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Sample records for gram-positive cocci isolated

  1. Changes of the Quinolones Resistance to Gram-positive Cocci Isolated during the Past 8 Years in the First Bethune Hospital

    NASA Astrophysics Data System (ADS)

    Xu, Jiancheng; Chen, Qihui; Yao, Hanxin; Zhou, Qi

    This study was to investigate the quinolones resistance to gram-positive cocci isolated in the First Bethune Hospital during the past 8 years. Disk diffusion test was used to study the antimicrobial resistance. The data were analyzed by WHONET 5 software according to Clinical and Laboratory Standards Institute (CLSI). The rates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCNS) were 50.8%∼83.3% and 79.4%∼81.5%during the past 8 years, respectively. In recent 8 years, the quinolones resistance to gram-positive cocci had increased. Monitoring of the quinolones resistance to gram-positive cocci should be strengthened. The change of the antimicrobial resistance should be investigated in order to guide rational drug usage in the clinic and prevent bacterial strain of drug resistance from being transmitted.

  2. Comparative evaluation of two matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems, Vitek MS and Microflex LT, for the identification of Gram-positive cocci routinely isolated in clinical microbiology laboratories.

    PubMed

    Lee, Miae; Chung, Hae-Sun; Moon, Hee-Won; Lee, Sun Hwa; Lee, Kyungwon

    2015-06-01

    We evaluated the performance of two MALDI-TOF MS systems for the identification of clinically important Gram-positive cocci. Vitek MS and Microflex LT correctly identified 97.2% and 94.7%, respectively. Both systems offer reliable and rapid identification of clinically important Gram-positive cocci isolated in clinical laboratories, including staphylococci, streptococci, and enterococci. Expanding the databases, especially of coagulase-negative staphylococci and viridans streptococci, would enhance performance.

  3. Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

    PubMed Central

    Rodríguez, M; Núñez, F; Córdoba, J J; Bermúdez, E; Asensio, M A

    1996-01-01

    Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer. PMID:8787389

  4. Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

    PubMed

    Rodríguez, M; Núñez, F; Córdoba, J J; Bermúdez, E; Asensio, M A

    1996-06-01

    Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.

  5. Prevalence of multidrug resistant Gram-positive cocci in a Chinese hospital over an 8-year period.

    PubMed

    Zhang, Ruiqin; Wang, Fengzhi; Kang, Jianbang; Wang, Xinchun; Yin, Donghong; Dang, Wen; Duan, Jinju

    2015-01-01

    Gram-positive cocci are common causes of bloodstream and nosocomial infections, and their multi-drug resistance is an increasingly serious problem. The present study aimed to assess the prevalence of multi-drug-resistant Gram-positive cocci in a Chinese population. In this retrospective study, data about Gram-positive cocci from in-patients (January 2006 and December 2013) at the Second Hospital of Shanxi Medical University, Taiyuan, China, were reviewed. Antimicrobial susceptibility profile of the isolated Gram-positive cocci was evaluated using the disk diffusion method. Antibiotic resistance was determined according to the Clinical and Laboratory Standards Institute 2009 guidelines. The prevalence of drug resistance was determined, as well as correlation coefficients for various drugs between the resistance rate and year of sample collection. A total of 7789 Gram-positive cocci isolates were found, including 2576 (33%) coagulase-negative Staphylococci, 1477 (19%) Staphylococci aureus, 1343 (17%) Enterococcus faecalis, and 1139 (15%) Enterococcus faecium. The proportions of methicillin-resistant Staphylococci aureus (MRSA) and methicillin-resistant Staphylococci (MRS) were 31.5% (465/1477) and 61.6% (1587/2576), respectively. Among all isolates, MRS had much higher drug resistance rate than methicillin-sensitive Staphylococci (P<0.05). E. faecalis had a higher multi-drug resistance rate than E. faecium (P<0.01). Interestingly, MRSA resistance rates declined over the years, showing a negative correlation coefficient for all drugs, with significance for levofloxacin, azithromycin, erythromycin, and clindamycin (P<0.05), but not sulphamethoxazole/trimethoprim (P=0.057) and gentamicin (P=0.186). These results indicated that Staphylococci were the predominant Gram-positive cocci isolated. There was a trend of decreasing MRSA in the population studied.

  6. Gene homogeneity for aminoglycoside-modifying enzymes in gram-positive cocci.

    PubMed Central

    Ounissi, H; Derlot, E; Carlier, C; Courvalin, P

    1990-01-01

    Aminoglycoside-resistant strains of Staphylococcus and Enterococcus, approximately 500 of each, were screened by dot blot hybridization for the presence of genes encoding aminoglycoside-modifying enzymes. The MICs of various aminoglycosides for the strains were determined, and the enzyme contents of the cells were inferred from the resistance phenotypes. The agreements (in percent) of the hybridization results with the deduced enzyme contents for Staphylococcus and Enterococcus species were, respectively, 80 and 87.6 for ANT(6) (aminoglycoside nucleotidyltransferase), 99.8 and 100 for both APH(3') (aminoglycoside phosphotransferase) and APH(2")-AAC(6') (aminoglycoside acetyltransferase), and 100 and 100 for ANT(4'). The weak correlation obtained with the probe for ANT(6) was due to the fact that gram-positive cocci can also be streptomycin resistant by synthesis of APH(3") or ANT(3")(9) and by ribosomal mutation. The remaining probes appeared to be specific: they hybridized with all the resistant clinical isolates but not with the susceptible strains. These results indicate that, except for streptomycin, nucleic acid hybridization is a valid approach for the detection and characterization of aminoglycoside resistance in gram-positive cocci. PMID:1963528

  7. Recovery of Gram-Positive Cocci and Candida albicans from Peroxygen/Silver-Based Disinfectants.

    PubMed

    Eissa, Mostafa E; Nouby, Ahmed S

    2016-01-01

    Neutralization method evaluation is an important first step in a disinfectant validation study program. It is also crucial in assessment of the efficiency of microbial recovery media in the presence of a residual biocidal agent. In the present study, four commercially available peroxygen/silver-based disinfectant formulae--intended to be used in a pharmaceutical facility sanitization program--were tested at two dilutions against three Gram-positive cocci and one yeast; Staphylococcus aureus (ATCC 6538), Kucoria rhizophila (ATCC 9341) and Candida albicans (ATCC 10231) and Staphylococcus capitis as an environmental monitoring (EM) isolate sample (identified by miniaturized biochemical identification system). Disinfectants preparation, dilutions and the test procedure were carried on in laboratory under conditions simulating the working environment of 20-25 degrees C and RH% 40-60. In-house made neutralizing broth was mixed with biocidal agents to make two dilutions of each disinfectant forming Peroxygen: neutralizing broth ratios of 1:10 and 1:100 (v/v). Three populations were established and two comparison ratio groups were examined, namely neutralizer efficacy and neutralizer toxicity. Two acceptance criteria were tested. One criterion showed higher rate of neutralization success than the other. S. aureus showed the highest rate of successful microbial recovery from neutralization process. The Disinfectant (Bixco) was exceptionally neutralized at all dilutions with all microorganisms. In conclusion, in-house made neutralizing broth effectively neutralized all disinfectants with all the tested microorganisms at 1:100 (v/v); thus, it can be used in sanitizer validation studies and EM media.

  8. Recovery of Gram-Positive Cocci and Candida albicans from Peroxygen/Silver-Based Disinfectants.

    PubMed

    Eissa, Mostafa E; Nouby, Ahmed S

    2016-01-01

    Neutralization method evaluation is an important first step in a disinfectant validation study program. It is also crucial in assessment of the efficiency of microbial recovery media in the presence of a residual biocidal agent. In the present study, four commercially available peroxygen/silver-based disinfectant formulae--intended to be used in a pharmaceutical facility sanitization program--were tested at two dilutions against three Gram-positive cocci and one yeast; Staphylococcus aureus (ATCC 6538), Kucoria rhizophila (ATCC 9341) and Candida albicans (ATCC 10231) and Staphylococcus capitis as an environmental monitoring (EM) isolate sample (identified by miniaturized biochemical identification system). Disinfectants preparation, dilutions and the test procedure were carried on in laboratory under conditions simulating the working environment of 20-25 degrees C and RH% 40-60. In-house made neutralizing broth was mixed with biocidal agents to make two dilutions of each disinfectant forming Peroxygen: neutralizing broth ratios of 1:10 and 1:100 (v/v). Three populations were established and two comparison ratio groups were examined, namely neutralizer efficacy and neutralizer toxicity. Two acceptance criteria were tested. One criterion showed higher rate of neutralization success than the other. S. aureus showed the highest rate of successful microbial recovery from neutralization process. The Disinfectant (Bixco) was exceptionally neutralized at all dilutions with all microorganisms. In conclusion, in-house made neutralizing broth effectively neutralized all disinfectants with all the tested microorganisms at 1:100 (v/v); thus, it can be used in sanitizer validation studies and EM media. PMID:27281990

  9. Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence

    PubMed Central

    Burnside, Kellie; Rajagopal, Lakshmi

    2011-01-01

    Living organisms adapt to the dynamic external environment for their survival. Environmental adaptation in prokaryotes is thought to be primarily accomplished by signaling events mediated by two-component systems, consisting of histidine kinases and response regulators. However, eukaryotic-like serine/threonine kinases (STKs) have recently been described to regulate growth, antibiotic resistance and virulence of pathogenic bacteria. This article summarizes the role of STKs and their cognate phosphatases (STPs) in Gram-positive cocci that cause invasive infections in humans. Given that a large number of inhibitors to eukaryotic STKs are approved for use in humans, understanding how serine/threonine phosphorylation regulates virulence and antibiotic resistance will be beneficial for the development of novel therapeutic strategies against bacterial infections. PMID:21797690

  10. Identification of pili on the surface of Finegoldia magna--a gram-positive anaerobic cocci.

    PubMed

    Murphy, Elizabeth C; Janulczyk, Robert; Karlsson, Christofer; Mörgelin, Matthias; Frick, Inga-Maria

    2014-06-01

    Pili have only been discovered in the major Gram-positive pathogens in the past decade and they have been found to play an important role in colonisation and virulence. Pili have been shown to have many important functions including attachment to host tissues, mediating bacterial aggregation, biofilm formation and binding to proteins in the extracellular matrix. In this study, sortase-dependent pili have been found to be expressed on the surface of Finegoldia magna ALB8. F. magna is a Gram-positive anaerobic coccus that, primarily, is a commensal of the skin and mucous membranes, but has also been isolated from various clinical infection sites and is associated with soft-tissue abscesses, wound infections and bone and prosthetic joint infections. In this study, F. magna ALB8 was found to harbour three sortases at the pilus locus, two of which bear high similarity to class C sortases in Streptococcus pneumoniae. Two putative sortase-dependent pili proteins were found in the locus, with one being identified as the major pilus subunit, Fmp1 (F. magna pilus subunit 1), due to its high similarity to other major pilus proteins in prominent Gram-positive pathogens. The presence of sortase-dependent pili was confirmed experimentally through recombinant production of Fmp1 and production of antiserum. The Fmp1 antiserum was used in Western blot to show the presence of a high molecular weight protein ladder, characteristic of the presence of pili, in trypsin released cell wall surface proteins from F. magna. The presence of sortase-dependent pili was visually confirmed by transmission electron microscopy, which showed the binding of gold labelled anti-Fmp1 to individual pilus proteins along the pilus. Furthermore, pili could also be found to bind and interact with keratinocytes in the epidermal layer of human skin, suggesting an adhesive role for pili on F. magna. Our work represents the first description of pilus structures in F. magna. This discovery further

  11. Accuracy of the VITEK® 2 system for a rapid and direct identification and susceptibility testing of Gramnegative rods and Gram-positive cocci in blood samples.

    PubMed

    Nimer, N A; Al-Saa'da, R J; Abuelaish, O

    2016-06-15

    The performance of the VITEK® 2 system for direct rapid identification and antimicrobial susceptibility testing of the bacteria responsible for blood infections was determined. The isolates studied included 166 Gram-negative rods and 74 Gram-positive cocci from inpatients. Specially treated monomicrobial samples from positive blood culture bottles were directly inoculated into the VITEK 2 system and the results were compared with those from cards inoculated with standardized bacterial suspensions. Compared with the standard method, 95.8% of Gram-negative rods were correctly identified by VITEK 2 and the overall level of agreement between the two methods in susceptibility testing was 92.0%. For Gram-positive bacteria, 89.2% were correctly identified by VITEK 2 and susceptibility testing revealed an overall agreement rate of 91.3%. These results suggest that VITEK 2 cards inoculated with fluids sampled directly from positive blood culture bottles are suitable for speedy identification and susceptibility testing of Gram-negative bacilli and Gram-positive cocci.

