI (including toxigenic and nontoxigenic strains, risk factors for infection, and recurrent infection), methods of diagnosis, existing first-line therapies in CDI, the role of fecal microbiota transplant for multiple recurrent CDIs, and the potential use of fecal microbial transplant for patients with severe or refractory infection. PMID:27959316

The safe enterocin DD14 is a leaderless two-peptide bacteriocin with anti-Clostridium perfringens activity.

PubMed

Caly, Delphine L; Chevalier, Mickaël; Flahaut, Christophe; Cudennec, Benoit; Al Atya, Ahmed Khassaf; Chataigné, Gabrielle; D'Inca, Romain; Auclair, Eric; Drider, Djamel

2017-03-01

Enterococcus faecalis 14, a strain previously isolated from meconium, displayed activity against four Clostridium perfringens isolates when co-cultured on agar plates. The anti-Clostridium activity was ascribed to the production of enterocin DD14, which was subsequently purified. The minimum inhibitory concentration (MIC) of enterocin DD14 against one collection strain and one clinical C. perfringens strain was determined at 50 µg/mL. Furthermore, using the intestinal epithelial cell line IPEC-1, it was shown that E. faecalis 14 was not cytotoxic after 24 h of contact, and no cytotoxicity was observed when IPEC-1 cells were incubated with pure enterocin DD14 for 4 h. Enterocin DD14 was characterised using mass spectrometry and was shown to consist of two small proteins of 5200.74 Da and 5206.41 Da, respectively. The two peptides (DD14A and DD14B) have highly similar amino acid sequences and no signal peptide, which classifies enterocin DD14 as a class IIb leaderless two-peptide bacteriocin. The genes encoding DD14A and DD14B were sequenced and were shown to be 100% identical to other previously described enterocins MR10A and MR10B, in contrast to the producing strains, which are different. Consequently, the present in vitro study supports the potential of this E. faecalis 14 strain and/or its purified enterocin DD14 as putative anti-C. perfringens compounds in chickens. Copyright © 2017. Published by Elsevier B.V.

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

PubMed

Rydzak, Thomas; Garcia, David; Stevenson, David M; Sladek, Margaret; Klingeman, Dawn M; Holwerda, Evert K; Amador-Noguez, Daniel; Brown, Steven D; Guss, Adam M

2017-05-01

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H 2 ), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum. Copyright © 2017. Published by Elsevier Inc.

Binary toxin and its clinical importance in Clostridium difficile infection, Belgium.

PubMed

Pilate, T; Verhaegen, J; Van Ranst, M; Saegeman, V

2016-11-01

Binary toxin-producing Clostridium difficile strains such as ribotypes 027 and 078 have been associated with increased Clostridium difficile infection (CDI) severity. Our objective was to investigate the association between presence of the binary toxin gene and CDI severity and recurrence. We performed a laboratory-based retrospective study including patients between January 2013 and March 2015 whose fecal samples were analyzed by polymerase chain reaction (PCR) for the presence of the genes for toxin B and binary toxin and a deletion in the tcdC gene, specific for ribotype 027. Clinical and epidemiological characteristics were compared between 33 binary toxin-positive CDI patients and 33 binary toxin-negative CDI patients. Subsequently, the characteristics of 66 CDI patients were compared to those of 66 diarrhea patients who were carriers of non-toxigenic C. difficile strains. Fifty-nine of 1034 (5.7 %) fecal samples analyzed by PCR were binary toxin-positive, belonging to 33 different patients. No samples were positive for ribotype 027. Binary toxin-positive CDI patients did not differ from binary toxin-negative CDI patients in terms of disease recurrence, morbidity, or mortality, except for a higher peripheral leukocytosis in the binary toxin-positive group (16.30 × 10 9 /L vs. 11.65 × 10 9 /L; p = 0.02). The second part of our study showed that CDI patients had more severe disease, but not a higher 30-day mortality rate than diarrhea patients with a non-toxicogenic C. difficile strain. In our setting with a low prevalence of ribotype 027, the presence of the binary toxin gene is not associated with poor outcome.

Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

DOE PAGES

Sander, Kyle; Asano, Keiji G.; Bhandari, Deepak; ...

2017-11-30

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging.Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing massmore » spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Lastly, our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.« less

Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sander, Kyle; Asano, Keiji G.; Bhandari, Deepak

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging.Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing massmore » spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Lastly, our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.« less

Strain-Engineered Multiferroicity in Pnma NaMnF_{3} Fluoroperovskite.

PubMed

Garcia-Castro, A C; Romero, A H; Bousquet, E

2016-03-18

In this study we show from first principles calculations the possibility to induce multiferroic and magnetoelectric functional properties in the Pnma NaMnF_{3} fluoroperovskite by means of epitaxial strain engineering. Surprisingly, we found a very strong nonlinear polarization-strain coupling that drives an atypical amplification of the ferroelectric polarization for either compression or expansion of the cell. This property is associated with a noncollinear antiferromagnetic ordering, which induces a weak ferromagnetism phase and makes the strained NaMnF_{3} fluoroperovskite multiferroic. The magnetoelectric response was calculated and it was found to be composed of linear and nonlinear components with amplitudes similar to the ones of Cr_{2}O_{3}. These findings show that it is possible to move the fluoride family toward functional applications with unique responses.

Strain-induced band engineering in monolayer stanene on Sb(111)

NASA Astrophysics Data System (ADS)

Gou, Jian; Kong, Longjuan; Li, Hui; Zhong, Qing; Li, Wenbin; Cheng, Peng; Chen, Lan; Wu, Kehui

2017-10-01

The two-dimensional (2D) allotrope of tin with low buckled honeycomb structure named stanene is proposed to be an ideal 2D topological insulator with a nontrivial gap larger than 0.1 eV. Theoretical works also pointed out the topological property of stanene amenability to strain tuning. In this paper we report the successful realization of high quality, monolayer stanene film as well as monolayer stanene nanoribbons on Sb(111) surface by molecular-beam epitaxy, providing an ideal platform to the study of stanene. More importantly, we observed a continuous evolution of the electronic bands of stanene across the nanoribbon, related to the strain field gradient in stanene. Our work experimentally confirmed that strain is an effective method for band engineering in stanene, which is important for fundamental research and application of stanene.

Computational methods in metabolic engineering for strain design.

PubMed

Long, Matthew R; Ong, Wai Kit; Reed, Jennifer L

2015-08-01

Metabolic engineering uses genetic approaches to control microbial metabolism to produce desired compounds. Computational tools can identify new biological routes to chemicals and the changes needed in host metabolism to improve chemical production. Recent computational efforts have focused on exploring what compounds can be made biologically using native, heterologous, and/or enzymes with broad specificity. Additionally, computational methods have been developed to suggest different types of genetic modifications (e.g. gene deletion/addition or up/down regulation), as well as suggest strategies meeting different criteria (e.g. high yield, high productivity, or substrate co-utilization). Strategies to improve the runtime performances have also been developed, which allow for more complex metabolic engineering strategies to be identified. Future incorporation of kinetic considerations will further improve strain design algorithms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigation of Clostridium botulinum group III's mobilome content.

PubMed

Woudstra, Cédric; Le Maréchal, Caroline; Souillard, Rozenn; Anniballi, Fabrizio; Auricchio, Bruna; Bano, Luca; Bayon-Auboyer, Marie-Hélène; Koene, Miriam; Mermoud, Isabelle; Brito, Roseane B; Lobato, Francisco C F; Silva, Rodrigo O S; Dorner, Martin B; Fach, Patrick

2018-02-01

Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Excessive by-product formation: A key contributor to low isobutanol yields of engineered Saccharomyces cerevisiae strains.

PubMed

Milne, N; Wahl, S A; van Maris, A J A; Pronk, J T; Daran, J M

2016-12-01

It is theoretically possible to engineer Saccharomyces cerevisiae strains in which isobutanol is the predominant catabolic product and high-yielding isobutanol-producing strains are already reported by industry. Conversely, isobutanol yields of engineered S. cerevisiae strains reported in the scientific literature typically remain far below 10% of the theoretical maximum. This study explores possible reasons for these suboptimal yields by a mass-balancing approach. A cytosolically located, cofactor-balanced isobutanol pathway, consisting of a mosaic of bacterial enzymes whose in vivo functionality was confirmed by complementation of null mutations in branched-chain amino acid metabolism, was expressed in S. cerevisiae . Product formation by the engineered strain was analysed in shake flasks and bioreactors. In aerobic cultures, the pathway intermediate isobutyraldehyde was oxidized to isobutyrate rather than reduced to isobutanol. Moreover, significant concentrations of the pathway intermediates 2,3-dihydroxyisovalerate and α-ketoisovalerate, as well as diacetyl and acetoin, accumulated extracellularly. While the engineered strain could not grow anaerobically, micro-aerobic cultivation resulted in isobutanol formation at a yield of 0.018±0.003 mol/mol glucose. Simultaneously, 2,3-butanediol was produced at a yield of 0.649±0.067 mol/mol glucose. These results identify massive accumulation of pathway intermediates, as well as overflow metabolites derived from acetolactate, as an important, previously underestimated contributor to the suboptimal yields of 'academic' isobutanol strains. The observed patterns of by-product formation is consistent with the notion that in vivo activity of the iron-sulphur-cluster-requiring enzyme dihydroxyacid dehydratase is a key bottleneck in the present and previously described 'academic' isobutanol-producing yeast strains.

Special Concerns for Seniors: Clostridium difficile

MedlinePlus

... and Drugs" Home | Contact Us Special Concerns for Seniors Clostridium difficile - an introduction Clostridium difficile (“C. diff”) ... see APUA’s contribution to CDC’s Vital Signs campaign . Seniors are especially at risk People over the age ...

CRISPR Diversity and Microevolution in Clostridium difficile.