  12. 18 GHz electromagnetic field induces permeability of Gram-positive cocci

    PubMed Central

    Nguyen, The Hong Phong; Shamis, Yury; Croft, Rodney J.; Wood, Andrew; McIntosh, Robert L.; Crawford, Russell J.; Ivanova, Elena P.

    2015-01-01

    The effect of electromagnetic field (EMF) exposures at the microwave (MW) frequency of 18 GHz, on four cocci, Planococcus maritimus KMM 3738, Staphylococcus aureus CIP 65.8T, S. aureus ATCC 25923 and S. epidermidis ATCC 14990T, was investigated. We demonstrate that exposing the bacteria to an EMF induced permeability in the bacterial membranes of all strains studied, as confirmed directly by transmission electron microscopy (TEM), and indirectly via the propidium iodide assay and the uptake of silica nanospheres. The cells remained permeable for at least nine minutes after EMF exposure. It was shown that all strains internalized 23.5 nm nanospheres, whereas the internalization of the 46.3 nm nanospheres differed amongst the bacterial strains (S. epidermidis ATCC 14990T~ 0%; Staphylococcus aureus CIP 65.8T S. aureus ATCC 25923, ~40%; Planococcus maritimus KMM 3738, ~80%). Cell viability experiments indicated that up to 84% of the cells exposed to the EMF remained viable. The morphology of the bacterial cells was not altered, as inferred from the scanning electron micrographs, however traces of leaked cytosolic fluids from the EMF exposed cells could be detected. EMF-induced permeabilization may represent an innovative, alternative cell permeability technique for applications in biomedical engineering, cell drug delivery and gene therapy. PMID:26077933

  13. 18 GHz electromagnetic field induces permeability of Gram-positive cocci.

    PubMed

    Nguyen, The Hong Phong; Shamis, Yury; Croft, Rodney J; Wood, Andrew; McIntosh, Robert L; Crawford, Russell J; Ivanova, Elena P

    2015-06-16

    The effect of electromagnetic field (EMF) exposures at the microwave (MW) frequency of 18 GHz, on four cocci, Planococcus maritimus KMM 3738, Staphylococcus aureus CIP 65.8(T), S. aureus ATCC 25923 and S. epidermidis ATCC 14990(T), was investigated. We demonstrate that exposing the bacteria to an EMF induced permeability in the bacterial membranes of all strains studied, as confirmed directly by transmission electron microscopy (TEM), and indirectly via the propidium iodide assay and the uptake of silica nanospheres. The cells remained permeable for at least nine minutes after EMF exposure. It was shown that all strains internalized 23.5 nm nanospheres, whereas the internalization of the 46.3 nm nanospheres differed amongst the bacterial strains (S. epidermidis ATCC 14990(T) ~  0%; Staphylococcus aureus CIP 65.8(T) S. aureus ATCC 25923, ~40%; Planococcus maritimus KMM 3738, ~ 80%). Cell viability experiments indicated that up to 84% of the cells exposed to the EMF remained viable. The morphology of the bacterial cells was not altered, as inferred from the scanning electron micrographs, however traces of leaked cytosolic fluids from the EMF exposed cells could be detected. EMF-induced permeabilization may represent an innovative, alternative cell permeability technique for applications in biomedical engineering, cell drug delivery and gene therapy.

  14. 18 GHz electromagnetic field induces permeability of Gram-positive cocci.

    PubMed

    Nguyen, The Hong Phong; Shamis, Yury; Croft, Rodney J; Wood, Andrew; McIntosh, Robert L; Crawford, Russell J; Ivanova, Elena P

    2015-01-01

    The effect of electromagnetic field (EMF) exposures at the microwave (MW) frequency of 18 GHz, on four cocci, Planococcus maritimus KMM 3738, Staphylococcus aureus CIP 65.8(T), S. aureus ATCC 25923 and S. epidermidis ATCC 14990(T), was investigated. We demonstrate that exposing the bacteria to an EMF induced permeability in the bacterial membranes of all strains studied, as confirmed directly by transmission electron microscopy (TEM), and indirectly via the propidium iodide assay and the uptake of silica nanospheres. The cells remained permeable for at least nine minutes after EMF exposure. It was shown that all strains internalized 23.5 nm nanospheres, whereas the internalization of the 46.3 nm nanospheres differed amongst the bacterial strains (S. epidermidis ATCC 14990(T) ~  0%; Staphylococcus aureus CIP 65.8(T) S. aureus ATCC 25923, ~40%; Planococcus maritimus KMM 3738, ~ 80%). Cell viability experiments indicated that up to 84% of the cells exposed to the EMF remained viable. The morphology of the bacterial cells was not altered, as inferred from the scanning electron micrographs, however traces of leaked cytosolic fluids from the EMF exposed cells could be detected. EMF-induced permeabilization may represent an innovative, alternative cell permeability technique for applications in biomedical engineering, cell drug delivery and gene therapy. PMID:26077933

  15. Use of Enzyme Tests in Characterization and Identification of Aerobic and Facultatively Anaerobic Gram-Positive Cocci

    PubMed Central

    Bascomb, Shoshana; Manafi, Mammad

    1998-01-01

    The contribution of enzyme tests to the accurate and rapid routine identification of gram-positive cocci is introduced. The current taxonomy of the genera of aerobic and facultatively anaerobic cocci based on genotypic and phenotypic characterization is reviewed. The clinical and economic importance of members of these taxa is briefly summarized. Tables summarizing test schemes and kits available for the identification of staphylococci, enterococci, and streptococci on the basis of general requirements, number of tests, number of taxa, test classes, and completion times are discussed. Enzyme tests included in each scheme are compared on the basis of their synthetic moiety. The current understanding of the activity of enzymes important for classification and identification of the major groups, methods of testing, and relevance to the ease and speed of identification are reviewed. Publications describing the use of different identification kits are listed, and overall identification successes and problems are discussed. The relationships between the results of conventional biochemical and rapid enzyme tests are described and considered. The use of synthetic substrates for the detection of glycosidases and peptidases is reviewed, and the advantages of fluorogenic synthetic moieties are discussed. The relevance of enzyme tests to accurate and meaningful rapid routine identification is discussed. PMID:9564566

  16. In Vitro Activities of Tedizolid and Linezolid against Gram-Positive Cocci Associated with Acute Bacterial Skin and Skin Structure Infections and Pneumonia

    PubMed Central

    Chen, Ko-Hung; Huang, Yu-Tsung; Liao, Chun-Hsing; Sheng, Wang-Hui

    2015-01-01

    Tedizolid is a novel, expanded-spectrum oxazolidinone with potent activity against a wide range of Gram-positive pathogens. A total of 425 isolates of Gram-positive bacteria were obtained consecutively from patients with acute bacterial skin and skin structure infections (ABSSSIs) or pneumonia. These isolates included methicillin-susceptible Staphylococcus aureus (MSSA) (n = 100), methicillin-resistant Staphylococcus aureus (MRSA) (n = 100), Streptococcus pyogenes (n = 50), Streptococcus agalactiae (n = 50), Streptococcus anginosus group (n = 75), Enterococcus faecalis (n = 50), and vancomycin-resistant enterococci (VRE) (Enterococcus faecium) (n = 50). The MICs of tedizolid and linezolid were determined by the agar dilution method. Tedizolid exhibited better in vitro activities than linezolid against MSSA (MIC90s, 0.5 versus 2 μg/ml), MRSA (MIC90s, 0.5 versus 2 μg/ml), S. pyogenes (MIC90s, 0.5 versus 2 μg/ml), S. agalactiae (MIC90s, 0.5 versus 2 μg/ml), Streptococcus anginosus group (MIC90s, 0.5 versus 2 μg/ml), E. faecalis (MIC90s, 0.5 versus 2 μg/ml), and VRE (MIC90s, 0.5 versus 2 μg/ml). The tedizolid MICs against E. faecalis (n = 3) and VRE (n = 2) intermediate to linezolid (MICs, 4 μg/ml) were 1 μg/ml and 0.5 μg/ml, respectively. The tedizolid MIC90s against S. anginosus, S. constellatus, and S. intermedius were 0.5, 1, and 0.5 μg/ml, respectively, and the rates of susceptibility based on the U.S. FDA MIC interpretive breakpoints to the isolates were 16%, 28%, and 72%, respectively. Tedizolid exhibited 2- to 4-fold better in vitro activities than linezolid against a variety of Gram-positive cocci associated with ABSSSIs and pneumonia. The lower susceptibilities of tedizolid against isolates of S. anginosus and S. constellatus than against those of S. intermedius in Taiwan were noted. PMID:26248355

  17. In Vitro Activities of Tedizolid and Linezolid against Gram-Positive Cocci Associated with Acute Bacterial Skin and Skin Structure Infections and Pneumonia.

    PubMed

    Chen, Ko-Hung; Huang, Yu-Tsung; Liao, Chun-Hsing; Sheng, Wang-Hui; Hsueh, Po-Ren

    2015-10-01

    Tedizolid is a novel, expanded-spectrum oxazolidinone with potent activity against a wide range of Gram-positive pathogens. A total of 425 isolates of Gram-positive bacteria were obtained consecutively from patients with acute bacterial skin and skin structure infections (ABSSSIs) or pneumonia. These isolates included methicillin-susceptible Staphylococcus aureus (MSSA) (n = 100), methicillin-resistant Staphylococcus aureus (MRSA) (n = 100), Streptococcus pyogenes (n = 50), Streptococcus agalactiae (n = 50), Streptococcus anginosus group (n = 75), Enterococcus faecalis (n = 50), and vancomycin-resistant enterococci (VRE) (Enterococcus faecium) (n = 50). The MICs of tedizolid and linezolid were determined by the agar dilution method. Tedizolid exhibited better in vitro activities than linezolid against MSSA (MIC90s, 0.5 versus 2 μg/ml), MRSA (MIC90s, 0.5 versus 2 μg/ml), S. pyogenes (MIC90s, 0.5 versus 2 μg/ml), S. agalactiae (MIC90s, 0.5 versus 2 μg/ml), Streptococcus anginosus group (MIC90s, 0.5 versus 2 μg/ml), E. faecalis (MIC90s, 0.5 versus 2 μg/ml), and VRE (MIC90s, 0.5 versus 2 μg/ml). The tedizolid MICs against E. faecalis (n = 3) and VRE (n = 2) intermediate to linezolid (MICs, 4 μg/ml) were 1 μg/ml and 0.5 μg/ml, respectively. The tedizolid MIC90s against S. anginosus, S. constellatus, and S. intermedius were 0.5, 1, and 0.5 μg/ml, respectively, and the rates of susceptibility based on the U.S. FDA MIC interpretive breakpoints to the isolates were 16%, 28%, and 72%, respectively. Tedizolid exhibited 2- to 4-fold better in vitro activities than linezolid against a variety of Gram-positive cocci associated with ABSSSIs and pneumonia. The lower susceptibilities of tedizolid against isolates of S. anginosus and S. constellatus than against those of S. intermedius in Taiwan were noted.

  18. [Consensus on antimicrobial sensitivity tests in gram-positive cocci. Subcommittee on Antimicrobials, SADEBAC (Argentinian Society of Clinical Bacteriology), Argentinian Association of Microbiology].

    PubMed

    Famiglietti, A; Quinteros, M; Predari, S C; Corso, A; Lopardo, H; Casellas, J M; Bantar, C; Couto, E; Galas, M; Goldberg, M; Gutkind, G; Kovensky Pupko, J; Marín, M; Nicola, F; Pasterán, F; Radice, M; Soloaga, R

    2003-01-01

    Antimicrobial susceptibility testing is mainly performed in Argentina by disk diffusion method, following National Committee for Clinical Laboratory Standards (NCCLS) recommendations. We worked out new recommendations for the reporting and interpretation of this test when dealing with gram-positive cocci, in accordance to local trends and epidemiology. General considerations for performing the diffusion assay, quality control, and an update on susceptibility testing for gram-positive cocci are reported in this first document. The present update should be considered as a group of recommendations summarized by Argentinean experts and as the result of a consensus meeting coordinated by the Subcomisión de Antimicrobianos of the Sociedad Argentina de Bacteriología Clínica (Asociación Argentina de Microbiología). Experts in antimicrobial agents were convened in order to prepare this final document. These recommendations take into account local needs, affordability and availability to be used in current practice, tending to contribute to the correct antimicrobial treatment election, according to the particular microorganism and the infection sites.

  19. Gram-positive bacteria of marine origin: a numerical taxonomic study on Mediterranean isolates.

    PubMed

    Ortigosa, M; Garay, E; Pujalte, M J

    1997-12-01

    A numerical taxonomic study was performed on 65 Gram-positive wild strains of heterotrophic, aerobic, marine bacteria, and 9 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterized by 96 morphological, biochemical, physiological and nutritional tests. Clustering yielded 13 phena at 0.62 similarity level (Sl coefficient). Only one of the seven phena containing wild isolates could be identified (Bacillus marinus). A pronounced salt requirement was found in most isolates.

  20. Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

    PubMed Central

    Dargis, Rimtas; Hammer, Monja; Justesen, Ulrik Stenz; Nielsen, Xiaohui C.; Kemp, Michael

    2012-01-01

    Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful. PMID:22403420

  1. Antimicrobial interactions (synergy) of teicoplanin with two broad-spectrum drugs (cefotaxime, ofloxacin) tested against gram-positive isolates from Germany and the United States.

    PubMed

    Jones, R N; Marshall, S A; Grimm, H

    1997-10-01

    Teicoplanin, a glycopeptide, has been widely used in some nations alone and in empiric therapy combinations to address infections caused by Gram-positive cocci. However, glycopeptide resistance and the increasing incidence of oxacillin-resistant staphylococci have compromised contemporary chemotherapy. In this study, teicoplanin was tested in combinations with ampicillin, cefotaxime with and without desacetylcefotaxime, and ofloxacin against 151 Gram-positive cocci to assess the potential for enhanced action. The strains included recent isolates from the United States and Germany having well-characterized resistance mechanisms (oxacillin-resistant staphylococci, vancomycin-resistant enterococci), each tested by NCCLS methods, checkerboard synergy tests, and kill-curves. Teicoplanin alone was active (MIC90s, 0.25-2 micrograms/mL) against all species except vanA enterococci. Drug interactions of teicoplanin with beta-lactams revealed synergy and partial synergy versus oxacillin-resistant Staphylococcus spp. (67-100%) and vancomycin-resistant enterococci (70-100%), many at clinically achievable drug concentrations. However, confirming kill-curve experiments showed static action and no significant bactericidal effect. Combinations of ofloxacin with teicoplanin or cefotaxime plus desacetylcefotaxime showed a dominant additive and indifferent interaction. Teicoplanin continues to be a viable alternative to vancomycin, especially in combination therapy with selected broad-spectrum cephalosporins or fluoroquinolones. Many emerging pathogens that test resistant to individual drugs appear to be inhibited by tested combinations, extending their potential clinical utility.

  2. Isolation and Characterization of Four Gram-Positive Nickel-Tolerant Microorganisms from Contaminated Sediments

    SciTech Connect

    Van Nostrand, J. D.; Khijniak, T. V.; Gentry, T. J.; Novak, M. T.; Sowder, A. G.; Zhou, J. Z.; Bertsch, P. M.; Morris, P. J.

    2007-01-01

    Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.

  3. Characterization of unusual alkaliphilic gram-positive bacteria isolated from degraded brown alga thalluses.

    PubMed

    Ivanova, E P; Wright, J P; Lysenko, A M; Zhukova, N V; Alexeeva, Y V; Buljan, V; Kalinovskaya, N I; Nicolau, D V; Christen, R; Mikhailov, V V

    2006-01-01

    Two orange-pigmented Gram-positive, aerobic bacteria were isolated from enrichment culture during degradation of brown alga Fucus evanescens thalluses. In this work, atomic force microscopy (AFM) has been used to study the cell morphology. The non-contact mode imaging revealed unusual irregular coccoid shape of cells, possessing a single flagellum. Bacteria produced carotenoid pigments, were chemo-organotrophic, alkaliphilic and halo-tolerant growing well on nutrient media containing up to 15% NaCl. Growth temperature ranged from 5 to 45 degrees C. The DNA base compositions were 48 mol% G + C and the level of DNA similarity of two strains was conspecific (98%). A comparative phylogenetic analysis of 16S rRNA gene sequences revealed that the strain KMM 3738 tightly clustered with recently described Planococcus maritimus (99.9% 16S rRNA gene sequence similarity). DNA-DNA hybridisation experiments revealed that DNA from the KMM 3738 showed 12-15% and 16-35% of genetic relatedness with the DNA of type strains of the genera Planomicrobium and Planococcus, respectively, and 87% with DNA from Planococcus maritimus, indicating that new isolates belong to the later species. PMID:17100323

  4. Isolating "Unknown" Bacteria in the Introductory Microbiology Laboratory: A New Selective Medium for Gram-Positives.

    ERIC Educational Resources Information Center

    McKillip, John L.; Drake, MaryAnne

    1999-01-01

    Describes the development, preparation, and use of a medium that can select against a wide variety of Gram-negative bacteria while still allowing growth and differentiation of a wide range of Gram-positives. (WRM)

  5. Bacillus daliensis sp. nov., an alkaliphilic, Gram-positive bacterium isolated from a soda lake.

    PubMed

    Zhai, Lei; Liao, Tingting; Xue, Yanfen; Ma, Yanhe

    2012-04-01

    A Gram-positive, alkaliphilic bacterium, designated strain DLS13T, was isolated from Dali Lake in Inner Mongolia Autonomous Region, China. The isolate was able to grow at pH 7.5-11.0 (optimum at pH 9), in 0-8 % (w/v) NaCl (optimum at 2 %, w/v) and at 10-45 °C (optimum at 30 °C). Cells of the isolate were facultatively anaerobic, spore-forming rods with peritrichous flagella. The predominant isoprenoid quinone was MK-7 and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were anteiso-C15:0, anteiso-C17:0 and iso-C15:0. The genomic DNA G+C content of the isolate was 43.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain DLS13T was a member of the genus Bacillus and most closely related to Bacillus saliphilus DSM 15402T (96.9 % similarity). The DNA-DNA relatedness value between strain DLS13T and B. saliphilus DSM 15402T was 38.7±1.9 %. Comparative analysis of genotypic and phenotypic features indicated that strain DLS13T represents a novel species of the genus Bacillus, for which the name Bacillus daliensis sp. nov. is proposed; the type strain is DLS13T (=CGMCC 1.10369T=JCM 17097T=NBRC 107572T).

  6. Isolation and Characterization of Gram-Positive Piezophilic Bacteria from Deep Marine Subsurface Sediment

    NASA Astrophysics Data System (ADS)

    Runko, G. M.; Fang, J.; Kato, C.

    2014-12-01

    The marine deep biosphere remains as the least studied of all of Earth's habitats and is inadequately understood, but is extremely important to understand the impacts that microbes have on global biogeochemical cycles. Sediment samples were obtained during IODP Expedition 337 in the western Pacific Ocean, from 1,498 meters below the seafloor (mbsf; samples 6R3), 1,951-1,999 mbsf (19R1), and 2,406 mbsf (29R7). These samples were initially mixed with marine broth and cultivated under anaerobic conditions at pressure of 35 MPa (megapascal) and temperatures of 35° C, 45° C, and 55° C for 3 months on board the Chikyu. Single colonies were isolated via plating on marine broth. Then, six strains of bacteria were identified, 6R3-1, 6R3-15, 19R1-5, 29R7-12B, 29R7-12M, and 29R7-12S. The six strains were then examined for optimal growth temperature and pressure. These organisms are Gram-positive, spore-forming, facultative anaerobic piezophilic bacteria. Major fatty acids are anteiso-15:0, anteiso-17:0 and iso-15:0. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolates are closely related to Virgibacillus pantothenticus, Robinsoniella peoriensis, and Bacillus subtilis. Because of their abundance in the deep marine subsurface, these microorganisms likely play an important role in sustaining the deep microbial ecosystem and influencing biogeochemical cycles in the deep biosphere.

  7. Paucisalibacillus globulus gen. nov., sp. nov., a Gram-positive bacterium isolated from potting soil.

    PubMed

    Nunes, Inês; Tiago, Igor; Pires, Ana Luísa; da Costa, Milton S; Veríssimo, António

    2006-08-01

    A Gram-positive bacterium, designated B22(T), was isolated from potting soil produced in Portugal. This organism is a catalase-positive, oxidase-negative, motile, spore-forming, aerobic rod that grows optimally at 37 degrees C and pH 8.0-8.5. Optimal growth occurs in media containing 1 % (w/v) NaCl, although the organism can grow in 0-8 % NaCl. The cell wall peptidoglycan is of the A4alpha type with a cross-linkage containing d-Asp. The major respiratory quinone is menaquinone 7 and the major fatty acids are anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0. The DNA G+C content is 37.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain B22(T) formed a new branch within the family Bacillaceae. The novel isolate is phylogenetically closely related to members of genera of moderately halophilic bacilli and formed a coherent cluster with species of the genera Salinibacillus, Virgibacillus, Oceanobacillus and Lentibacillus, supported by bootstrap analysis at a confidence level of 71 %. Strain B22(T) exhibited 16S rRNA gene pairwise sequence similarity values of 94.7-94.3 % with members of the genus Salinibacillus, 95.1-92.8 % with members of the genus Virgibacillus, 94.7-93.2 % with members of the genus Oceanobacillus and 93.1-92.3 % with members of the genus Lentibacillus. On the basis of phylogenetic analysis and physiological and biochemical characteristics, it is proposed that strain B22(T) represents a novel species in a new genus, Paucisalibacillus globulus gen. nov., sp. nov. Strain B22(T) (=LMG 23148(T)=CIP 108857(T)) is the type strain of Paucisalibacillus globulus.

  8. Paucisalibacillus globulus gen. nov., sp. nov., a Gram-positive bacterium isolated from potting soil.

    PubMed

    Nunes, Inês; Tiago, Igor; Pires, Ana Luísa; da Costa, Milton S; Veríssimo, António

    2006-08-01

    A Gram-positive bacterium, designated B22(T), was isolated from potting soil produced in Portugal. This organism is a catalase-positive, oxidase-negative, motile, spore-forming, aerobic rod that grows optimally at 37 degrees C and pH 8.0-8.5. Optimal growth occurs in media containing 1 % (w/v) NaCl, although the organism can grow in 0-8 % NaCl. The cell wall peptidoglycan is of the A4alpha type with a cross-linkage containing d-Asp. The major respiratory quinone is menaquinone 7 and the major fatty acids are anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0. The DNA G+C content is 37.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain B22(T) formed a new branch within the family Bacillaceae. The novel isolate is phylogenetically closely related to members of genera of moderately halophilic bacilli and formed a coherent cluster with species of the genera Salinibacillus, Virgibacillus, Oceanobacillus and Lentibacillus, supported by bootstrap analysis at a confidence level of 71 %. Strain B22(T) exhibited 16S rRNA gene pairwise sequence similarity values of 94.7-94.3 % with members of the genus Salinibacillus, 95.1-92.8 % with members of the genus Virgibacillus, 94.7-93.2 % with members of the genus Oceanobacillus and 93.1-92.3 % with members of the genus Lentibacillus. On the basis of phylogenetic analysis and physiological and biochemical characteristics, it is proposed that strain B22(T) represents a novel species in a new genus, Paucisalibacillus globulus gen. nov., sp. nov. Strain B22(T) (=LMG 23148(T)=CIP 108857(T)) is the type strain of Paucisalibacillus globulus. PMID:16902018

  9. In vitro activity of paldimycin (U-70138F) against gram-positive bacteria isolated from patients with cancer.

    PubMed Central

    Rolston, K V; LeBlanc, B; Ho, D H; Bodey, G P

    1987-01-01

    The in vitro activity of paldimycin, a novel antimicrobial agent, was compared with that of vancomycin against 306 gram-positive isolates (representing 12 bacterial species) obtained from patients with cancer. Paldimycin had lower MICs for 90% of isolates than vancomycin did against most isolates tested. Its activity, however, was medium and pH dependent, being greatest in Nutrient broth at a pH of 6.8. PMID:3606069

  10. Isolation and Characterization of Gram-Positive Biosurfactant-Producing Halothermophilic Bacilli From Iranian Petroleum Reservoirs

    PubMed Central

    Zargari, Saeed; Ramezani, Amin; Ostvar, Sassan; Rezaei, Rasool; Niazi, Ali; Ayatollahi, Shahab