PubMed

Andersen, Joakim M; Shoup, Madelyn; Robinson, Cathy; Britton, Robert; Olsen, Katharina E P; Barrangou, Rodolphe

2016-09-19

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Crystallization engineering as a route to epitaxial strain control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akbashev, Andrew R.; Plokhikh, Aleksandr V.; Barbash, Dmitri

2015-10-01

The controlled synthesis of epitaxial thin films offers opportunities for tuning their functional properties via enabling or suppressing strain relaxation. Examining differences in the epitaxial crystallization of amorphous oxide films, we report on an alternate, low-temperature route for strain engineering. Thin films of amorphous Bi–Fe–O were grown on (001)SrTiO{sub 3} and (001)LaAlO{sub 3} substrates via atomic layer deposition. In situ X-ray diffraction and X-ray photoelectron spectroscopy studies of the crystallization of the amorphous films into the epitaxial (001)BiFeO{sub 3} phase reveal distinct evolution profiles of crystallinity with temperature. While growth on (001)SrTiO{sub 3} results in a coherently strained film, themore » same films obtained on (001)LaAlO{sub 3} showed an unstrained, dislocation-rich interface, with an even lower temperature onset of the perovskite phase crystallization than in the case of (001)SrTiO{sub 3}. Our results demonstrate how the strain control in an epitaxial film can be accomplished via its crystallization from the amorphous state.« less

Fully printable, strain-engineered electronic wrap for customizable soft electronics

PubMed Central

Byun, Junghwan; Lee, Byeongmoon; Oh, Eunho; Kim, Hyunjong; Kim, Sangwoo; Lee, Seunghwan; Hong, Yongtaek

2017-01-01

Rapid growth of stretchable electronics stimulates broad uses in multidisciplinary fields as well as industrial applications. However, existing technologies are unsuitable for implementing versatile applications involving adaptable system design and functions in a cost/time-effective way because of vacuum-conditioned, lithographically-predefined processes. Here, we present a methodology for a fully printable, strain-engineered electronic wrap as a universal strategy which makes it more feasible to implement various stretchable electronic systems with customizable layouts and functions. The key aspects involve inkjet-printed rigid island (PRI)-based stretchable platform technology and corresponding printing-based automated electronic functionalization methodology, the combination of which provides fully printed, customized layouts of stretchable electronic systems with simplified process. Specifically, well-controlled contact line pinning effect of printed polymer solution enables the formation of PRIs with tunable thickness; and surface strain analysis on those PRIs leads to the optimized stability and device-to-island fill factor of strain-engineered electronic wraps. Moreover, core techniques of image-based automated pinpointing, surface-mountable device based electronic functionalizing, and one-step interconnection networking of PRIs enable customized circuit design and adaptable functionalities. To exhibit the universality of our approach, multiple types of practical applications ranging from self-computable digital logics to display and sensor system are demonstrated on skin in a customized form. PMID:28338055

Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system.

PubMed

Chen, Jianming; Rood, Julian I; McClane, Bruce A

2011-01-01

Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the

Direct observation of local magnetic properties in strain engineered lanthanum cobaltate thin films

NASA Astrophysics Data System (ADS)

Park, S.; Wu, Weida; Freeland, J. W.; Ma, J. X.; Shi, J.

2009-03-01

Strain engineered thin film devices with emergent properties have significant impacts on both technical application and material science. We studied strain-induced modification of magnetic properties (Co spin state) in epitaxially grown lanthanum cobaltate (LaCoO3) thin films with a variable temperature magnetic force microscopy (VT-MFM). The real space observation confirms long range magnetic ordering on a tensile-strained film and non-magnetic low-spin configuration on a low-strained film at low temperature. Detailed study of local magnetic properties of these films under various external magnetic fields will be discussed. Our results also demonstrate that VT-MFM is a very sensitive tool to detect the nanoscale strain induced magnetic defects.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum.

PubMed

Oguntimein, Gbekeloluwa B; Rodriguez, Miguel; Dumitrache, Alexandru; Shollenberger, Todd; Decker, Stephen R; Davison, Brian H; Brown, Steven D

2018-02-01

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

DOE PAGES

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru; ...

2017-11-09

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic engineering of strains: from industrial-scale to lab-scale chemical production.

PubMed

Sun, Jie; Alper, Hal S

2015-03-01

A plethora of successful metabolic engineering case studies have been published over the past several decades. Here, we highlight a collection of microbially produced chemicals using a historical framework, starting with titers ranging from industrial scale (more than 50 g/L), to medium-scale (5-50 g/L), and lab-scale (0-5 g/L). Although engineered Escherichia coli and Saccharomyces cerevisiae emerge as prominent hosts in the literature as a result of well-developed genetic engineering tools, several novel native-producing strains are gaining attention. This review catalogs the current progress of metabolic engineering towards production of compounds such as acids, alcohols, amino acids, natural organic compounds, and others.

Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications

PubMed Central

Freedman, John C.; Shrestha, Archana; McClane, Bruce A.

2016-01-01

Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B ▿ †

PubMed Central

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

Control wafer bow of InGaP on 200 mm Si by strain engineering

NASA Astrophysics Data System (ADS)

Wang, Bing; Bao, Shuyu; Made, Riko I.; Lee, Kwang Hong; Wang, Cong; Eng Kian Lee, Kenneth; Fitzgerald, Eugene A.; Michel, Jurgen

2017-12-01

When epitaxially growing III-V compound semiconductors on Si substrates the mismatch of coefficients of thermal expansion (CTEs) between III-V and Si causes stress and wafer bow. The wafer bow is deleterious for some wafer-scale processing especially when the wafer size is large. Strain engineering was applied in the epitaxy of InGaP films on 200 mm silicon wafers having high quality germanium buffers. By applying compressive strain in the InGaP films to compensate the tensile strain induced by CTE mismatch, wafer bow was decreased from about 100 μm to less than 50 μm. X-ray diffraction studies show a clear trend between the decrease of wafer bow and the compensation of CTE mismatch induced tensile strain in the InGaP layers. In addition, the anisotropic strain relaxation in InGaP films resulted in anisotropic wafer bow along two perpendicular (110) directions. Etch pit density and plane-view transmission electron microscopy characterizations indicate that threading dislocation densities did not change significantly due to the lattice-mismatch applied in the InGaP films. This study shows that strain engineering is an effective method to control wafer bow when growing III-V semiconductors on large size Si substrates.

Epidemiology of Clostridium difficile infection.

PubMed

Depestel, Daryl D; Aronoff, David M

2013-10-01

There has been dramatic change in the epidemiology of Clostridium difficile infection (CDI) since the turn of the 21st century noted by a marked increase in incidence and severity, occurring at a disproportionately higher frequency in older patients. Historically considered a nosocomial infection associated with antibiotic exposure, CDI has now also emerged in the community in populations previously considered low risk. Emerging risk factors and disease recurrence represent continued challenges in the management of CDI. The increased incidence and severity associated with CDI has coincided with the emergence and rapid spread of a previously rare strain, ribotype 027. Recent data from the United States and Europe suggest that the incidence of CDI may have reached a crescendo in the recent years and is perhaps beginning to plateau. The acute care direct costs of CDI were estimated to be US$4.8 billion in 2008. However, nearly all the published studies have focused on CDI diagnosed and treated in the acute care hospital setting and fail to measure the burden outside the hospital, including recently discharged patients, outpatients, and those in long-term care facilities. Enhanced surveillance methods are needed to monitor the incidence, to identify populations at risk, and to characterize the molecular epidemiology of strains causing CDI.

Epidemiology of Clostridium difficile Infection

PubMed Central

DePestel, Daryl D.; Aronoff, David M.

2014-01-01

There has been dramatic change in the epidemiology of Clostridium difficile infection (CDI) since the turn of the 21st Century noted by a marked increase in incidence and severity, occurring at a disproportionately higher frequency in older patients. Historically considered a nosocomial infection associated with antibiotic exposure, CDI has now also emerged in the community in populations previously considered low risk. Emerging risk factors and disease recurrence represent continued challenges in the management of CDI. The increased incidence and severity associated with CDI has coincided with the emergence and rapid spread of a previously rare strain, ribotype 027. Recent data from the U.S. and Europe suggest the incidence of CDI may have reached a crescendo in recent years and is perhaps beginning to plateau. The acute-care direct costs of CDI were estimated to be $4.8 billion in 2008. However, nearly all the published studies have focused on CDI diagnosed and treated in acute-care hospital setting and fail to measure the burden outside the hospital, including recently discharged patients, outpatients, and those in long-term care facilities. Enhanced surveillance methods are needed to monitor the incidence, identify populations at risk, and characterize the molecular epidemiology of strains causing CDI. PMID:24064435

Epidemiology of Clostridium difficile in infants in Oxfordshire, UK: Risk factors for colonization and carriage, and genetic overlap with regional C. difficile infection strains.

PubMed

Stoesser, Nicole; Eyre, David W; Quan, T Phuong; Godwin, Heather; Pill, Gemma; Mbuvi, Emily; Vaughan, Alison; Griffiths, David; Martin, Jessica; Fawley, Warren; Dingle, Kate E; Oakley, Sarah; Wanelik, Kazimierz; Finney, John M; Kachrimanidou, Melina; Moore, Catrin E; Gorbach, Sherwood; Riley, Thomas V; Crook, Derrick W; Peto, Tim E A; Wilcox, Mark H; Walker, A Sarah

2017-01-01

Approximately 30-40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized. Predominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases. 338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0-10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%). In predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic overlap between some infant strains and

Epidemiology of Clostridium difficile in infants in Oxfordshire, UK: Risk factors for colonization and carriage, and genetic overlap with regional C. difficile infection strains

PubMed Central

Godwin, Heather; Pill, Gemma; Mbuvi, Emily; Vaughan, Alison; Griffiths, David; Martin, Jessica; Fawley, Warren; Dingle, Kate E.; Oakley, Sarah; Wanelik, Kazimierz; Finney, John M.; Kachrimanidou, Melina; Moore, Catrin E.; Gorbach, Sherwood; Riley, Thomas V.; Crook, Derrick W.; Peto, Tim E. A.; Wilcox, Mark H.; Walker, A. Sarah

2017-01-01

Background Approximately 30–40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized. Methods Predominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases. Results 338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0–10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%). Conclusions In predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic

Emerging therapies for Clostridium difficile infections.

PubMed

McFarland, Lynne V

2011-09-01

Clostridium difficile infection (CDI) is the leading identifiable gastrointestinal disease in healthcare institutions, but the response rates to the two standard therapies for CDI are declining and so innovative therapies are being developed for CDI. The purpose of this paper is to review the data on the efficacy and safety of emerging therapies for CDI and assess their potential for effectiveness based on the clinical phase of development and marketing challenges. Emerging therapies for CDI are reviewed including new antibiotics, peptides, immune regulators, probiotics and toxin binders. PubMed, Medline and Google Scholar and online clinical trial registers are searched from 1976 to 2010 for articles unrestricted by language. Secondary searches by author, manufacturing companies and FDA websites are also performed. Of the emerging therapies for CDI, several may ultimately reduce the incidence of CDI and the economic burden of this disease on the healthcare system. Several emerging treatments (fidaxomicin, rifaximin and mAbs) show the most promise, although only one is currently being actively developed. Use of other clostridial strains, probiotic strains and immune enhancers have great potential as therapies, but require further development.