    2014-01-01

    Background: Petroleum reservoirs have long been known as the hosts of extremophilic microorganisms. Some of these microorganisms are known for their potential biotechnological applications, particularly production of extra and intracellular polymers and enzymes. Objectives: Here, 14 petroleum liquid samples from southern Iranian oil reservoirs were screened for presence of biosurfactant‐producing halothermophiles. Materials and Methods: Mixture of the reservoir fluid samples with a minimal growth medium was incubated under an N2 atmosphere in 40°C; 0.5 mL samples were transferred from the aqueous phase to agar plates after 72 hours of incubation; 100 mL cell cultures were prepared using the MSS-1 (mineral salt solution 1) liquid medium with 5% (w/v) NaCl. The time-course samples were analyzed by recording the absorbance at 600 nm using a spectrophotometer. Incubation was carried out in 40°C with mild shaking in aerobic conditions. Thermotolerance was evaluated by growing the isolates at 40, 50, 60 and 70°C with varying NaCl concentrations of 5% and 10% (w/v). Halotolerance was evaluated using NaCl concentrations of 5%, 10%, 12.5% and 15% (w/v) and incubating them at 40°C under aerobic and anaerobic conditions. Different phenotypic characteristics were evaluated, as outlined in Bergey's manual of determinative bacteriology. Comparing 16S rDNA sequences is one of the most powerful tools for classification of microorganisms. Results: Among 34 isolates, 10 demonstrated biosurfactant production and growth at temperatures between 40°C and 70°C in saline media containing 5%‐15% w/v NaCl. Using partial 16S rDNA sequencing (and amplified ribosomal DNA restriction analysis [ARDRA]) and biochemical tests (API tests 20E and 50 CHB), all the 10 isolates proved to be facultative anaerobic, Gram-positive moderate thermohalophiles of the genus Bacillus (B. thermoglucosidasius, B. thermodenitrificans, B. thermoleovorans, B. stearothermophilus and B. licheniformis

  11. Thalassobacillus pellis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from salted hides.

    PubMed

    Sánchez-Porro, Cristina; Yilmaz, Pinar; de la Haba, Rafael R; Birbir, Meral; Ventosa, Antonio

    2011-05-01

    A Gram-positive, moderately halophilic and endospore-forming bacterium, designated strain 18OM(T), was isolated from salted animal hides. The cells were rods and produced ellipsoidal endospores at a terminal position. Strain 18OM(T) was motile, strictly aerobic and grew at 0.5-25 % (w/v) NaCl [optimal growth at 10 % (w/v) NaCl], at between pH 5.0 and 9.0 (optimal growth at pH 7.5) and at temperatures between 15 and 45 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain 18OM(T) was closely related to species of the genus Thalassobacillus within the phylum Firmicutes. The closest phylogenetic similarity was with Thalassobacillus devorans G-19.1(T) (98.4 %), Thalassobacillus cyri HS286(T) (97.9 %) and Thalassobacillus hwangdonensis AD-1(T) (97.4 %). The major cellular fatty acids were anteiso-C(15 : 0) (57.9 %), anteiso-C(17 : 0) (14.0 %), iso-C(15 : 0) (10.8 %) and iso-C(16 : 0) (8.1 %). The respiratory isoprenoid quinones were MK-7 (98.5 %) and MK-6 (1.5 %). The DNA G+C content was 42.9 mol%. These features confirmed the placement of strain 18OM(T) within the genus Thalassobacillus. The DNA-DNA hybridization values between strain 18OM(T) and T. devorans G-19.1(T), T. cyri HS286(T) and T. hwangdonensis AD-1(T) were 49 %, 9 % and 15 %, respectively, showing unequivocally that strain 18OM(T) constituted a novel genospecies. On the basis of phylogenetic analysis and phenotypic, genotypic and chemotaxonomic characteristics, strain 18OM(T) is considered to represent a novel species of the genus Thalassobacillus, for which the name Thalassobacillus pellis sp. nov. is proposed. The type strain is 18OM(T) ( = CECT 7566(T) = DSM 22784(T) = JCM 16412(T)).

  12. In vitro activities of a new lipopeptide, HMR 1043, against susceptible and resistant gram-positive isolates.

    PubMed

    Bemer, Pascale; Juvin, Marie-Emmanuelle; Bryskier, Andre; Drugeon, Henri

    2003-09-01

    The purpose of this study was to compare the activity of HMR 1043 with those of daptomycin and teicoplanin against gram-positive isolates. Susceptibility tests were performed for 52 strains, 26 parental strains, including staphylococcal, streptococcal, enterococcal, and listerial strains, and 26 HMR 1043-resistant mutants obtained from parental strains by using the Szybalski method. Agar dilution and disk diffusion susceptibility tests were performed by the procedures outlined by the NCCLS. HMR 1043 demonstrated good activity against susceptible and resistant gram-positive bacteria. The activity of HMR 1043 in vitro was less influenced by the presence of calcium ions than that of daptomycin. Susceptibility test breakpoints were not defined because of the poor correlation coefficients obtained with the different disks tested.

  13. In Vitro Activities of a New Lipopeptide, HMR 1043, against Susceptible and Resistant Gram-Positive Isolates

    PubMed Central

    Bemer, Pascale; Juvin, Marie-Emmanuelle; Bryskier, Andre; Drugeon, Henri

    2003-01-01

    The purpose of this study was to compare the activity of HMR 1043 with those of daptomycin and teicoplanin against gram-positive isolates. Susceptibility tests were performed for 52 strains, 26 parental strains, including staphylococcal, streptococcal, enterococcal, and listerial strains, and 26 HMR 1043-resistant mutants obtained from parental strains by using the Szybalski method. Agar dilution and disk diffusion susceptibility tests were performed by the procedures outlined by the NCCLS. HMR 1043 demonstrated good activity against susceptible and resistant gram-positive bacteria. The activity of HMR 1043 in vitro was less influenced by the presence of calcium ions than that of daptomycin. Susceptibility test breakpoints were not defined because of the poor correlation coefficients obtained with the different disks tested. PMID:12937020

  14. Bactericidal effect of ultraviolet C (UVC), direct and filtered through transparent plastic, on gram-positive cocci: an in vitro study.

    PubMed

    Rao, Bhamini K; Kumar, Pramod; Rao, Sugandhi; Gurung, Bimala

    2011-07-01

    The prevalence of wound infections caused by multidrug-resistant (MDR) bacteria is increasing along with concern about widespread use of antibiotics. In vitro studies have shown that ultraviolet radiation, especially UVC, is both an effective bactericidal and antifungal. However, evidence about its bactericidal effect on wounds covered with transparent dressings remains inconclusive. Transparent dressings are used to retain moisture over the wound as part of an intermittent negative pressure dressing-the Limited Access Dressing (LAD) technique. Because this dressing is designed to remain in place for a number of days, an in vitro study was conducted to explore the bactericidal effect of direct and indirect UVR through a transparent 0.15-mm thick transparent polythene sheet on Gram-positive cocci. Six bacterial strains were inoculated to sheep blood agar (SBA) plates and exposed to direct and filtered UVC (254 nm) for 5, 10, 15, 20, 25, and 30 seconds with one media serving as a control (no UVC exposure). Plates were subsequently incubated for 24 hours and bacterial growth observed. Each set of experiment was repeated three times. Direct UVC was shown to have good bactericidal effect (100% eradication of organisms inoculated) at durations ranging from a minimum of 5 seconds (methicillin-resistant, coagulase-negative Staphylococcus and Streptococcus pyogenes) to a maximum of 15 seconds (methicillin-susceptible Staphylococcus aureus and Enterococci species). No bactericidal effect was observed when UVC was filtered through a 0.15-mm thick transparent polythene sheet. The results confirm the bactericidal effect of UVC in vitro and suggest that this effect can be achieved after a very short period of time. At the same time, film dressings appear to filter UVC. This may help protect skin from exposure to UVC but also limits its utility for use with the LAD technique. In vivo studies to evaluate the shortest effective UVC treatment duration and follow-up clinical studies

  15. Evidence for Direct Electron Transfer by a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell▿†

    PubMed Central

    Wrighton, K. C.; Thrash, J. C.; Melnyk, R. A.; Bigi, J. P.; Byrne-Bailey, K. G.; Remis, J. P.; Schichnes, D.; Auer, M.; Chang, C. J.; Coates, J. D.

    2011-01-01

    Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction. PMID:21908627

  16. Micropruina glycogenica gen. nov., sp. nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge.

    PubMed

    Shintani, T; Liu, W T; Hanada, S; Kamagata, Y; Miyaoka, S; Suzuki, T; Nakamura, K

    2000-01-01

    A new Gram-positive non-spore-forming bacterium, strain Lg2T, was isolated from an activated sludge reactor showing enhanced biological phosphorus removal activity. The new isolate was a slowly growing organism and was capable of accumulating large amounts of intracellular glycogen from substrate taken up. Both oxidase and catalase were produced. The new isolate contained meso-diaminopimelic acid (DAP) in the cell wall. Complex fatty acid patterns with iso-C14:0, anteiso-C15:0, C16:0, iso-C16:0 and four other minor saturated or unsaturated straight-chain fatty acids were detected. The isolate contained a high genomic G+C content (70.5 mol%). Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate in the high G+C Gram-positive group with Microlunatus phosphovorus and Friedmanniella antarctica as the closest relatives (sequence similarities are 93 and 92 %, respectively). These three organisms shared common features in morphology, but strain Lg2T could be differentiated from the other species by its peptidoglycan type (meso-DAP), fatty acid composition, carbon source utilization profile and G+C content. On the basis of these findings, it is proposed that a new genus and species, Micropruina glycogenica, should be created for the new isolate; the type strain is strain Lg2T (= JCM 10248T).

  17. [GEIPC-SEIMC (Study Group for Infections in the Critically Ill Patient of the Spanish Society for Infectious Diseases and Clinical Microbiology) and GTEI-SEMICYUC ( Working Group on Infectious Diseases of the Spanish Society of Intensive Medicine, Critical Care, and Coronary Units) recommendations for antibiotic treatment of gram-positive cocci infections in the critical patient].

    PubMed

    Astigarraga, P M Olaechea; Montero, J Garnacho; Cerrato, S Grau; Colomo, O Rodríguez; Martínez, M Palomar; Crespo, R Zaragoza; García-Paredes, P Muñoz; Cerdá, E Cerdá; Lerma, F Alvarez

    2007-01-01

    In recent years, an increment of infections caused by gram-positive cocci has been documented in nosocomial and hospital-acquired-infections. In diverse countries, a rapid development of resistance to common antibiotics against gram-positive cocci has been observed. This situation is exceptional in Spain but our country might be affected in the near future. New antimicrobials active against these multi-drug resistant pathogens are nowadays available. It is essential to improve our current knowledge about pharmacokinetic properties of traditional and new antimicrobials to maximize its effectiveness and to minimize toxicity. These issues are even more important in critically ill patients because inadequate empirical therapy is associated with therapeutic failure and a poor outcome. Experts representing two scientific societies (Grupo de estudio de Infecciones en el Paciente Crítico de la SEIMC and Grupo de trabajo de Enfermedades Infecciosas de la SEMICYUC) have elaborated a consensus document based on the current scientific evidence to summarize recommendations for the treatment of serious infections caused by gram-positive cocci in critically ill patients.

  18. Production of a bacteriocin by a poultry derived Campylobacter jejuni isolate with antimicrobial activity against Clostridium perfringens and other Gram positive bacteria.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have purified a bacteriocin peptide (termed CUV-3), produced by a poultry cecal isolate of Campylobacter jejuni (strain CUV-3) with inhibitory activity against Gram positive bacteria including Clostridium perfringens (38 strains), Staphylococcus aureus, Staphylococcus epidermidis and Listeria mon...

  19. Isolation and Characterization of Four Gram-PositiveNickel-Tolerant Microorganisms from Contaminated Riparian Sediments

    SciTech Connect

    Van Nostrand, Joy D.; Khijniak, Tatiana V.; Gentry, Terry J.; Novak, Michelle T.; Sowder, Andrew G.; Zhou, Jizhong Z.; Bertsch, PaulM.; Morris, Pamela J.