Strain-engineered optoelectronic properties of 2D transition metal dichalcogenide lateral heterostructures

DOE PAGES

Lee, Jaekwang; Huang, Jingsong; Sumpter, Bobby G.; ...

2017-02-17

Compared with their bulk counterparts, 2D materials can sustain much higher elastic strain at which optical quantities such as bandgaps and absorption spectra governing optoelectronic device performance can be modified with relative ease. Using first-principles density functional theory and quasiparticle GW calculations, we demonstrate how uniaxial tensile strain can be utilized to optimize the electronic and optical properties of transition metal dichalcogenide lateral (in-plane) heterostructures such as MoX 2/WX 2 (X = S, Se, Te). We find that these lateral-type heterostructures may facilitate efficient electron–hole separation for light detection/harvesting and preserve their type II characteristic up to 12% of uniaxialmore » strain. Based on the strain-dependent bandgap and band offset, we show that uniaxial tensile strain can significantly increase the power conversion efficiency of these lateral heterostructures. Our results suggest that these strain-engineered lateral heterostructures are promising for optimizing optoelectronic device performance by selectively tuning the energetics of the bandgap.« less

Effect of continuous sub-culturing on infectivity of Clostridium perfringens ATCC13124 in mouse gas gangrene model.

PubMed

Kumar, Ravi Bhushan; Alam, Syed Imteyaz

2017-07-01

Clostridium perfringens is a Validated Biological Agent and a pathogen of medical, veterinary, and military significance. Gas gangrene is the most destructive of all the clostridial diseases and is caused by C. perfringens type A strains wherein the infection spreads quickly (several inches per hour) with production of gas. Influence of repeated in vitro cultivation on the infectivity of C. perfringens was investigated by comparing the surface proteins of laboratory strain and repository strains of the bacterium using 2DE-MS approach. In order to optimize host-pathogen interaction during experimental gas gangrene infection, we also explored the role of particulate matrix on ability of C. perfringens to cause gas gangrene.

Spirotetronate antibiotics with anti-Clostridium activity from Actinomadura sp. 2EPS.

PubMed

Euanorasetr, Jirayut; Intra, Bungonsiri; Mongkol, Phayungsak; Chankhamhaengdecha, Surang; Tuchinda, Patoomratana; Mori, Mihoko; Shiomi, Kazuro; Nihira, Takuya; Panbangred, Watanalai

2015-02-01

The rare actinomycetes strain 2EPS was isolated from soil and analysis of cultural, morphological characteristics, diaminopimelic acid content of its cell wall, and 16S rRNA gene sequence indicates that 2EPS belongs to genus Actinomadura. In addition, neighbor-joining phylogenetic tree also confirmed the relationships of this strain to other members of Actinomadura. A butanol extract with antibacterial activity was purified by reversed-phase chromatography to obtain three bioactive compounds, designated as compounds 1, 2 and 3. The structures of these compounds were determined using spectroscopic analysis ((1)H-NMR and (13)C-NMR) and mass spectrometric analysis (HR-TOF-MS). Compounds 1-3 were identified and found to be the same as those included in the Japanese patent number JP 09227587 for spirotetronate antibiotics and are BE-45722A (1), BE-45722B (2) and BE-45722C (3), respectively. All compounds were active against Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579, and B. subtilis ATCC 6633) with low MIC values between 0.08 and 5.0 µg/ml. Moreover, both 1 and 3 also exhibited strong activity, with similar MIC values, against Clostridium perfringens S107 at 0.63 µg/ml and C. difficile 630 at 0.08 µg/ml. These results suggest the identified spirotetronate compounds may have potential in the treatment of Clostridium infections. Overall, this analysis demonstrates that rare actinomycetes are a promising source for discovery of antimicrobial compounds.

Isolation of Clostridium perfringens type A from wild bharals (Pseudois nayaur) following sudden death in Tibet, China.

PubMed

Zhu, Lingwei; Zhou, Wei; Wang, Tiecheng; Xiang, Haiyang; Ji, Xue; Han, Yixiao; Tian, Yuan; Sun, Yang; Liu, Jun; Guo, Xuejun

2017-04-01

Dozens of wild bharals died suddenly in Tibet. Necropsy showed severe congestion and hemorrhage in multiple organs, with large numbers of Gram-positive bacilli. Strains of Clostridium perfringens type A were isolated from the different organs and the intestinal contents. The other possible pathogens were ruled out by PCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production

PubMed Central

Hawkins, Aaron B.; Lian, Hong; Zeldes, Benjamin M.; Loder, Andrew J.; Lipscomb, Gina L.; Schut, Gerrit J.; Keller, Matthew W.; Adams, Michael W.W.; Kelly, Robert M.

2015-01-01

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus(Topt 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/hr to 11 mg/L/hr, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures. PMID:25753826

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain.

PubMed

Song, Hyohak; Huh, Yun Suk; Lee, Sang Yup; Hong, Won Hi; Hong, Yeon Ki

2007-12-01

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.

Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection.

PubMed

Sun, Ping; Liang, Jing-Long; Kang, Lin-Zhi; Huang, Xiao-Yan; Huang, Jia-Jun; Ye, Zhi-Wei; Guo, Li-Qiong; Lin, Jun-Fang

2015-01-01

Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4-coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p-coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans-resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermentation. © 2015 American Institute of Chemical Engineers.

Escherichia coli strains engineered for homofermentative production of D-lactic acid from glycerol.

PubMed

Mazumdar, Suman; Clomburg, James M; Gonzalez, Ramon

2010-07-01

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (DeltapflB) and fumarate reductase (DeltafrdA) (strain LA01) and (ii) inactivation of fumarate reductase (DeltafrdA), phosphate acetyltransferase (Deltapta), and alcohol/acetaldehyde dehydrogenase (DeltaadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Deltadld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Deltadld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.

Clostridium difficile associated diarrhoea: An increased problem.

PubMed

Urbina Soto, Leticia; García Ávila, Sara; Córdoba Alonso, Ana Isabel; Roiz Mesones, M Pía; Arnaiz García, Ana M; Valero Díaz de Lamadrid, M Carmen

2016-12-16

Clostridium difficile associated diarrhoea is a major health problem that seems to be on the increase. In our study, we analyse the changes in the incidence of this infection over the last 11 years. A descriptive study in hospitalised patients with Clostridium difficile associated diarrhoea in University Hospital Marqués de Valdecilla (Santander, Spain) from 2004 to 2014. A total of 244 adults were identified [53% men; 66 (SD 15) years]. The cases of nosocomial acquisition (80%), with respect to community acquired Clostridium difficile infection, were older [67 (SD 15) years vs. 63 (19) years; P=.01), high comorbidity (86% vs. 75%; P=.01), use of antibiotics (95% vs. 75%; P<.001) and proton pump inhibitors (87% vs. 48% P<.001). There has been an increasing incidence of Clostridium difficile associated diarrhoea in our hospital over an 11-year period. The clinical profile of patients with Clostridium difficile diarrhoea varies by place of acquisition of infection. The prevalence of this disease is increasing. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Antibacterial activity against Clostridium genus and antiradical activity of the essential oils from different origin.

PubMed

Kačániová, Miroslava; Vukovič, Nenad; Horská, Elena; Salamon, Ivan; Bobková, Alica; Hleba, Lukáš; Fiskelová, Martina; Vatľák, Alexander; Petrová, Jana; Bobko, Marek

2014-01-01

In the present study, the antimicrobial and antiradical activities of 15 essential oils were investigated. The antimicrobial activities were determined by using agar disc diffusion and broth microdilution methods against Clostridium genus and antioxidant properties of essential oils by testing their scavenging effect on DPPH radicals activities. We determined the antibacterial activity of Clostridium butyricum, Clostridium hystoliticum, Clostridium intestinale, Clostridium perfringens and Clostridium ramosum. We obtained the original commercial essential oils samples of Lavandula angustifolia, Carum carvi, Pinus montana, Mentha piperita, Foeniculum vulgare Mill., Pinus sylvestris, Satureia montana, Origanum vulgare L. (2 samples), Pimpinella anisum, Rosmarinus officinalis L., Salvia officinalis L., Abies alba Mill., Chamomilla recutita L. Rausch and Thymus vulgaris L. produced in Slovakia (Calendula a.s., Nova Lubovna, Slovakia). The results of the disk diffusion method showed very high essential oils activity against all tested strains of microorganisms. The best antimicrobial activity against C. butyricum was found at Pimpinella anisum, against C. hystoliticum was found at Pinus sylvestris, against C. intestinale was found at Satureia hortensis L., against C. perfringens was found at Origanum vulgare L. and against C. ramosum was found at Pinus sylvestris. The results of broth microdilution assay showed that none of the essential oils was active against C. hystoliticum. The best antimicrobial activity against C. butyricum was found at Abies alba Mill., against C. intestinale was found at Abies alba Mill., against C. perfringens was found at Satureia montana and against C. ramosum was found at Abius alba and Carum carvi. Antioxidant DPPH radical scavenging activity was determined at several solutions of oil samples (50 μL.mL(-1)-0.39 μL.mL(-1)) and the best scavenging effect for the highest concentration (50 μL.mL(-1)) was observed. The antioxidant properties

Clostridium difficile infection in the twenty-first century

PubMed Central

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

A mesophilic Clostridium species that produces butanol from monosaccharides and hydrogen from polysaccharides.

PubMed

Bramono, Sandhi Eko; Lam, Yuen Sean; Ong, Say Leong; He, Jianzhong

2011-10-01

A unique mesophilic Clostridium species strain BOH3 is obtained in this study, which is capable of fermenting monosaccharides to produce butanol and hydrolyzing polysaccharides to produce hydrogen (H(2)) and volatile fatty acids (VFAs). From 30 g/L of glucose and xylose each, batch culture BOH3 was able to produce 4.67 and 4.63 g/L of butanol. Enhancement treatments by increasing the inoculated cells improved butanol production to 7.05 and 7.41 g/L, respectively. Hydrogen production (2.47 and 1.93 mmol) was observed when cellulose and xylan (10 g/L each) were used, suggesting that strain BOH3 possesses xylanolytic and cellulolytic capabilities. These unique features reveal the strain's novelty as most wild-type solventogenic strains have not been reported to have such properties. Therefore, culture BOH3 is promising in generating butanol and hydrogen from renewable feedstock. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Extending the spectral range of CdSe/ZnSe quantum wells by strain engineering

NASA Astrophysics Data System (ADS)

Finke, A.; Ruth, M.; Scholz, S.; Ludwig, A.; Wieck, A. D.; Reuter, D.; Pawlis, A.

2015-01-01

We demonstrate efficient room-temperature photoluminescence and spectral tuning of epitaxially grown ZnSe/CdSe quantum well structures almost over the whole visible spectrum (470-600 nm wavelength). The key element to achieve the observed high quantum efficiency and enormous tuning range was the implementation of a special strain engineering technique, which allows us to suppress substantial lattice relaxation of CdSe on ZnSe. Previous studies indicated that a CdSe coverage exceeding 3 ML on ZnSe results in the formation of extensive lattice defects and complete quenching of the photoluminescence at low and room temperature. In contrast, our approach of strain engineering enables the deposition of planar CdSe quantum wells with a thickness ranging from 1 to 6 ML with excellent optical properties. We attribute the observed experimental features to a controllable strain compensation effect that is present in an alternating system of tensile and compressively strained epitaxial layers and supported this model by calculations of the transition energies of the ZnSe/CdSe quantum wells.

Antimonide-based membranes synthesis integration and strain engineering

PubMed Central

Anwar, Farhana; Klein, Brianna A.; Rasoulof, Amin; Dawson, Noel M.; Schuler-Sandy, Ted; Deneke, Christoph F.; Ferreira, Sukarno O.; Cavallo, Francesca; Krishna, Sanjay

2017-01-01

Antimonide compounds are fabricated in membrane form to enable materials combinations that cannot be obtained by direct growth and to support strain fields that are not possible in the bulk. InAs/(InAs,Ga)Sb type II superlattices (T2SLs) with different in-plane geometries are transferred from a GaSb substrate to a variety of hosts, including Si, polydimethylsiloxane, and metal-coated substrates. Electron microscopy shows structural integrity of transferred membranes with thickness of 100 nm to 2.5 μm and lateral sizes from 24×24μm2 to 1×1 cm2. Electron microscopy reveals the excellent quality of the membrane interface with the new host. The crystalline structure of the T2SL is not altered by the fabrication process, and a minimal elastic relaxation occurs during the release step, as demonstrated by X-ray diffraction and mechanical modeling. A method to locally strain-engineer antimonide-based membranes is theoretically illustrated. Continuum elasticity theory shows that up to ∼3.5% compressive strain can be induced in an InSb quantum well through external bending. Photoluminescence spectroscopy and characterization of an IR photodetector based on InAs/GaSb bonded to Si demonstrate the functionality of transferred membranes in the IR range. PMID:27986953

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly.

PubMed

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

Observations on the distribution and ecology of Clostridium botulinum type E in Alaska.

PubMed

Miller, L G

1975-06-01

Environmental samples collected along the coastline and from the interior of Alaska were examined for the presence of Clostridium botulinum. Clostridium botulinum type E was detected in soils from 5 of 12 beaches; in 7 of 115 non-coastal soil samples; in sediments from six of eight locales; in gills of salmon from two fishing areas; and in the feces of 1 of 44 colonic samples from marine mammals. The basic biochemical characteristics of the isolates were determined. Tube tests for demonstrating gelatin liquefaction proved insensitive with these strains, whereas a plate test detected gelatinase in all isolates. The presence of multiple nidi and the continual discharge of organic materials into the environment may contribute to the perpetuation of botulinum spores by which foods prepared form marine animals become contaminated. An emphasis should be placed upon the need for measures to reduce environmental contamination, to reduce contamination during food preparation, and to alert continually the population of the hazard wherever botulism is endemic.

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin.

PubMed

Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Oda, Masataka; Sakaguchi, Yoshihiko; Hisatsune, Junzo; Ochi, Sadayuki; Kobayashi, Keiko; Nagahama, Masahiro

2017-08-11

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

Strain response of thermal barrier coatings captured under extreme engine environments through synchrotron X-ray diffraction

NASA Astrophysics Data System (ADS)

Knipe, Kevin; Manero, Albert; Siddiqui, Sanna F.; Meid, Carla; Wischek, Janine; Okasinski, John; Almer, Jonathan; Karlsson, Anette M.; Bartsch, Marion; Raghavan, Seetha

2014-07-01

The mechanical behaviour of thermal barrier coatings in operation holds the key to understanding durability of jet engine turbine blades. Here we report the results from experiments that monitor strains in the layers of a coating subjected to thermal gradients and mechanical loads representing extreme engine environments. Hollow cylindrical specimens, with electron beam physical vapour deposited coatings, were tested with internal cooling and external heating under various controlled conditions. High-energy synchrotron X-ray measurements captured the in situ strain response through the depth of each layer, revealing the link between these conditions and the evolution of local strains. Results of this study demonstrate that variations in these conditions create corresponding trends in depth-resolved strains with the largest effects displayed at or near the interface with the bond coat. With larger temperature drops across the coating, significant strain gradients are seen, which can contribute to failure modes occurring within the layer adjacent to the interface.

Evaluation of a commercial latex test for Clostridium difficile for reactivity with C. difficile and cross-reactions with other bacteria.

PubMed Central

Miles, B L; Siders, J A; Allen, S D

1988-01-01

Seventy-eight species of bacteria (739 isolates) were tested for reactivity with a commercial latex test for Clostridium difficile. All noncytotoxic as well as cytotoxic strains of C. difficile reacted positively. Immuno-specific cross-reactions were found only with C. sporogenes, proteolytic C. botulinum, and Peptostreptococcus anaerobius. PMID:3235677

Human alpha-defensin-1 protects cells from intoxication with Clostridium perfringens iota toxin.

PubMed

Fischer, Stephan; Popoff, Michel R; Barth, Holger

2018-03-01

Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.

A case of multiple recurrence of Clostridium difficile infection with severe hematochezia in an immunocompromised host.

PubMed

Zhang, Xuewu; Chen, Yunbo; Gu, Silan; Zheng, Beiwen; Lv, Tao; Lou, Yinjun; Jin, Jie

2016-12-01

Clostridium difficile infection (CDI) is increasing in incidence and severity. Clinically, diarrhea frequently occurs, but severe hematochezia is rarely seen with CDI. We describe here a hematopoietic stem cell transplantation (HSCT) recipient who experienced life-threatening gastrointestinal bleeding due to severe CDI. Subsequent stool surveillance and molecular typing observed the patient who had two episodes of recurrence with a new strain of C. difficile distinct from the initial infection. We analyze C. difficile strains obtained from the patient, and also discuss the diagnosis and treatment of this case. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO 2-based 3-hydroxypropionate production

DOE PAGES

Hawkins, Aaron B.; Lian, Hong; Zeldes, Benjamin M.; ...

2015-06-11

In this paper, metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T opt 95–100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO 2 using enzymes from the Metallosphaera sedula (T opt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formationmore » catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO 2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. Lastly, the results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.« less

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO 2-based 3-hydroxypropionate production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawkins, Aaron B.; Lian, Hong; Zeldes, Benjamin M.

In this paper, metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T opt 95–100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO 2 using enzymes from the Metallosphaera sedula (T opt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formationmore » catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO 2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. Lastly, the results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.« less

Pharmacodynamic studies of vancomycin, metronidazole and fusidic acid against Clostridium difficile.

PubMed

Odenholt, Inga; Walder, Mats; Wullt, Marlene

2007-01-01

Pharmacodynamic studies of antibiotics have attracted great interest in recent years. However, studies on the pharmacodynamics of different antibiotics against Clostridium difficile are scarce. The postantibiotic effects (PAE) and the postantibiotic sub-minimum inhibitory concentration (MIC) effects (PA SME) of vancomycin, metronidazole and fusidic acid were investigated by viable counts against three different strains of C. difficile. The killing rate and extent of the three antibiotics against the same strains were also studied by adding 2, 4, 8, 16 and 32x MIC of the three antibiotics, respectively. Metronidazole exerted a very rapid bactericidal effect at concentrations of 8x MIC and above against all three strains investigated. Vancomycin gave overall less kill in comparison to metronidazole and was bacteriostatic against two of the three strains. Fusidic acid exerted a concentration-dependent killing against two of the strains. Vancomycin exerted short PAEs and PA SMEs against all three strains. Significantly longer PAEs and PA SMEs were noted for fusidic acid. Metronidazole gave similar short PAEs like vancomycin but longer PA SMEs were noted against two of the investigated strains. Metronidazole exerted the most prominent bactericidal effect greater than fusidic acid and greater than vancomycin. Fusidic acid gave overall the longest PAEs and PA SMEs greater than metronidazole and greater than vancomycin. Copyright 2007 S. Karger AG, Basel.

Mortality and Clostridium difficile infection in an Australian setting.

PubMed

Mitchell, Brett G; Gardner, Anne; Hiller, Janet E

2013-10-01

To quantify the risk of death associated with Clostridium difficile infection, in an Australian tertiary hospital. Two reviews examining Clostridium difficile infection and mortality indicate that Clostridium difficile infection is associated with increased mortality in hospitalized patients. Studies investigating the mortality of Clostridium difficile infection in settings outside of Europe and North America are required, so that the epidemiology of Clostridium difficile infection in these regions can be understood and appropriate prevention strategies made. An observational non-concurrent cohort study design was used. Data from all persons who had (exposed) and a matched sample of persons who did not have Clostridium difficile infection, for the calendar years 2007-2010, were analysed. The risk of dying within 30, 60, 90 and 180 days was compared using the two groups. Kaplan-Meier survival analysis and conditional logistic regression models were applied to the data to examine time to death and mortality risk adjusted for comorbidities using the Charlson Comorbidity Index. One hundred and fifty-eight cases of infection were identified. A statistically significant difference in all-cause mortality was identified between exposed and non-exposed groups at 60 and 180 days. In a conditional regression model, mortality in the exposed group was significantly higher at 180 days. In this Australian study, Clostridium difficile infection was associated with increased mortality. In doing so, it highlights the need for nurses to immediately instigate contact precautions for persons suspected of having Clostridium difficile infection and to facilitate a timely faecal collection for testing. Our findings support ongoing surveillance of Clostridium difficile infection and associated prevention and control activities. © 2013 Blackwell Publishing Ltd.