    2006-08-30

    Microbial communities from riparian sediments contaminatedwith high levels of Ni and U were examined for metal-tolerantmicroorganisms. Isolation of four aerobic Ni-tolerant, Gram-positiveheterotrophic bacteria indicated selection pressure from Ni. Theseisolates were identified as Arthrobacter oxydans NR-1, Streptomycesgalbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatosporacystarginea NR-4 based on partial 16S rDNA sequences. A functional genemicroarray containing gene probes for functions associated withbiogeochemical cycling, metal homeostasis, and organic contaminantdegradation showed little overlap among the four isolates. Fifteen of thegenes were detected in all four isolates with only two of these relatedto metal resistance, specifically to tellurium. Each of the four isolatesalso displayed resistance to at least one of six antibiotics tested, withresistance to kanamycin, gentamycin, and ciprofloxacin observed in atleast two of the isolates. Further characterization of S. aureofaciensNR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Nitolerance constitutively. In addition, both were able to grow in higherconcentrations of Ni at pH 6 as compared to pH 7 (42.6 and 8.5 mM Ni atpH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examinedin these two isolates; a similar pH-dependent metal tolerance wasobserved when grown with Co and Zn. Neither isolate was tolerant to Cd.These findings suggest that Ni is exerting a selection pressure at thissite for metal-resistant actinomycetes.

  20. [Estimation of activity of pharmakopeal disinfectants and antiseptics against Gram-negative and Gram-positive bacteria isolated from clinical specimens, drugs and environment].

    PubMed

    Grzybowska, Wanda; Młynarczyk, Grazyna; Młynarczyk, Andrzej; Bocian, Ewa; Luczak, Mirosław; Tyski, Stefan

    2007-01-01

    The MIC of nine different disinfectants and antiseptics were determined for the Gram-negative and Gram-positive bacteria. Strains originated from clinical specimens, drugs and environment. A sensitivity was determined against chlorhexidinum digluconate (Gram-negative: 0,625-80 mg/L, Gram-positive: 0,3-10 mg/L), benzalconium chloride (Gram-negative: 2,5-1280 mg/L, Gram-positive: 1,25-20 mg/L), salicilic acid (Gram-negative and Gram-positive: 400-1600 mg/L), benzoic acid (Gram-negative: 800-1600 mg/L, Gram-positive: 400-1 600 mg/L), boric acid (Gram-negative: 800-12 800 mg/L, Gram-positive: 1 600-6400 mg/L), chloramine B (Gram-negative: 1600-6400 mg/L, Gram-positive:800- 6400 mg/L), jodine (Gram-negative: 200-1600 mg/L, Gram-positive: 200-1600 mg/L), etacridine lactate (Gram-negative: 40 do > 20480 mg/L, Gram-positive: 40-1280 mg/L) and resorcine (Gram-negative: 1600-6400 mg/L, Gram-positive: 800-6400 mg/L). Diversified values of MIC for different strains were obtained, especially in the case of benzalconium chloride, etacridine lactate, chlorhexidinum digluconate, boric acid and iodine. Strains isolated from environment were usually more susceptible to examined compounds than clinical strains. The biggest diversification of sensitivity was observed among strains originated from drugs where besides sensitive appeared strains characterizing by very high MIC values of some substances, eg. boric acid.

  1. Virgibacillus subterraneus sp. nov., a moderately halophilic Gram-positive bacterium isolated from subsurface saline soil.

    PubMed

    Wang, Xiaowei; Xue, Yanfen; Ma, Yanhe

    2010-12-01

    A Gram reaction-positive, moderately halophilic bacterium, designated H57B72(T), was isolated from subsurface saline soil of Qaidam basin in the Qinghai province, China. Cells were rod-shaped, strictly aerobic, spore-forming and motile. The isolate grew optimally at 9 % (w/v) NaCl, pH7.5 and 30°C. The cell-wall peptidoglycan of strain H57B72(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was MK-7. The major cellular fatty acids were anteiso-C(15 : 0) (59.97 %) and anteiso-C(17 : 0) (17.14 %). Phosphatidylglycerol, diphosphatidylglycerol and a glycolipid were found to be the predominant polar lipids. The genomic DNA G+C content of strain H57B72(T) was 37.1mol%. 16S rRNA gene sequence analysis showed that strain H57B72(T) was a member of the genus Virgibacillus and was most closely related to Virgibacillus salinus DSM 21756(T) (98.3 % gene sequence similarity). The level of DNA-DNA relatedness between strain H57B72(T) and V. salinus DSM 21756(T) was 8.5 %. Based on the phenotypic, genotypic and phylogenetic data presented, strain H57B72(T) represents a novel species, for which the name Virgibacillus subterraneus sp. nov. is proposed. The type strain is H57B72(T) (=DSM 22441(T) =CGMCC 1.7734(T)). PMID:20061492

  2. Application of different molecular techniques for characterization of catalase-positive cocci isolated from sucuk.

    PubMed

    Kesmen, Zülal; Yarimcam, Burcu; Aslan, Hakiye; Ozbekar, Esra; Yetim, Hasan

    2014-02-01

    This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.

  3. Application of different molecular techniques for characterization of catalase-positive cocci isolated from sucuk.

    PubMed

    Kesmen, Zülal; Yarimcam, Burcu; Aslan, Hakiye; Ozbekar, Esra; Yetim, Hasan

    2014-02-01

    This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level. PMID:24410408

  4. A poultry-intestinal isolate of Campylobacter jejuni produces a bacteriocin (CUV-3) active against a range of Gram positive bacterial pathogens including Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A newly isolated bacteriocin, CUV-3, produced by a poultry cecal isolate of Campylobacter jejuni strain CUV-3 had inhibitory activity against several Gram positive bacteria including Clostridium perfringens (38 strains), Staphylococcus aureus, Staph.epidermidis and Listeria monocytogenes. The pept...

  5. Microcella putealis gen. nov., sp. nov., a gram-positive alkaliphilic bacterium isolated from a nonsaline alkaline groundwater.

    PubMed

    Tiago, Igor; Pires, Carlos; Mendes, Vítor; Morais, Paula V; da Costa, Milton; Veríssimo, António

    2005-08-01

    Three Gram-positive bacteria designated CV-2T, CV-40 and AC-30 were isolated from a highly alkaline groundwater environment (pH 11.4). These organisms formed very small rod-shaped cells, are aerobic, non-spore-forming, catalase-positive, oxidase-negative, with an optimum growth temperature of 35 degrees C and optimum pH value of growth between 8.5 and 9.0. The strains possessed a novel B-type cell-wall peptidoglycan structure with lysine as the diamino acid; the major respiratory quinones were menaquinone 12 (MK12) and MK13. The G + C content of DNA was between 67.1 and 70.7 mol%. The phylogenetic analyses of the sequences of the 16S rRNA genes reveled that they formed a deep branch within the family Microbacteriaceae, with the highest similarity of approximately 95.6% with members of the genera Agreia, Agrococcus, Cryobacterium, Clavibacter, Frigoribacterium, Leifsonia, Mycetocola, Rhodoglobus, Salinibacterium and Subtercola. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strains CV-2T, CV-40 and AC-30, represent a new species of a novel genus within the family Microbacteriaceae for which we propose the name Microcella putealis gen. nov., sp. nov.

  6. Bacillus alkalicola sp. nov., an alkaliphilic, gram-positive bacterium isolated from Zhabuye Lake in Tibet, China.

    PubMed

    Zhai, Lei; Ma, Yiwei; Xue, Yanfen; Ma, Yanhe

    2014-09-01

    A Gram-positive, alkaliphilic bacterium, designated strain Zby6(T), was isolated from Zhabuye Lake in Tibet, China. The strain was able to grow at pH 8.0-11.0 (optimum at pH 10.0), in 0-8 % (w/v) NaCl (optimum at 3 %, w/v) and at 10-45 °C (optimum at 37 °C). Cells of the isolate were facultatively anaerobic and spore-forming rods with polar flagellum. The predominant isoprenoid quinone was MK-7, and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C(15:0), C(16:0) and anteiso-C(15:0). The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the isolate was 38.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Zby6(T) was a member of the genus Bacillus and most closely related to Bacillus cellulosilyticus DSM 2522(T) (97.7 % similarity). The DNA-DNA relatedness value between strain Zby6(T) and B. cellulosilyticus DSM 2522(T) was 59.2 ± 1.8 %. Comparative analysis of genotypic and phenotypic features indicated that strain Zby6(T) represents a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed; the type strain is Zby6(T) (=CGMCC 1.10368(T) = JCM 17098(T) = NBRC 107743(T)).

  7. Phenotypic antimicrobial susceptibility and occurrence of selected resistance genes in gram-positive mastitis pathogens isolated from Wisconsin dairy cows.

    PubMed

    Ruegg, P L; Oliveira, L; Jin, W; Okwumabua, O

    2015-07-01

    . uberis carried multiple resistance genes. Results indicate that most gram-positive mastitis organisms were susceptible to most antimicrobials used for intramammary administration, but some resistance to drugs used for systemic treatment of mastitis was noted. The presence of selected resistance genes was not proportional to the occurrence of phenotypic resistance. PMID:25912858

  8. Differential toxicity of Al2O3 particles on Gram-positive and Gram-negative sediment bacterial isolates from freshwater.

    PubMed

    Bhuvaneshwari, M; Bairoliya, Sakcham; Parashar, Abhinav; Chandrasekaran, N; Mukherjee, Amitava

    2016-06-01

    The current study was aimed to explore the differential effects on Gram-positive and Gram-negative freshwater sediment bacterial isolates upon exposure to nano-particles and bulk particles of Al2O3 at low concentrations (0.25, 0.5, and 1 mg/L). The Gram-negative Pseudomonas aeruginosa was more susceptible to both the nano-forms and bulk forms than the Gram-positive Bacillus altitudinis. The generation of reactive oxygen species (ROS) and release of lipopolysaccharide due to membrane damage were dependent on the dose of nano-Al2O3. The Fourier transform infrared spectroscopy (FT-IR) studies confirmed the attachment of nano-Al2O3 on bacterial cells, which may lead to subsequent changes in the cell membrane composition and integrity. Internalization of nano-Al2O3 was estimated to be more for P. aeruginosa than for B. altitudinis cells. As a role of defense mechanism, the biofilm formation and production of extracellular polymeric substances (EPSs; polysaccharide and protein) were increased with respect to the concentration of toxicant. Nano-Al2O3 was estimated to cause more DNA damage than the bulk particles in both Gram-positive and Gram-negative bacterial strains.

  9. Salirhabdus euzebyi gen. nov., sp. nov., a Gram-positive, halotolerant bacterium isolated from a sea salt evaporation pond.

    PubMed

    Albuquerque, Luciana; Tiago, Igor; Rainey, Fred A; Taborda, Marco; Nobre, M Fernanda; Veríssimo, António; da Costa, Milton S

    2007-07-01

    A low-G+C, Gram-positive bacterium, designated CVS-14(T), was recovered from a sea salt evaporation pond on the island of Sal in the Cape Verde Archipelago. This organism was catalase- and oxidase-positive. Cells were motile, spore-forming aerobic rods, with an optimum growth temperature of about 35-40 degrees C and optimum pH between 7.0 and 8.5. Optimal growth occurred in media containing 4-6 % (w/v) NaCl, although the organism was able to grow in medium without added NaCl and in medium containing 16 % NaCl. The cell-wall peptidoglycan was of A1 gamma type and the major respiratory quinone was menaquinone 7 (MK-7). Major fatty acids were iso-15 : 0, anteiso-15 : 0, iso-17 : 0 and anteiso-17 : 0. The DNA G+C content was 37.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-14(T) formed a distinct new branch within the radiation of the moderately halophilic bacilli group, forming a separate lineage from species of the genera Salinibacillus, Paucisalibacillus, Oceanobacillus, Lentibacillus and Virgibacillus. Strain CVS-14(T) showed 16S rRNA gene pairwise similarity values of approximately 95 % with species of the genus Salinibacillus. On the basis of morphological, physiological, chemotaxonomic and phylogenetic characteristics, strain CVS-14(T) is considered to represent a novel species in a new genus, for which the name Salirhabdus euzebyi gen. nov., sp. nov. is proposed. The type strain is CVS-14(T) (=LMG 22839(T)=CIP 108577(T)).

  10. Salirhabdus euzebyi gen. nov., sp. nov., a Gram-positive, halotolerant bacterium isolated from a sea salt evaporation pond.