Wrinkling instability in nanoparticle-supported graphene: implications for strain engineering

NASA Astrophysics Data System (ADS)

Cullen, William; Yamamoto, Mahito; Pierre-Louis, Olivier; Huang, Jia; Fuhrer, Michael; Einstein, Theodore

2013-03-01

We have carried out a systematic study of the wrinkling instability of graphene membranes supported on SiO2 substrates with randomly placed silica nanoparticles. At small nanoparticle density, monolayer graphene adheres to the substrate and is highly conformal over the nanoparticles. With increasing nanoparticle density, and decreasing nanoparticle separation to ~100 nm, graphene's elastic response dominates substrate adhesion, and elastic stretching energy is reduced by the formation of wrinkles which connect protrusions. Above a critical nanoparticle density, the wrinkles form a percolating network through the sample. As the graphene membrane is made thicker, delamination from the substrate is observed. Since the wrinkling instability acts to remove inhomogeneous in-plane elastic strains through out-of-plane buckling, our results can be used to place limits on the possible in-plane strain magnitudes that may be created in graphene to realized strain-engineered electronic structures.[2] Supported by the UMD NSF-MRSEC under Grant No. DMR 05-20471, the US ONR MURI and UMD CNAM.

Clostridium subterminale septicemia in an immunocompetent patient.

PubMed

Daganou, Maria; Kyriakoudi, Ann; Moraitou, Helen; Pontikis, Konstantinos; Avgeropoulou, Stavrina; Tripolitsioti, Paraskevi; Koutsoukou, Antonia

2016-01-01

Clostridium subterminale is a Clostridium species that has been rarely isolated in the blood of immunocompromised patients. We report a case of C. subterminale septicemia in an immunocompetent patient who presented with acute mediastinitis following spontaneous esophageal rupture.

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Oda, Masataka; Sakaguchi, Yoshihiko; Hisatsune, Junzo; Ochi, Sadayuki; Kobayashi, Keiko; Nagahama, Masahiro

2017-01-01

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins. PMID:28800062

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

PubMed Central

Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

2015-01-01

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718.

PubMed

Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A

2011-12-01

Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.

CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

DOE Office of Scientific and Technical Information (OSTI.GOV)

SWAMINATHAN,S.; ESWARAMOORTHY,S.

2001-11-19

The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxinsmore » are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].« less

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation.

PubMed

Liu, Xiao-Bo; Gu, Qiu-Ya; Yu, Xiao-Bin; Luo, Wei

2012-12-01

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

PubMed

Veetil, Vinod Puthan; Angermayr, S Andreas; Hellingwerf, Klaas J

2017-02-23

Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene. This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background. Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

PubMed

Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

2016-06-20

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.

Retaining large and adjustable elastic strains of kilogram-scale Nb nanowires [Better Superconductor by Elastic Strain Engineering: Kilogram-scale Free-Standing Niobium Metal Composite with Large Retained Elastic Strains

DOE PAGES

Hao, Shijie; Cui, Lishan; Wang, Hua; ...

2016-02-10

Crystals held at ultrahigh elastic strains and stresses may exhibit exceptional physical and chemical properties. Individual metallic nanowires can sustain ultra-large elastic strains of 4-7%. However, retaining elastic strains of such magnitude in kilogram-scale nanowires is challenging. Here, we find that under active load, ~5.6% elastic strain can be achieved in Nb nanowires in a composite material. Moreover, large tensile (2.8%) and compressive (-2.4%) elastic strains can be retained in kilogram-scale Nb nanowires when the composite is unloaded to a free-standing condition. It is then demonstrated that the retained tensile elastic strains of Nb nanowires significantly increase their superconducting transitionmore » temperature and critical magnetic fields, corroborating ab initio calculations based on BCS theory. This free-standing nanocomposite design paradigm opens new avenues for retaining ultra-large elastic strains in great quantities of nanowires and elastic-strain-engineering at industrial scale.« less

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

PubMed

Irikura, Daisuke; Monma, Chie; Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

2015-01-01

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

PubMed

Zhang, Rong; Xu, Xingjian; Chen, Wenli; Huang, Qiaoyun

2016-02-01

A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

Fatal neutropenic enterocolitis due to clostridium septicum.

PubMed

Shah, B K; KC, R

2011-10-01

We describe a case of Clostridium septicum enterocolitis in a patient with pre-B acute lymphoblastic leukaemia undergoing autologous stem cell transplant. In the setting of neutropenia, Clostridium septicum should be suspected in patients who develop signs and symptoms of acute abdomen.

Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

PubMed

Romaní, Aloia; Pereira, Filipa; Johansson, Björn; Domingues, Lucília

2015-03-01

In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield. Copyright © 2014 Elsevier Ltd. All rights reserved.

Coherent, atomically thin transition-metal dichalcogenide superlattices with engineered strain

NASA Astrophysics Data System (ADS)

Xie, Saien; Tu, Lijie; Han, Yimo; Huang, Lujie; Kang, Kibum; Lao, Ka Un; Poddar, Preeti; Park, Chibeom; Muller, David A.; DiStasio, Robert A.; Park, Jiwoong

2018-03-01

Epitaxy forms the basis of modern electronics and optoelectronics. We report coherent atomically thin superlattices in which different transition metal dichalcogenide monolayers—despite large lattice mismatches—are repeated and laterally integrated without dislocations within the monolayer plane. Grown by an omnidirectional epitaxy, these superlattices display fully matched lattice constants across heterointerfaces while maintaining an isotropic lattice structure and triangular symmetry. This strong epitaxial strain is precisely engineered via the nanoscale supercell dimensions, thereby enabling broad tuning of the optical properties and producing photoluminescence peak shifts as large as 250 millielectron volts. We present theoretical models to explain this coherent growth and the energetic interplay governing the ripple formation in these strained monolayers. Such coherent superlattices provide building blocks with targeted functionalities at the atomically thin limit.

Strain engineering of graphene nanoribbons: pseudomagnetic versus external magnetic fields

NASA Astrophysics Data System (ADS)

Prabhakar, Sanjay; Melnik, Roderick; Bonilla, Luis

2017-05-01

Bandgap opening due to strain engineering is a key architect for making graphene's optoelectronic, straintronic, and spintronic devices. We study the bandgap opening due to strain induced ripple waves and investigate the interplay between pseudomagnetic fields and externally applied magnetic fields on the band structures and spin relaxation in graphene nanoribbons (GNRs). We show that electron-hole bands of GNRs are highly influenced (i.e. level crossing of the bands are possible) by coupling two combined effects: pseudomagnetic fields (PMF) originating from strain tensor and external magnetic fields. In particular, we show that the tuning of the spin-splitting band extends to large externally applied magnetic fields with increasing values of pseudomagnetic fields. Level crossings of the bands in strained GNRs can also be observed due to the interplay between pseudomagnetic fields and externally applied magnetic fields. We also investigate the influence of this interplay on the electromagnetic field mediated spin relaxation mechanism in GNRs. In particular, we show that the spin hot spot can be observed at approximately B = 65 T (the externally applied magnetic field) and B0 = 53 T (the magnitude of induced pseudomagnetic field due to ripple waves) which may not be considered as an ideal location for the design of straintronic devices. Our analysis might be used for tuning the bandgaps in strained GNRs and utilized to design the optoelectronic devices for straintronic applications.

Application of serological typing to the investigation of outbreaks of Clostridium perfringens food poisoning, 1970-1978.

PubMed Central

Stringer, M. F.; Turnbull, P. C.; Gilbert, R. J.

1980-01-01

Serological typing was used as an epidemiological tool in the investigation of 524 outbreaks of Clostridium perfringens food poisoning in the United Kingdom and 37 outbreaks in other countries. Five thousand five hundred and fifty-four (77%) of 7245 strains of C. perfringens associated with the 561 outbreaks were typable with the 75 Food Hygiene Laboratory antisera; in 354 (63%) of these outbreaks a specific serotype was established as being responsible for the outbreak. An assessment is made of the ability of two additional sets of antisera, prepared against 34 American and 34 Japanese strains of C. perfringens, to increase the number of strains which can be typed. The extent of cross-reaction between the three sets of antisera was determined and the results are discussed in relation to the source and history of the type strains. PMID:6300225

Production of prebiotic-xylooligosaccharides from alkali pretreated mahogany and mango wood sawdust by using purified xylanase of Clostridium strain BOH3.

PubMed

Rajagopalan, Gobinath; Shanmugavelu, Kavitha; Yang, Kun-Lin

2017-07-01

Xylooligosaccharides (XOS) are emerging prebiotics which can be produced from lignocellulosic biomass including agro-residues and hardwood. In this study, we report the production of XOS from thermal-alkali pretreated hardwood such as mahogany and mango by using a purified xylanase from Clostridium strain BOH3. In the first approach, pure xylan is extracted from mahogany and mango hardwood and then the pure xylan is hydrolyzed by using the xylanase. In this case, 572 and 504mg XOS/g pure xylan were obtained from mahogany and mango woods, respectively. In the second approach, the same xylanase is employed to hydrolyze sawdust of hardwood after different types of pretreatments. After a thermal (121°C for 15min) pretreatment under a mild alkaline (0.05N NaOH) condition, the pretreated mahogany and mango sawdust can be utilized directly to produce 89.5 and 67.6mg XOS/g pretreated sawdust, respectively. XOS produced from the pretreated sawdust show strong prebiotic effects on Bifidobacteria and Lactobacilli. This report shows the possibility of producing XOS from pretreated woody wastes without using pure xylan as a substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

PubMed

Wu, Pengfei; Wang, Genyu; Wang, Gehua; Børresen, Børre Tore; Liu, Hongjuan; Zhang, Jianan

2016-01-14

One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models.

PubMed

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

Discrimination of clostridium species using a magnetic bead based hybridization assay

NASA Astrophysics Data System (ADS)

Pahlow, Susanne; Seise, Barbara; Pollok, Sibyll; Seyboldt, Christian; Weber, Karina; Popp, Jürgen

2014-05-01

Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5

Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains.