    PubMed

    Albuquerque, Luciana; Tiago, Igor; Rainey, Fred A; Taborda, Marco; Nobre, M Fernanda; Veríssimo, António; da Costa, Milton S

    2007-07-01

    A low-G+C, Gram-positive bacterium, designated CVS-14(T), was recovered from a sea salt evaporation pond on the island of Sal in the Cape Verde Archipelago. This organism was catalase- and oxidase-positive. Cells were motile, spore-forming aerobic rods, with an optimum growth temperature of about 35-40 degrees C and optimum pH between 7.0 and 8.5. Optimal growth occurred in media containing 4-6 % (w/v) NaCl, although the organism was able to grow in medium without added NaCl and in medium containing 16 % NaCl. The cell-wall peptidoglycan was of A1 gamma type and the major respiratory quinone was menaquinone 7 (MK-7). Major fatty acids were iso-15 : 0, anteiso-15 : 0, iso-17 : 0 and anteiso-17 : 0. The DNA G+C content was 37.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-14(T) formed a distinct new branch within the radiation of the moderately halophilic bacilli group, forming a separate lineage from species of the genera Salinibacillus, Paucisalibacillus, Oceanobacillus, Lentibacillus and Virgibacillus. Strain CVS-14(T) showed 16S rRNA gene pairwise similarity values of approximately 95 % with species of the genus Salinibacillus. On the basis of morphological, physiological, chemotaxonomic and phylogenetic characteristics, strain CVS-14(T) is considered to represent a novel species in a new genus, for which the name Salirhabdus euzebyi gen. nov., sp. nov. is proposed. The type strain is CVS-14(T) (=LMG 22839(T)=CIP 108577(T)). PMID:17625195

  11. A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.

    PubMed

    Sung, Kidon; Khan, Saeed A; Nawaz, Mohamed S; Khan, Ashraf A

    2003-12-01

    A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments. PMID:14659548

  12. Emergence of Carbapenem resistant Gram negative and vancomycin resistant Gram positive organisms in bacteremic isolates of febrile neutropenic patients: A descriptive study

    PubMed Central

    Irfan, Seema; Idrees, Faiza; Mehraj, Vikram; Habib, Faizah; Adil, Salman; Hasan, Rumina

    2008-01-01

    Background This study was conducted to evaluate drug resistance amongst bacteremic isolates of febrile neutropenic patients with particular emphasis on emergence of carbapenem resistant Gram negative bacteria and vancomycin resistant Enterococcus species. Methods A descriptive study was performed by reviewing the blood culture reports from febrile neutropenic patients during the two study periods i.e., 1999–00 and 2001–06. Blood cultures were performed using BACTEC 9240 automated system. Isolates were identified and antibiotic sensitivities were done using standard microbiological procedures. Results Seven twenty six febrile neutropenic patients were admitted during the study period. A total of 5840 blood cultures were received, off these 1048 (18%) were culture positive. Amongst these, 557 (53%) grew Gram positive bacteria, 442 (42%) grew Gram negative bacteria, 43 (4%) fungi and 6 (1%) anaerobes. Sixty (5.7%) out of 1048 positive blood cultures were polymicrobial. In the Gram negative bacteria, Enterobacteriaceae was the predominant group; E. coli was the most frequently isolated organism in both study periods. Amongst non- Enterobacteriaceae group, Pseudomonas aeruginosa was the commonest organism isolated during first study period followed by Acinetobacter spp. However, during the second period Acinetobacter species was the most frequent pathogen. Enterobacteriaceae group showed higher statistically significant resistance in the second study period against ceftriaxone, quinolone and piperacillin/tazobactam, whilst no resistance observed against imipenem/meropenem. The susceptibility pattern of Acinetobacter species shifted from sensitive to highly resistant one with significant p values against ceftriaxone, quinolone, piperacillin/tazobactam and imipenem/meropenem. Amongst Gram positive bacteria, MRSA isolation rate remained static, vancomycin resistant Enterococcus species emerged in second study period while no Staphylococcus species resistant to

  13. In vitro activity of tedizolid against gram-positive bacteria in patients with skin and skin structure infections and hospital-acquired pneumonia: a Korean multicenter study.

    PubMed

    Lee, Yangsoon; Hong, Sung Kuk; Choi, Sunghak; Im, Weonbin; Yong, Dongeun; Lee, Kyungwon

    2015-09-01

    We compared the activities of tedizolid to those of linezolid and other commonly used antimicrobial agents against gram-positive cocci recovered from patients with skin and skin structure infections (SSSIs) and hospital-acquired pneumonia (HAP) in Korean hospitals. Gram-positive isolates were collected from 356 patients with SSSIs and 144 patients with HAP at eight hospitals in Korea from 2011 to 2014. SSSIs included impetigo, cellulitis, erysipelas, furuncles, abscesses, and infected burns. Antimicrobial susceptibility was tested by using the CLSI agar dilution method. All of the gram-positive isolates were inhibited by ≤1 μg/mL tedizolid. The minimum inhibitory concentration [MIC]₉₀ of tedizolid was 0.5 μg/mL for methicillin-resistant Staphylococcus aureus, which was 4-fold lower than that of linezolid. Tedizolid may become a useful option for the treatment of SSSIs and HAP caused by gram-positive bacteria. PMID:26206690

  14. In vitro activity of tedizolid against gram-positive bacteria in patients with skin and skin structure infections and hospital-acquired pneumonia: a Korean multicenter study.

    PubMed

    Lee, Yangsoon; Hong, Sung Kuk; Choi, Sunghak; Im, Weonbin; Yong, Dongeun; Lee, Kyungwon

    2015-09-01

    We compared the activities of tedizolid to those of linezolid and other commonly used antimicrobial agents against gram-positive cocci recovered from patients with skin and skin structure infections (SSSIs) and hospital-acquired pneumonia (HAP) in Korean hospitals. Gram-positive isolates were collected from 356 patients with SSSIs and 144 patients with HAP at eight hospitals in Korea from 2011 to 2014. SSSIs included impetigo, cellulitis, erysipelas, furuncles, abscesses, and infected burns. Antimicrobial susceptibility was tested by using the CLSI agar dilution method. All of the gram-positive isolates were inhibited by ≤1 μg/mL tedizolid. The minimum inhibitory concentration [MIC]₉₀ of tedizolid was 0.5 μg/mL for methicillin-resistant Staphylococcus aureus, which was 4-fold lower than that of linezolid. Tedizolid may become a useful option for the treatment of SSSIs and HAP caused by gram-positive bacteria.

  15. Streptohalobacillus salinus gen. nov., sp. nov., a moderately halophilic, Gram-positive, facultative anaerobe isolated from subsurface saline soil.

    PubMed

    Wang, Xiaowei; Xue, Yanfen; Ma, Yanhe

    2011-05-01

    A Gram-stain-positive, rod-shaped, non-sporulating, motile and moderately halophilic bacterium, designated strain H96B60(T), was isolated from a saline soil sample of the Qaidam basin, China. The strain was facultatively anaerobic. Major end products formed from glucose fermentation were acetate, ethanol and lactic acid. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The isoprenoid quinone component was menaquinone-6 (MK-6). The predominant cellular fatty acids were C(16: 0), anteiso-C(13 : 0) and anteiso-C(15 : 0). The genomic DNA G+C content of strain H96B60(T) was 36.2 mol%. Phylogenetic analysis based on comparative 16S rRNA gene sequences indicated that strain H96B60(T) represented a novel phyletic lineage within the family Bacillaceae and was related most closely to Halolactibacillus species (96.1-96.4 % similarity). Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain H96B60(T) is considered to represent a novel species of a new genus, for which the name Streptohalobacillus salinus gen. nov., sp. nov. is proposed. The type strain of Streptohalobacillus salinus is H96B60(T) ( = DSM 22440(T)  = CGMCC 1.7733(T)).

  16. In vitro activity of ceftazidime, ceftaroline and aztreonam alone and in combination with avibactam against European Gram-negative and Gram-positive clinical isolates.

    PubMed

    Testa, Raymond; Cantón, Rafael; Giani, Tommaso; Morosini, María-Isabel; Nichols, Wright W; Seifert, Harald; Stefanik, Danuta; Rossolini, Gian Maria; Nordmann, Patrice

    2015-06-01

    Recent clinical isolates of key Gram-negative and Gram-positive bacteria were collected in 2012 from hospitalised patients in medical centres in four European countries (France, Germany, Italy and Spain) and were tested using standard broth microdilution methodology to assess the impact of 4 mg/L avibactam on the in vitro activities of ceftazidime, ceftaroline and aztreonam. Against Enterobacteriaceae, addition of avibactam significantly enhanced the level of activity of these antimicrobials. MIC(90) values (minimum inhibitory concentration that inhibits 90% of the isolates) of ceftazidime, ceftaroline and aztreonam for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Morganella morganii were reduced up to 128-fold or greater when combined with avibactam. A two-fold reduction in the MIC(90) of ceftazidime to 8 mg/L was noted in Pseudomonas aeruginosa isolates when combined with avibactam, whereas little effect of avibactam was noted on the MIC values of the test compounds when tested against Acinetobacter baumannii isolates. Avibactam had little effect on the excellent activity of ceftazidime, ceftaroline and aztreonam against Haemophilus influenzae. It had no impact on the in vitro activity of ceftazidime and ceftaroline against staphylococci and streptococci. This study demonstrates that addition of avibactam enhances the activities of ceftazidime, ceftaroline and aztreonam against Enterobacteriaceae and P. aeruginosa but not against A. baumannii.

  17. Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria.

    PubMed

    Snapper, Clifford M

    2016-06-24

    Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines.

  18. Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria.

    PubMed

    Snapper, Clifford M

    2016-06-24

    Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines. PMID:27133879

  19. Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients.

    PubMed

    Miteva, Vanya; Lantz, Sarah; Brenchley, Jean

    2008-05-01

    Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.

  20. Postantibiotic effect of ceftaroline against gram-positive organisms.

    PubMed

    Pankuch, G A; Appelbaum, P C

    2009-10-01

    The postantibiotic effects (PAEs), postantibiotic sub-MIC effects (PA-SMEs), and sub-MIC effects (SMEs) of ceftaroline, a novel injectable cephalosporin, were determined for 15 gram-positive organisms. The pneumococcal, staphylococcal, and enterococcal PAEs were 0.8 to 1.8 h, 0.7 to 2.2 h, and 0.2 to 1.1 h, respectively. The corresponding PA-SMEs (0.4 times the MIC) were 2.5 to 6.7 h, 2.9 to >0.0 h, and 7.9 to >10.3 h, respectively. The PA-SMEs were longer than the PAEs, suggesting that sub-MIC levels extend the PAE of ceftaroline against gram-positive cocci.

  1. Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram-Positive Bacteria

    PubMed Central

    Yang, Xu; Yuan, Chunhua; Zhang, Liwen

    2016-01-01

    A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporus via morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance liquid chromatography, and structurally analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of 13 amino acids and an N-terminal C6 fatty acid (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA-Dhb-Leu-Orn-Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu-valinol, where Dhb is α,β-didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1,583.0794 Da and contains three modified amino acid residues: α,β-didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin, was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant bacteria, brevibacillin also exhibited low MICs (1 to 8 μg/ml) against selected foodborne pathogenic and spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus, and Alicyclobacillus acidoterrestris. Purified brevibacillin showed no sign of degradation when it was held at 80°C for 60 min, and it retained at least 50% of its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings, brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens and foodborne pathogenic and spoilage bacteria. PMID:26921428

  2. Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram-Positive Bacteria.

    PubMed

    Yang, Xu; Huang, En; Yuan, Chunhua; Zhang, Liwen; Yousef, Ahmed E

    2016-05-01

    A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporusvia morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance liquid chromatography, and structurally analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of 13 amino acids and an N-terminal C6 fatty acid (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA-Dhb-Leu-Orn-Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu-valinol, where Dhb is α,β-didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1,583.0794 Da and contains three modified amino acid residues: α,β-didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin, was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant bacteria, brevibacillin also exhibited low MICs (1 to 8 μg/ml) against selected foodborne pathogenic and spoilage bacteria, such as Listeria monocytogenes,Bacillus cereus, and Alicyclobacillus acidoterrestris Purified brevibacillin showed no sign of degradation when it was held at 80 °C for 60 min, and it retained at least 50% of its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings, brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens and foodborne pathogenic and spoilage bacteria. PMID:26921428

  3. Transformation of gram positive bacteria by sonoporation

    DOEpatents

    Yang, Yunfeng; Li, Yongchao

    2014-03-11

    The present invention provides a sonoporation-based method that can be universally applied for delivery of compounds into Gram positive bacteria. Gram positive bacteria which can be transformed by sonoporation include, for example, Bacillus, Streptococcus, Acetobacterium, and Clostridium. Compounds which can be delivered into Gram positive bacteria via sonoporation include nucleic acids (DNA or RNA), proteins, lipids, carbohydrates, viruses, small organic and inorganic molecules, and nano-particles.