PubMed

Seo, Mi-Ran; Kim, Jieun; Lee, Yangsoon; Lim, Dong-Gyun; Pai, Hyunjoo

2018-05-01

Clostridium difficile infection (CDI) is a major healthcare-associated infection. The aim of this study was to investigate the genetic relatedness of the endemic C. difficile PCR ribotype 018 strains in an institution and changes to their characteristics during a five-year period. A total of 207 isolates from inpatients at Hanyang University Hospital from 2009 to 2013 were analysed using multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations (MICs) of several antibiotics were determined. In total, 204 (98.6%) were genetically related, with a summed tandem-repeat distance (STRD) ≤ 10. Minimum-spanning-tree analysis identified 78 MLVA types, categorized into six clonal complexes (CCs). The largest cluster, CC-I, included 51 MLVA types from 148 isolates (71.5%) and the second largest cluster, CC-II, included 10 MLVA types from 36 isolates (17.4%). Resistance rates for antibiotics were: clindamycin (CLI), 97.6%; moxifloxacin (MXF), 98.6%; vancomycin (VAN), 1.4%; and rifaximin (RFX), 8.2%. All isolates were susceptible to piperacillin/tazobactam (TZP) and metronidazole (MTZ). Comparing the MICs of antibiotics for the isolates each year from 2009 to 2013, MICs of antibiotics that promote CDI, such as CLI, MXF, TZP and RFX, increased over the five-year period (P-value by Kruskal-Wallis test: < 0.0001, <0.0001, <0.0001, and <0.0001 respectively); however, MICs of VAN or MTZ, antibiotics for treatment of CDI, did not increase or decreased over the same time period (P-value by Kruskal-Wallis test: 0.166, <0.0001). C. difficile RT018 isolates in a tertiary hospital over a five-year period presented a close clonal relationship. MICs of antibiotics promoting CDI increased with this clonal expansion. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Development of Clostridium septicum gas gangrene as an adverse effect of clindamycin-induced Clostridium difficile infection in a pediatric patient.

PubMed

Kiser, Casey J; Urish, Kenneth L; Boateng, Henry A

2014-09-01

Clostridium myonecrosis or gas gangrene is a life-threatening infection characterized by either traumatic or atraumatic etiology. It has been widely described in patients with traumatic open wounds and in immunocompromised patients, including malignancy. A third source can result from natural flora in the gastrointestinal tract after bowel ischemia. This is a rare occurrence and is even less commonly described in the pediatric population. We present a pediatric patient who developed Clostridium septicum myonecrosis as an iatrogenic complication from clindamycin-induced Clostridium difficile ischemic colitis.

Updates on the sporulation process in Clostridium species.

PubMed

Talukdar, Prabhat K; Olguín-Araneda, Valeria; Alnoman, Maryam; Paredes-Sabja, Daniel; Sarker, Mahfuzur R

2015-05-01

Sporulation is an important strategy for certain bacterial species within the phylum Firmicutes to survive longer periods of time in adverse conditions. All spore-forming bacteria have two phases in their life; the vegetative form, where they can maintain all metabolic activities and replicate to increase numbers, and the spore form, where no metabolic activities exist. Although many essential components of sporulation are conserved among the spore-forming bacteria, there are differences in the regulation and the pathways among different genera, even at the species level. While we have gained much information from the most studied spore-forming bacterial genus, Bacillus, we still lack an in-depth understanding of spore formation in the genus Clostridium. Clostridium and Bacillus share the master regulator of sporulation, Spo0A, and its downstream pathways, but there are differences in the activation of the Spo0A pathway. While Bacillus species use a multi-component phosphorylation pathway for phosphorylation of Spo0A, termed phosphorelay, such a phosphorelay system is absent in Clostridium. On the other hand, a number of genes regulated by the different sporulation-specific transcription factors are conserved between different Clostridium and Bacillus species. In this review, we discuss the recent findings on Clostridium sporulation and compare the sporulation mechanism in Clostridium and Bacillus. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

PubMed Central

Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

2015-01-01

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

PubMed

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Systems and synthetic metabolic engineering for amino acid production - the heartbeat of industrial strain development.

PubMed

Becker, Judith; Wittmann, Christoph

2012-10-01

With a world market of more than four million tons per year, l-amino acids are among the most important products in industrial biotechnology. The recent years have seen a tremendous progress in the development of tailor-made strains for such products, intensively driven from systems metabolic engineering, which upgrades strain engineering into a concept of optimization on a global scale. This concept seems especially valuable for efficient amino acid production, demanding for a global modification of pathway fluxes - a challenge with regard to the high complexity of the underlying metabolism, superimposed by various layers of metabolic and transcriptional control. Copyright © 2011 Elsevier Ltd. All rights reserved.

CLOSTRIDIUM RUBRUM SP. N. AND OTHER PECTINOLYTIC CLOSTRIDIA FROM SOIL1

PubMed Central

Ng, Henry; Vaughn, Reese H.

1963-01-01

Ng, Henry (University of California, Davis) and Reese H. Vaughn. Clostridium rubrum sp. n. and other pectinolytic clostridia from soil. J. Bacteriol. 85:1104–1113. 1963.—Reports in the literature and results of experiments described herein suggest that pectinolytic anaerobes constitute a very heterogeneous group. The cultures isolated in this study all belonged to the genus Clostridium. The following species were identified: C. butyricum, C. fallax, C. multifermentans, and C. indolis. In addition, a species believed to be previously undescribed was named C. rubrum sp. n. The ability to ferment galacturonic acid was found to be adaptive. Some cultures fermented pectin and pectic acid to the same degree, whereas others fermented pectin only partially. The partial fermentation was attributed to the lack of a pectinesterase. On the basis of fermentation balances, it was concluded that the four strains of galacturonic acid fermenters selected for study yielded identical end products in approximately the same proportions. Per mole of galacturonic acid fermented, about 2 moles of CO2, 1.5 moles of H2, 1.5 moles of acetic acid, and 0.25 mole of butyric acid were produced. PMID:14044001

Switchgrass (Panicum virgatum) fermentation by sequential culture of Clostridium thermocellum and Clostridium beijerinckii: effect of particle size on gas production

USDA-ARS?s Scientific Manuscript database

Fuel alcohols can be produced by fermenting cellulosic biomass. Clostridium beijerinckii produces both ethanol and butanol, but it is non-cellulolytic. Cellulose requires saccharification prior to fermentation by C. beijerinckii. In contrast, the thermophile, Clostridium thermocellum, is highly ce...

Presence of Clostridium difficile in pig faecal samples and wild animal species associated with pig farms.

PubMed

Andrés-Lasheras, S; Bolea, R; Mainar-Jaime, R C; Kuijper, E; Sevilla, E; Martín-Burriel, I; Chirino-Trejo, M

2017-02-01

To determine the presence of Clostridium difficile on fattening pig farms in north-eastern Spain. Twenty-seven farms were sampled. Pools of pig faecal samples (n = 210), samples of intestinal content from common farm pest species (n = 95) and environment-related samples (n = 93) were collected. Isolates were tested for toxin genes of C. difficile, and typed by PCR-ribotyping and toxinotyping. The minimal inhibitory concentrations of six antimicrobial agents were determined using Etest. Thirty-four isolates were obtained from 12 farms, and 30 (88·2%) had toxin genes. Seven ribotypes were identified. Ribotype 078 and its variant 126 were predominant (52·9%). The same ribotypes were isolated from different animal species on the same farm. None of the isolates were resistant to metronidazole or vancomycin. Clostridium difficile was common within the pig farm environment. Most of the positive samples came from pest species or were pest-related environmental samples. Pest species were colonized with toxigenic and antimicrobial-resistant C. difficile strains of the same ribotypes that are found in humans and pigs. Rodents and pigeons may transmit toxigenic and antimicrobial-resistant C. difficile strains that are of the same ribotypes as those occuring in humans. © 2016 The Society for Applied Microbiology.

[Spontaneous gas gangrene in a diabetic patient with Clostridium septicum].

PubMed

Mischke, A; Besier, S; Walcher, F; Waibel, H; Brade, V; Brandt, C

2005-10-01

Atraumatic infections due to Clostridium septicum are known to be associated with immunosuppression or even malignancy. In this case report, we present a patient with severe Clostridium septicum infection related to advanced colon cancer that had not previously been diagnosed. The case demonstrates the strong association between Clostridium septicum infections and malignancy, particularly in the presence of other predisposing diseases such as diabetes mellitus. It strongly suggests excluding malignant neoplasms, especially of the gastrointestinal tract, when severe Clostridium septicum infections occur. Moreover, if patients with known colorectal or other malignancy develop septicaemia or spontaneous gas gangrene, clinicians should be aware of Clostridium septicum as one of the main causative agents, as early diagnosis and aggressive treatment are important to improve prognosis.

A Novel Dual-cre Motif Enables Two-Way Autoregulation of CcpA in Clostridium acetobutylicum.

PubMed

Zhang, Lu; Liu, Yanqiang; Yang, Yunpeng; Jiang, Weihong; Gu, Yang

2018-04-15

The master regulator CcpA (catabolite control protein A) manages a large and complex regulatory network that is essential for cellular physiology and metabolism in Gram-positive bacteria. Although CcpA can affect the expression of target genes by binding to a cis -acting catabolite-responsive element ( cre ), whether and how the expression of CcpA is regulated remain poorly explored. Here, we report a novel dual- cre motif that is employed by the CcpA in Clostridium acetobutylicum , a typical solventogenic Clostridium species, for autoregulation. Two cre sites are involved in CcpA autoregulation, and they reside in the promoter and coding regions of CcpA. In this dual- cre motif, cre P , in the promoter region, positively regulates ccpA transcription, whereas cre ORF , in the coding region, negatively regulates this transcription, thus enabling two-way autoregulation of CcpA. Although CcpA bound cre P more strongly than cre ORF in vitro , the in vivo assay showed that cre ORF -based repression dominates CcpA autoregulation during the entire fermentation. Finally, a synonymous mutation of cre ORF was made within the coding region, achieving an increased intracellular CcpA expression and improved cellular performance. This study provides new insights into the regulatory role of CcpA in C. acetobutylicum and, moreover, contributes a new engineering strategy for this industrial strain. IMPORTANCE CcpA is known to be a key transcription factor in Gram-positive bacteria. However, it is still unclear whether and how the intracellular CcpA level is regulated, which may be essential for maintaining normal cell physiology and metabolism. We discovered here that CcpA employs a dual- cre motif to autoregulate, enabling dynamic control of its own expression level during the entire fermentation process. This finding answers the questions above and fills a void in our understanding of the regulatory network of CcpA. Interference in CcpA autoregulation leads to improved cellular

Risk factors for Clostridium difficile infection in HIV-infected patients.