  4. Antibacterial activity of Lactobacillus acidophilus strains isolated from honey marketed in Malaysia against selected multiple antibiotic resistant (MAR) Gram-positive bacteria.

    PubMed

    Aween, Mohamed Mustafa; Hassan, Zaiton; Muhialdin, Belal J; Eljamel, Yossra A; Al-Mabrok, Asma Saleh W; Lani, Mohd Nizam

    2012-07-01

    A total of 32 lactic acid bacteria (LAB) were isolated from 13 honey samples commercially marketed in Malaysia, 6 strains identified as Lactobacillus acidophilus by API CHL50. The isolates had antibacterial activities against multiple antibiotic resistant's Staphylococcus aureus (25 to 32 mm), Staphylococcus epidermis (14 to 22 mm) and Bacillus subtilis (12 to 19 mm) in the agar overlay method after 24 h incubation at 30 °C. The crude supernatant was heat stable at 90 °C and 121 °C for 1 h. Treatment with proteinase K and RNase II maintained the antimicrobial activity of all the supernatants except sample H006-A and H010-G. All the supernatants showed antimicrobial activities against target bacteria at pH 3 and pH 5 but not at pH 6 within 72 h incubation at 30 °C. S. aureus was not inhibited by sample H006-A isolated from Libyan honey and sample H008-D isolated from Malaysian honey at pH 5, compared to supernatants from other L. acidophilus isolates. The presence of different strains of L. acidophilus in honey obtained from different sources may contribute to the differences in the antimicrobial properties of honey. PMID:22757710

  5. In vitro activity of linezolid against clinical Gram-positive bacterial isolates from Taiwan: an area with a high prevalence of antibiotic resistance.

    PubMed

    Fang, C T; Chang, S C; Chen, Y C; Hsieh, S M; Hsieh, W C

    2001-09-01

    The prevalence of antibiotic-resistant bacteria in Taiwan is due to the heavy use of antimicrobial agents in both animal husbandry and clinical practice over the past decades. Minimum inhibitory concentrations (MICs) of linezolid were established for 371 clinical isolates of staphylococci, pneumococci, enterococci and group A streptococci from Taiwan. All isolates tested including those resistant to beta-lactams, erythromycin, vancomycin and quinupristin-dalfopristin were uniformly susceptible to linezolid, with MICs ranging from 0.125 to 2 mg/l. Our data support the observation that there is no cross-resistance between linezolid and other classes of antimicrobial substances.

  6. Isolation and Purification of Enteroicin E-760 with a Broad Antimicrobial Activity Against Gram-positive and Gram-negative Bacteria (AAC01569-06 Version 3)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strain NRRL B-30745, isolated from chicken ceca, and identified as an Enterococcus spp. of the durans/faecium/hirae group, produces a 5362 Da bacteriocin that inhibits the growth of Salmonella enteritidis, S. choleraesuis, S. typhimurium, S. gallinarum, Escherichia coli O157:H7, Yersinia enterocolit...

  7. Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

    PubMed

    Valle, Demetrio L; Cabrera, Esperanza C; Puzon, Juliana Janet M; Rivera, Windell L

    2016-01-01

    Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria. PMID

  8. Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

    PubMed

    Valle, Demetrio L; Cabrera, Esperanza C; Puzon, Juliana Janet M; Rivera, Windell L

    2016-01-01

    Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

  9. Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance

    PubMed Central

    Valle, Demetrio L.; Cabrera, Esperanza C.; Puzon, Juliana Janet M.; Rivera, Windell L.

    2016-01-01

    Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria. PMID

  10. Naumannella halotolerans gen. nov., sp. nov., a Gram-positive coccus of the family Propionibacteriaceae isolated from a pharmaceutical clean room and from food.

    PubMed

    Rieser, Gernot; Scherer, Siegfried; Wenning, Mareike

    2012-12-01

    Four Gram-stain-positive, aerobic bacterial strains isolated from a pharmaceutical clean room (strain WS4616(T)), a dessert milk product (strain WS4617) and from raw milk (strains WS4623 and WS4624) were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences showed that they formed a distinct lineage within the family Propionibacteriaceae. Similarity values between 16S rRNA gene sequences of the four novel strains and the type species of all genera belonging to the family Propionibacteriaceae were 89.2-94.1%. The major cellular fatty acid was anteiso-C(15:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Respiratory quinones were MK-8(H(4)) and MK-9(H(4)). The cell-wall peptidoglycan of type A3γ contained ll-diaminopimelic acid, alanine, glycine and glutamic acid. The G+C content of the genomic DNA of strain WS4616(T) was 67.7 mol%. The whole-cell sugar pattern contained ribose, mannose, arabinose, glucose and galactose. On the basis of phenotypic and genetic data, strains WS4616(T), WS4617, WS4623 and WS4624 are classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Naumannella halotolerans gen. nov., sp. nov. is proposed. The type strain is WS4616(T) ( = DSM 24323(T) = LMG 26184(T)) and three additional strains are WS4617, WS4623 and WS4624.

  11. Evolving resistance among Gram-positive pathogens.

    PubMed

    Munita, Jose M; Bayer, Arnold S; Arias, Cesar A

    2015-09-15

    Antimicrobial therapy is a key component of modern medical practice and a cornerstone for the development of complex clinical interventions in critically ill patients. Unfortunately, the increasing problem of antimicrobial resistance is now recognized as a major public health threat jeopardizing the care of thousands of patients worldwide. Gram-positive pathogens exhibit an immense genetic repertoire to adapt and develop resistance to virtually all antimicrobials clinically available. As more molecules become available to treat resistant gram-positive infections, resistance emerges as an evolutionary response. Thus, antimicrobial resistance has to be envisaged as an evolving phenomenon that demands constant surveillance and continuous efforts to identify emerging mechanisms of resistance to optimize the use of antibiotics and create strategies to circumvent this problem. Here, we will provide a broad perspective on the clinical aspects of antibiotic resistance in relevant gram-positive pathogens with emphasis on the mechanistic strategies used by these organisms to avoid being killed by commonly used antimicrobial agents.

  12. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, L.O.; Barbosa-Alleyne, M.D.F.

    1996-01-09

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

  13. Ethanol production in gram-positive microbes

    DOEpatents

    Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

    1999-01-01

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

  14. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

    1996-01-01

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

  15. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, L.O.; Barbosa-Alleyne, M.D.F.

    1999-06-29

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

  16. Evolving Resistance Among Gram-positive Pathogens

    PubMed Central

    Munita, Jose M.; Bayer, Arnold S.; Arias, Cesar A.

    2015-01-01

    Antimicrobial therapy is a key component of modern medical practice and a cornerstone for the development of complex clinical interventions in critically ill patients. Unfortunately, the increasing problem of antimicrobial resistance is now recognized as a major public health threat jeopardizing the care of thousands of patients worldwide. Gram-positive pathogens exhibit an immense genetic repertoire to adapt and develop resistance to virtually all antimicrobials clinically available. As more molecules become available to treat resistant gram-positive infections, resistance emerges as an evolutionary response. Thus, antimicrobial resistance has to be envisaged as an evolving phenomenon that demands constant surveillance and continuous efforts to identify emerging mechanisms of resistance to optimize the use of antibiotics and create strategies to circumvent this problem. Here, we will provide a broad perspective on the clinical aspects of antibiotic resistance in relevant gram-positive pathogens with emphasis on the mechanistic strategies used by these organisms to avoid being killed by commonly used antimicrobial agents. PMID:26316558

  17. Virulence Plasmids of Nonsporulating Gram-Positive Pathogens.

    PubMed

    Van Tyne, Daria; Gilmore, Michael S

    2014-10-01

    Gram-positive bacteria are leading causes of many types of human infection, including pneumonia, skin and nasopharyngeal infections, as well as urinary tract and surgical wound infections among hospitalized patients. These infections have become particularly problematic because many of the species causing them have become highly resistant to antibiotics. The role of mobile genetic elements, such as plasmids, in the dissemination of antibiotic resistance among Gram-positive bacteria has been well studied; less well understood is the role of mobile elements in the evolution and spread of virulence traits among these pathogens. While these organisms are leading agents of infection, they are also prominent members of the human commensal ecology. It appears that these bacteria are able to take advantage of the intimate association between host and commensal, via virulence traits that exacerbate infection and cause disease. However, evolution into an obligate pathogen has not occurred, presumably because it would lead to rejection of pathogenic organisms from the host ecology. Instead, in organisms that exist as both commensal and pathogen, selection has favored the development of mechanisms for variability. As a result, many virulence traits are localized on mobile genetic elements, such as virulence plasmids and pathogenicity islands. Virulence traits may occur within a minority of isolates of a given species, but these minority populations have nonetheless emerged as a leading problem in infectious disease. This chapter reviews virulence plasmids in nonsporulating Gram-positive bacteria, and examines their contribution to disease pathogenesis.

  18. Evaluation of a Microarray-Based Assay for Rapid Identification of Gram-Positive Organisms and Resistance Markers in Positive Blood Cultures

    PubMed Central

    Tibbetts, Robert J.; Agotesku, Adam; Fey, Margaret; Hensley, Rhonda; Meier, Frederick A.

    2013-01-01

    Rapid identification of pathogens directly from positive blood cultures can play a major role in reducing patient mortality rates. We evaluated the performance of the Verigene Gram-Positive Blood Culture (BC-GP) assay (Nanosphere Inc., Northbrook, IL) for detection of commonly isolated Gram-positive organisms as well as associated resistance markers from positive blood cultures. Positive blood cultures (VersaTREK; Trek Diagnostic Systems, Independence, OH) from 203 patients with Gram-positive organism infections were analyzed using the BC-GP assay within 12 h for the detection of 12 different organisms, including staphylococci, streptococci, and enterococci, as well as for the presence of 3 resistance markers (mecA, vanA, and vanB). Results were compared to those of routine laboratory methods for identification and susceptibility testing. For identification of organisms and detection of resistance markers in 178 monomicrobial positive blood cultures, the BC-GP assay showed 94% and 97% concordance, respectively, with routine methods. After 25 polymicrobial cultures were included, the results showed 92% and 96% agreement for identification and resistance markers, respectively, for a total of 203 positive cultures. In 6/25 polymicrobial cultures, at least 1 isolate was not detected. Concordance levels for detection of major pathogens such Staphylococcus aureus (n = 45) and enterococci (n = 19) were 98% and 95%, respectively. Agreement levels for detection of resistance markers such as mecA and vanA/B were 92% and 100%, respectively. The BC-GP assay is capable of providing rapid identification of Gram-positive cocci as well as detection of resistance markers directly from positive blood cultures at least 24 to 48 h earlier than conventional methods. PMID:23363838

  19. Bacteriocins of gram-positive bacteria.

    PubMed Central

    Jack, R W; Tagg, J R; Ray, B

    1995-01-01

    In recent years, a group of antibacterial proteins produced by gram-positive bacteria have attracted great interest in their potential use as food preservatives and as antibacterial agents to combat certain infections due to gram-positive pathogenic bacteria. They are ribosomally synthesized peptides of 30 to less than 60 amino acids, with a narrow to wide antibacterial spectrum against gram-positive bacteria; the antibacterial property is heat stable, and a producer strain displays a degree of specific self-protection against its own antibacterial peptide. In many respects, these proteins are quite different from the colicins and other bacteriocins produced by gram-negative bacteria, yet customarily they also are grouped as bacteriocins. Although a large number of these bacteriocins (or bacteriocin-like inhibitory substances) have been reported, only a few have been studied in detail for their mode of action, amino acid sequence, genetic characteristics, and biosynthesis mechanisms. Nevertheless, in general, they appear to be translated as inactive prepeptides containing an N-terminal leader sequence and a C-terminal propeptide component. During posttranslational modifications, the leader peptide is removed. In addition, depending on the particular type, some amino acids in the propeptide components may undergo either dehydration and thioether ring formation to produce lanthionine and beta-methyl lanthionine (as in lantibiotics) or thio ester ring formation to form cystine (as in thiolbiotics). Some of these steps, as well as the translocation of the molecules through the cytoplasmic membrane and producer self-protection against the homologous bacteriocin, are mediated through specific proteins (enzymes). Limited genetic studies have shown that the structural gene for such a bacteriocin and the genes encoding proteins associated with immunity, translocation, and processing are present in a cluster in either a plasmid, the chromosome, or a transposon. Following

  20. Antimicrobial Peptides Targeting Gram-Positive Bacteria.

    PubMed

    Malanovic, Nermina; Lohner, Karl

    2016-01-01

    Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. PMID:27657092

  1. Antimicrobial Peptides Targeting Gram-Positive Bacteria

    PubMed Central

    Malanovic, Nermina; Lohner, Karl

    2016-01-01

    Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. PMID:27657092

  2. [Epidemiology of the infection by resistant Gram-positive microorganisms].