PubMed

Imlay, Hannah; Kaul, Daniel; Rao, Krishna

2016-01-01

Clostridium difficile infection is a healthcare-associated infection resulting in significant morbidity. Although immunosuppression is associated with Clostridium difficile infection acquisition and adverse outcomes, the epidemiology of Clostridium difficile infection in HIV-infected patients has been little studied in the era of antiretroviral therapy. This study identifies the risk factors for acquisition of Clostridium difficile infection in HIV-infected patients. A retrospective, propensity score-matched case-control study design was employed, with patients selected from our institution's outpatient HIV clinic. Clostridium difficile infection cases were defined as having positive stool testing plus an appropriate clinical presentation. The propensity score was generated via multiple logistic regression from year of HIV diagnosis, age at first contact, duration of follow-up, gender, and initial CD4 count. The 46 cases included were matched to a total of 180 controls. Prior antibiotic treatment was a significant predictor of Clostridium difficile infection (odds ratio: 13, 95% confidence interval: 3.49-48.8, p  < .001) as was number of hospital admissions in the preceding year (odds ratio: 4.02, confidence interval: 1.81-8.94, p  < .001). Having both proton pump inhibitor use and CD4 count <200 cells/µL significantly increased odds of Clostridium difficile infection in the multivariable model (odds ratio: 15.17, confidence interval: 1.31-175.9, p  = .021). As in the general population, frequent hospitalizations and exposure to antimicrobials are independent predictors of Clostridium difficile infection acquisition in patients with HIV. Additionally, low CD4 count and proton pump inhibitor use are new potentially modifiable variables that can be targeted for prevention of Clostridium difficile infection in future interventional studies.

Mutant strain of C. acetobutylicum and process for making butanol

DOEpatents

Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

1993-01-01

A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

Morphogenesis and Biomechanics of Engineered Skin Cultured Under Uniaxial Strain.

PubMed

Blackstone, Britani N; Powell, Heather M

2012-04-01

Split-thickness autograft is the standard wound treatment for full-thickness burns. In large burns, sparse availability of uninjured skin prevents rapid closure of the wound, resulting in increased scar tissue formation or mortality. Tissue-engineered skin (ES) offers promise when autografts are not available. ES, constructed from a polymeric scaffold and skin cells, has been shown to reduce donor site area required to permanently close wounds, mortality, and morbidity from scarring but cannot restore all skin functions. Current generations of ES are orders of magnitude weaker than normal human skin, leading to difficulty in surgical application, greater susceptibility to mechanical damage during fabrication and application, and less elasticity and strength once engrafted. Previous studies to improve ES biomechanics focus on altering the scaffolding material, which resulted in modest improvements but often inhibited proper skin development. As the skin is naturally under static strain, adding these mechanical cues to the culture environment is hypothesized to improve ES biomechanics. ES was cultured under applied static strains ranging from 0% to 40% strain for a total of 10 days. Strain magnitudes of 10% and 20% strain resulted in significantly stronger ES than unstrained controls, showed upregulation of many genes encoding structural extracellular matrix proteins, and exhibited increased epidermal cell proliferation and differentiation. Enhanced biomechanical properties of ES can allow for facile surgical application and less damage during dressing changes. These findings suggest that mechanical cues play a significant role in skin development and should be further explored.

Clostridium difficile Toxins A and B: Insights into Pathogenic Properties and Extraintestinal Effects

PubMed Central

Di Bella, Stefano; Ascenzi, Paolo; Siarakas, Steven; Petrosillo, Nicola; di Masi, Alessandra

2016-01-01

Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight junctions of target cells causing cell rounding and ultimately cell death. Detectable C. difficile toxemia is strongly associated with fulminant disease. However, besides the well-known intestinal damage, recent animal and in vitro studies have suggested a more far-reaching role for these toxins activity including cardiac, renal, and neurologic impairment. The creation of C. difficile strains with mutations in the genes encoding toxin A and B indicate that toxin B plays a major role in overall CDI pathogenesis. Novel insights, such as the role of a regulator protein (TcdE) on toxin production and binding interactions between albumin and C. difficile toxins, have recently been discovered and will be described. Our review focuses on the toxin-mediated pathogenic processes of CDI with an emphasis on recent studies. PMID:27153087

Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

PubMed Central

2010-01-01

Background Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405) is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. Conclusions By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum and highlight remaining gaps

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks

PubMed Central

Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

2015-01-01

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium. PMID:26584048

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin.

PubMed

Popoff, M R; Boquet, P

1988-05-16

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Contamination of ready-to-eat raw vegetables with Clostridium difficile in France.

PubMed

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.

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.

Clostridium Difficile Infections

MedlinePlus

Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Loss of appetite Nausea Abdominal pain or tenderness C. difficile is more common in people who need ...

Improving itaconic acid production through genetic engineering of an industrial Aspergillus terreus strain.

PubMed

Huang, Xuenian; Lu, Xuefeng; Li, Yueming; Li, Xia; Li, Jian-Jun

2014-08-11

Itaconic acid, which has been declared to be one of the most promising and flexible building blocks, is currently used as monomer or co-monomer in the polymer industry, and produced commercially by Aspergillus terreus. However, the production level of itaconic acid hasn't been improved in the past 40 years, and mutagenesis is still the main strategy to improve itaconate productivity. The genetic engineering approach hasn't been applied in industrial A. terreus strains to increase itaconic acid production. In this study, the genes closely related to itaconic acid production, including cadA, mfsA, mttA, ATEG_09969, gpdA, ATEG_01954, acoA, mt-pfkA and citA, were identified and overexpressed in an industrial A. terreus strain respectively. Overexpression of the genes cadA (cis-aconitate decarboxylase) and mfsA (Major Facilitator Superfamily Transporter) enhanced the itaconate production level by 9.4% and 5.1% in shake flasks respectively. Overexpression of other genes showed varied effects on itaconate production. The titers of other organic acids were affected by the introduced genes to different extent. Itaconic acid production could be improved through genetic engineering of the industrially used A. terreus strain. We have identified some important genes such as cadA and mfsA, whose overexpression led to the increased itaconate productivity, and successfully developed a strategy to establish a highly efficient microbial cell factory for itaconate protuction. Our results will provide a guide for further enhancement of the itaconic acid production level through genetic engineering in future.

Flagellar glycosylation in Clostridium botulinum.

PubMed

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Cellulosilyticum ruminicola gen. nov., sp. nov., isolated from the rumen of yak, and reclassification of Clostridium lentocellum as Cellulosilyticum lentocellum comb. nov.

PubMed

Cai, Shichun; Dong, Xiuzhu

2010-04-01

An obligate anaerobic, Gram-staining-negative, mesophilic, cellulolytic bacterium, strain H1(T), was isolated from the rumen content of yak. Cells were straight to slightly curved rods, 0.8-1.0 x 3.0-4.0 microm in size, non-motile and encapsulated with mucous materials. Elliptical and terminal spores that swelled the cells were produced occasionally. The strain grew at 25-45 degrees C (optimum, 38 degrees C) and pH 6.0-7.8 (optimum, pH 6.7). Cellulose, cellobiose, xylan, xylose and maltose were used as carbon and energy sources, but not glucose. Products from cellulose and cellobiose fermentation were formic acid, acetic acid, carbon dioxide and trace amounts of ethanol, lactic acid and succinic acid. The genomic DNA G+C content was 33.7+/-1.2 mol%. The predominant fatty acids were C(16 : 0) (27.1 %), C(14 : 0) (9.2 %) and iso-C( 16 : 0) (6.4%). Based on the 16S rRNA gene sequence analysis, strain H1(T) was affiliated to the clostridial rRNA cluster XIVb and showed the highest 16S rRNA gene sequence similarity to Clostridium lentocellum DSM 5427(T) (96.0 %). These two strains formed a distinct lineage of the family 'Lachnospiraceae '. Based on data from this polyphasic taxonomic study, a new genus, Cellulosilyticum gen. nov., is proposed. Cellulosilyticum ruminicola sp. nov. is proposed for strain H1(T). The type strain of Cellulosilyticum ruminicola sp. nov. is strain H1(T) (=CGMCC 1.5065(T)=JCM 14822(T)). Clostridium lentocellum was reclassified in the new genus as Cellulosilyticum lentocellum comb. nov. (type strain RHM5(T)=ATCC 49066( T)=DSM 5427(T)=NCIMB 11756(T)).

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

PubMed

Llanco, Luis A; Nakano, Viviane; Moraes, Claudia T P de; Piazza, Roxane M F; Avila-Campos, Mario J

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Metronidazole-triazole conjugates: Activity against Clostridium difficile and parasites

PubMed Central

Jarrad, Angie M.; Karoli, Tomislav; Debnath, Anjan; Tay, Chin Yen; Huang, Johnny X.; Kaeslin, Geraldine; Elliott, Alysha G.; Miyamoto, Yukiko; Ramu, Soumya; Kavanagh, Angela M.; Zuegg, Johannes; Eckmann, Lars; Blaskovich, Mark A.T.; Cooper, Matthew A.

2015-01-01

Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window. PMID:26117821

Clostridial Strain-Specific Characteristics Associated with Necrotizing Enterocolitis.

PubMed

Schönherr-Hellec, Sophia; Klein, Geraldine L; Delannoy, Johanne; Ferraris, Laurent; Rozé, Jean Christophe; Butel, Marie José; Aires, Julio

2018-04-01

We aimed at identifying potential bacterial factors linking clostridia with necrotizing enterocolitis (NEC). We compared the phenotypic traits, stress responses, cellular cytotoxicity, and inflammatory capabilities of the largest collection of Clostridium butyricum and Clostridium neonatale strains isolated from fecal samples of NEC preterm neonates (PN) and control PNs. When strain characteristics were used as explanatory variables, a statistical discriminant analysis allowed the separation of NEC and control strains into separate groups. Strains isolated from NEC PN were characterized by a higher viability at 30°C ( P = 0.03) and higher aerotolerance ( P = 0.01), suggesting that NEC strains may have a competitive and/or survival advantage in the environmental gastrointestinal tract conditions of NEC PN. Heat-treated NEC bacteria induced higher production of interleukin-8 in Caco-2 cells ( P = 0.03), suggesting proinflammatory activity. In vitro , bacteria, bacterial components, and fecal filtrates showed variable cytotoxic effects affecting the cellular network and/or cell viability, without specific association with NEC or control samples. Altogether, our data support the existence of a specific clostridial strain signature associated with NEC. IMPORTANCE Clostridia are part of the commensal microbiota in preterm neonates (PN). However, microbiota analyses by culture and metagenomics have linked necrotizing enterocolitis (NEC) and intestinal colonization with clostridial species. Nevertheless, little is known about the specific characteristics that may be shared by clostridia associated with NEC compared to commensal clostridia. Therefore, our goal was to identify specific bacterial factors linking clostridial strains with NEC. We report the existence of a specific bacterial signature associated with NEC and propose that activation of the innate immune response may be a unifying causative mechanism for the development of NEC independent of a specific pathogenic

Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.

PubMed

Martin, Jessica S H; Monaghan, Tanya M; Wilcox, Mark H

2016-04-01

Clostridium difficile infection (CDI) continues to affect patients in hospitals and communities worldwide. The spectrum of clinical disease ranges from mild diarrhoea to toxic megacolon, colonic perforation and death. However, this bacterium might also be carried asymptomatically in the gut, potentially leading to 'silent' onward transmission. Modern technologies, such as whole-genome sequencing and multi-locus variable-number tandem-repeat analysis, are helping to track C. difficile transmission across health-care facilities, countries and continents, offering the potential to illuminate previously under-recognized sources of infection. These typing strategies have also demonstrated heterogeneity in terms of CDI incidence and strain types reflecting different stages of epidemic spread. However, comparison of CDI epidemiology, particularly between countries, is challenging due to wide-ranging approaches to sampling and testing. Diagnostic strategies for C. difficile are complicated both by the wide range of bacterial targets and tests available and the need to differentiate between toxin-producing and non-toxigenic strains. Multistep diagnostic algorithms have been recommended to improve sensitivity and specificity. In this Review, we describe the latest advances in the understanding of C. difficile epidemiology, transmission and diagnosis, and discuss the effect of these developments on the clinical management of CDI.

Structure-Function Analysis of Peptide Signaling in the Clostridium perfringens Agr-Like Quorum Sensing System

PubMed Central

Ma, Menglin; Li, Jihong

2015-01-01

ABSTRACT The accessory growth regulator (Agr)-like quorum sensing (QS) system of Clostridium perfringens controls the production of many toxins, including beta toxin (CPB). We previously showed (J. E. Vidal, M. Ma, J. Saputo, J. Garcia, F. A. Uzal, and B. A. McClane, Mol Microbiol 83:179–194, 2012, http://dx.doi.org/10.1111/j.1365-2958.2011.07925.x) that an 8-amino-acid, AgrD-derived peptide named 8-R upregulates CPB production by this QS system. The current study synthesized a series of small signaling peptides corresponding to sequences within the C. perfringens AgrD polypeptide to investigate the C. perfringens autoinducing peptide (AIP) structure-function relationship. When both linear and cyclic ring forms of these peptides were added to agrB null mutants of type B strain CN1795 or type C strain CN3685, the 5-amino-acid peptides, whether in a linear or ring (thiolactone or lactone) form, induced better signaling (more CPB production) than peptide 8-R for both C. perfringens strains. The 5-mer thiolactone ring peptide induced faster signaling than the 5-mer linear peptide. Strain-related variations in sensing these peptides were detected, with CN3685 sensing the synthetic peptides more strongly than CN1795. Consistent with those synthetic peptide results, Transwell coculture experiments showed that CN3685 exquisitely senses native AIP signals from other isolates (types A, B, C, and D), while CN1795 barely senses even its own AIP. Finally, a C. perfringens AgrD sequence-based peptide with a 6-amino-acid thiolactone ring interfered with CPB production by several C. perfringens strains, suggesting potential therapeutic applications. These results indicate that AIP signaling sensitivity and responsiveness vary among C. perfringens strains and suggest C. perfringens prefers a 5-mer AIP to initiate Agr signaling. IMPORTANCE Clostridium perfringens possesses an Agr-like quorum sensing (QS) system that regulates virulence, sporulation, and toxin production. The

Morphogenesis and Biomechanics of Engineered Skin Cultured Under Uniaxial Strain

PubMed Central

Blackstone, Britani N.; Powell, Heather M.

2012-01-01

Background Split-thickness autograft is the standard wound treatment for full-thickness burns. In large burns, sparse availability of uninjured skin prevents rapid closure of the wound, resulting in increased scar tissue formation or mortality. Tissue-engineered skin (ES) offers promise when autografts are not available. The Problem ES, constructed from a polymeric scaffold and skin cells, has been shown to reduce donor site area required to permanently close wounds, mortality, and morbidity from scarring but cannot restore all skin functions. Current generations of ES are orders of magnitude weaker than normal human skin, leading to difficulty in surgical application, greater susceptibility to mechanical damage during fabrication and application, and less elasticity and strength once engrafted. Basic/Clinical Science Advances Previous studies to improve ES biomechanics focus on altering the scaffolding material, which resulted in modest improvements but often inhibited proper skin development. As the skin is naturally under static strain, adding these mechanical cues to the culture environment is hypothesized to improve ES biomechanics. ES was cultured under applied static strains ranging from 0% to 40% strain for a total of 10 days. Strain magnitudes of 10% and 20% strain resulted in significantly stronger ES than unstrained controls, showed upregulation of many genes encoding structural extracellular matrix proteins, and exhibited increased epidermal cell proliferation and differentiation. Clinical Care Relevance Enhanced biomechanical properties of ES can allow for facile surgical application and less damage during dressing changes. Conclusion These findings suggest that mechanical cues play a significant role in skin development and should be further explored. PMID:24527283

Dentists, antibiotics and Clostridium difficile-associated disease.

PubMed

Beacher, N; Sweeney, M P; Bagg, J

2015-09-25

Dentists prescribe significant volumes of antimicrobial drugs within primary care settings. There is good evidence that many of the prescriptions are not justified by current clinical guidance and that that there is considerable misuse of these drugs in dentistry. One of the risks associated with antibiotic administration is Clostridium difficile-associated disease (CDAD), an entity of which many healthcare workers, including dentists, have little knowledge or understanding. This review seeks to identify the extent and nature of the problem and provides an up to date summary of current views on CDAD, with particular reference to community acquired disease. As for all healthcare workers, scrupulous attention to standard infection control procedures and reducing inappropriate antibiotic prescribing are essential to reduce the risks of CDAD, prevent emergence of further resistant strains of microorganisms and maintain the value of the arsenal of antibiotics currently available to us.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes.

PubMed

Zhang, Dong-Xiu; Nuss, Donald L

2016-02-23

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

Prevalence and Characterization of a Binary Toxin (Actin-Specific ADP-Ribosyltransferase) from Clostridium difficile

PubMed Central

Gonçalves, Carina; Decré, Dominique; Barbut, Frédéric; Burghoffer, Béatrice; Petit, Jean-Claude

2004-01-01

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated. PMID:15131151

Application of long sequence reads to improve genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7

DOE PAGES

Utturkar, Sagar M.; Bayer, Edward A.; Borovok, Ilya; ...

2016-09-29

Here, we and others have shown the utility of long sequence reads to improve genome assembly quality. In this study, we generated PacBio DNA sequence data to improve the assemblies of draft genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7.

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

PubMed

Yang, Jiangang; Zhu, Yueming; Men, Yan; Sun, Shangshang; Zeng, Yan; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

2016-12-21

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

Novel fermentation: the production of R(-)-1,2-propanediol and acetol by Clostridium thermosaccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cameron, D.C.; Cooney, C.L.

1986-07-01

Three strains of Clostridium thermosaccharolyticum were found that produce R(-)-1,2-propanediol from a variety of sugars, including D-glucose and D-xylose. The fermentation of glucose by strain HG-8 (ATCC 31960) gave 7.9 g/l of R(-)-1,2-propanediol with a best yield of 0.27 g/g glucose and an enantiomeric excess of greater than 99%. Acetol accumulated to 1.47 g/l. Product formation was not affected by phosphate concentrations up to 113 mM. A possible pathway to these products involves a variation of the methylglyoxal bypass. Methylglyoxal is reduced to acetol, which is further reduced to %(-)-1,2-propanediol. This fermentation provides a unique route to R(-)-1,2-propanediol and acetolmore » from inexpensive, readily available substrates.« less

Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum.

PubMed Central

Wiegel, J; Ljungdahl, L G; Rawson, J R

1979-01-01

Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment. PMID:39062

The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

PubMed Central

Akinosho, Hannah; Yee, Kelsey; Close, Dan; Ragauskas, Arthur

2014-01-01

First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing (CBP) applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes toward the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum's role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum's amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community. PMID:25207268

The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

NASA Astrophysics Data System (ADS)

Ragauskas, Arthur; Akinosho, Hannah; Yee, Kelsey; Close, Dan

2014-08-01

First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes towards the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum’s role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum’s amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

PubMed

Alimolaei, Mojtaba; Golchin, Mehdi; Daneshvar, Hamid

2016-06-01

Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Strain-Engineered Multiferroicity in P n m a NaMnF3 Fluoroperovskite

NASA Astrophysics Data System (ADS)

Garcia-Castro, A. C.; Romero, A. H.; Bousquet, E.

2016-03-01

In this study we show from first principles calculations the possibility to induce multiferroic and magnetoelectric functional properties in the P n m a NaMnF3 fluoroperovskite by means of epitaxial strain engineering. Surprisingly, we found a very strong nonlinear polarization-strain coupling that drives an atypical amplification of the ferroelectric polarization for either compression or expansion of the cell. This property is associated with a noncollinear antiferromagnetic ordering, which induces a weak ferromagnetism phase and makes the strained NaMnF3 fluoroperovskite multiferroic. The magnetoelectric response was calculated and it was found to be composed of linear and nonlinear components with amplitudes similar to the ones of Cr2O3. These findings show that it is possible to move the fluoride family toward functional applications with unique responses.

Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum.

PubMed

Daifas, Daphne Phillips; Smith, James P; Blanchfield, Burke; Sanders, Greg; Austin, John W; Koukoutisis, John

2004-08-01

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.

In Vitro Activities of Faropenem against 579 Strains of Anaerobic Bacteria

PubMed Central

Wexler, Hannah M.; Molitoris, Denise; St. John, Shahera; Vu, Ann; Read, Erik K.; Finegold, Sydney M.

2002-01-01

The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 μg/ml and imipenem MICs of >32 μg/ml. Faropenem had an MIC of 16 μg/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 μg/ml). Both faropenem and imipenem had MICs of ≤4 μg/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 μg/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were ≤4 μg/ml. Faropenem had MICs of 8 and 16 μg/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 μg/ml). MICs were ≤4 μg/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested. PMID:12384389

Spore Formation and Toxin Production in Clostridium difficile Biofilms

PubMed Central

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

Spore formation and toxin production in Clostridium difficile biofilms.