    PubMed

    Cercenado, E

    2016-09-01

    Resistance among Gram-positive microorganisms to classical and new antimicrobials is a therapeutic threat. In Spain, methicillin resistance among Staphylococcus aureus (25-30%) and coagulase-negative staphylococci (50-60%) seems to have stabilized in the last decade. Among enterococci, vancomycin resistance is less than 5%. Both linezolid and daptomycin, in general, show good activity against these microorganisms. However, the resistance rates of Staphylococcus epidermidis to linezolid (20.9%), and of Enterococcus faecium to daptomycin (10.5%) in isolates from intensive care units are a worrying. PMID:27608305

  3. Pili in gram-positive pathogens.

    PubMed

    Telford, John L; Barocchi, Michèle A; Margarit, Immaculada; Rappuoli, Rino; Grandi, Guido

    2006-07-01

    Most bacterial pathogens have long filamentous structures known as pili or fimbriae extending from their surface. These structures are often involved in the initial adhesion of the bacteria to host tissues during colonization. In gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunits. By contrast, the recently discovered pili in gram-positive pathogens are formed by covalent polymerization of adhesive pilin subunits. Evidence from studies of pili in the three principal streptococcal pathogens of humans indicates that the genes that encode the pilin subunits and the enzymes that are required for the assembly of these subunits into pili have been acquired en bloc by the horizontal transfer of a pathogenicity island.

  4. [GEIPC-SEIMC and GTEI-SEMICYUC recommendations for antibiotic treatment of gram positive coccal infections in the critical patient].

    PubMed

    Olaechea Astigarraga, P M; Garnacho Montero, J; Grau Cerrato, S; Rodríguez Colomo, O; Palomar Martínez, M; Zaragoza Crespo, R; Muñoz García-Paredes, P; Cerdá Cerdá, E; Alvarez Lerma, F

    2007-01-01

    In recent years, an increment of infections caused by gram-positive cocci has been documented in nosocomial and hospital-acquired infections. In diverse countries, a rapid development of resistance to common antibiotics against gram-positive cocci has been observed. This situation is exceptional in Spain but our country might be affected in the near future. New antimicrobials active against these multi-drug resistant pathogens are nowadays available. It is essential to improve our current knowledge about pharmacokinetic properties of traditional and new antimicrobials to maximize its effectiveness and to minimize toxicity. These issues are even more important in critically ill patients because inadequate empirical therapy is associated with therapeutic failure and a poor outcome. Experts representing two scientific societies (Grupo de estudio de Infecciones en el Paciente Critico de la SEIMC and Grupo de trabajo de Enfermedades Infecciosas de la SEMICYUC) have elaborated a consensus document based on the current scientific evidence to summarize recommendations for the treatment of serious infections caused by gram-positive cocci in critically ill patients.

  5. Gram-Positive Uropathogens, Polymicrobial Urinary Tract Infection, and the Emerging Microbiota of the Urinary Tract.

    PubMed

    Kline, Kimberly A; Lewis, Amanda L

    2016-04-01

    Gram-positive bacteria are a common cause of urinary-tract infection (UTI), particularly among individuals who are elderly, pregnant, or who have other risk factors for UTI. Here we review the epidemiology, virulence mechanisms, and host response to the most frequently isolated Gram-positive uropathogens: Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae. We also review several emerging, rare, misclassified, and otherwise underreported Gram-positive pathogens of the urinary tract including Aerococcus, Corynebacterium, Actinobaculum, and Gardnerella. The literature strongly suggests that urologic diseases involving Gram-positive bacteria may be easily overlooked due to limited culture-based assays typically utilized for urine in hospital microbiology laboratories. Some UTIs are polymicrobial in nature, often involving one or more Gram-positive bacteria. We herein review the risk factors and recent evidence for mechanisms of bacterial synergy in experimental models of polymicrobial UTI. Recent experimental data has demonstrated that, despite being cleared quickly from the bladder, some Gram-positive bacteria can impact pathogenic outcomes of co-infecting organisms. When taken together, the available evidence argues that Gram-positive bacteria are important uropathogens in their own right, but that some can be easily overlooked because they are missed by routine diagnostic methods. Finally, a growing body of evidence demonstrates that a surprising variety of fastidious Gram-positive bacteria may either reside in or be regularly exposed to the urinary tract and further suggests that their presence is widespread among women, as well as men. Experimental studies in this area are needed; however, there is a growing appreciation that the composition of bacteria found in the bladder could be a potentially important determinant in urologic disease, including susceptibility to UTI.

  6. Gram-Positive Uropathogens, Polymicrobial Urinary Tract Infection, and the Emerging Microbiota of the Urinary Tract

    PubMed Central

    Kline, Kimberly A.; Lewis, Amanda L.

    2015-01-01

    Gram-positive bacteria are a common cause of urinary tract infection (UTI), particularly among individuals who are elderly, pregnant, or who have other risk factors for UTI. Here we review the epidemiology, virulence mechanisms, and host response to the most frequently isolated Gram-positive uropathogens: Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae. We also review several emerging, rare, misclassified, and otherwise underreported Gram-positive pathogens of the urinary tract including Aerococcus, Corynebacterium, Actinobaculum, and Gardnerella. The literature strongly suggests that urologic diseases involving Gram-positive bacteria may be easily overlooked due to limited culture-based assays typically utilized for urine in hospital microbiology laboratories. Some UTIs are polymicrobial in nature, often involving one or more Gram-positive bacteria. We herein review the risk factors and recent evidence for mechanisms of bacterial synergy in experimental models of polymicrobial UTI. Recent experimental data has demonstrated that, despite being cleared quickly from the bladder, some Gram-positive bacteria can impact pathogenic outcomes of co-infecting organisms. When taken together, the available evidence argues that Gram-positive bacteria are important uropathogens in their own right, but that some can be easily overlooked because they are missed by routine diagnostic methods. Finally, a growing body of evidence demonstrates that a surprising variety of fastidious Gram-positive bacteria may either reside in or be regularly exposed to the urinary tract and further suggests that their presence is widespread among women, as well as men. Experimental studies in this area are needed; however, there is a growing appreciation that the composition of bacteria found in the bladder could be a potentially important determinant in urologic disease, including susceptibility to UTI. PMID:27227294

  7. Gram-Positive Uropathogens, Polymicrobial Urinary Tract Infection, and the Emerging Microbiota of the Urinary Tract.

    PubMed

    Kline, Kimberly A; Lewis, Amanda L

    2016-04-01

    Gram-positive bacteria are a common cause of urinary-tract infection (UTI), particularly among individuals who are elderly, pregnant, or who have other risk factors for UTI. Here we review the epidemiology, virulence mechanisms, and host response to the most frequently isolated Gram-positive uropathogens: Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae. We also review several emerging, rare, misclassified, and otherwise underreported Gram-positive pathogens of the urinary tract including Aerococcus, Corynebacterium, Actinobaculum, and Gardnerella. The literature strongly suggests that urologic diseases involving Gram-positive bacteria may be easily overlooked due to limited culture-based assays typically utilized for urine in hospital microbiology laboratories. Some UTIs are polymicrobial in nature, often involving one or more Gram-positive bacteria. We herein review the risk factors and recent evidence for mechanisms of bacterial synergy in experimental models of polymicrobial UTI. Recent experimental data has demonstrated that, despite being cleared quickly from the bladder, some Gram-positive bacteria can impact pathogenic outcomes of co-infecting organisms. When taken together, the available evidence argues that Gram-positive bacteria are important uropathogens in their own right, but that some can be easily overlooked because they are missed by routine diagnostic methods. Finally, a growing body of evidence demonstrates that a surprising variety of fastidious Gram-positive bacteria may either reside in or be regularly exposed to the urinary tract and further suggests that their presence is widespread among women, as well as men. Experimental studies in this area are needed; however, there is a growing appreciation that the composition of bacteria found in the bladder could be a potentially important determinant in urologic disease, including susceptibility to UTI. PMID:27227294

  8. Methods for targetted mutagenesis in gram-positive bacteria

    SciTech Connect

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  9. Invasive gram-positive bacterial infection in cancer patients.

    PubMed

    Holland, Thomas; Fowler, Vance G; Shelburne, Samuel A

    2014-11-15

    Systematic studies have shown that gram-positive organisms are the leading cause of invasive bacterial disease in patients with cancer. A broad range of gram-positive bacteria cause serious infections in the cancer patient with the greatest burden of disease being due to staphylococci, streptococci, and enterococci. The evolution of cancer therapy and the changing epidemiology of major gram-positive pathogens mean that ongoing efforts are needed to understand and mitigate the impact of these bacteria in patients with malignancy. The development of novel antibacterials, optimization of treatment approaches, implementation of improved vaccines, and manipulation of the microbiome are all active areas of investigation in the goal of improving the survival of the cancer patient through amelioration of the disease burden of gram-positive bacteria.

  10. Isolation, cultivation and genomic analysis of magnetosome biomineralization genes of a new genus of South-seeking magnetotactic cocci within the Alphaproteobacteria.

    PubMed

    Morillo, Viviana; Abreu, Fernanda; Araujo, Ana C; de Almeida, Luiz G P; Enrich-Prast, Alex; Farina, Marcos; de Vasconcelos, Ana T R; Bazylinski, Dennis A; Lins, Ulysses

    2014-01-01

    Although magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats, they are still considered fastidious microorganisms with regard to growth and cultivation with only a relatively low number of axenic cultures available to date. Here, we report the first axenic culture of an MTB isolated in the Southern Hemisphere (Itaipu Lagoon in Rio de Janeiro, Brazil). Cells of this new isolate are coccoid to ovoid in morphology and grow microaerophilically in semi-solid medium containing an oxygen concentration ([O2]) gradient either under chemoorganoheterotrophic or chemolithoautotrophic conditions. Each cell contains a single chain of approximately 10 elongated cuboctahedral magnetite (Fe3O4) magnetosomes. Phylogenetic analysis based on the 16S rRNA gene sequence shows that the coccoid MTB isolated in this study represents a new genus in the Alphaproteobacteria; the name Magnetofaba australis strain IT-1 is proposed. Preliminary genomic data obtained by pyrosequencing shows that M. australis strain IT-1 contains a genomic region with genes involved in biomineralization similar to those found in the most closely related magnetotactic cocci Magnetococcus marinus strain MC-1. However, organization of the magnetosome genes differs from M. marinus.

  11. Isolation, cultivation and genomic analysis of magnetosome biomineralization genes of a new genus of South-seeking magnetotactic cocci within the Alphaproteobacteria

    SciTech Connect

    Morillo, Viviana; Abreu, Fernanda; Araujo, Ana C; de Almeida, Luiz G; Enrich-Prast, Alex; Farina, Marcos; de Vasconcelos, Ana T; Bazylinski, Dennis A; Lins, Ulysses

    2014-01-01

    Although magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats, they are still considered fastidious microorganisms with regard to growth and cultivation with only a relatively low number of axenic cultures available to date. Here, we report the first axenic culture of an MTB isolated in the Southern Hemisphere (Itaipu Lagoon in Rio de Janeiro, Brazil). Cells of this new isolate are coccoid to ovoid in morphology and grow microaerophilically in semi-solid medium containing an oxygen concentration ([O2]) gradient either under chemoorganoheterotrophic or chemolithoautotrophic conditions. Each cell contains a single chain of approximately 10 elongated cuboctahedral magnetite (Fe3O4) magnetosomes. Phylogenetic analysis based on the 16S rRNA gene sequence shows that the coccoid MTB isolated in this study represents a new genus in the Alphaproteobacteria; the name Magnetofaba australis strain IT-1 is proposed. Preliminary genomic data obtained by pyrosequencing shows that M. australis strain IT-1 contains a genomic region with genes involved in biomineralization similar to those found in the most closely related magnetotactic cocci Magnetococcus marinus strain MC-1. However, organization of the magnetosome genes differs from M. marinus.

  12. Isolation, cultivation and genomic analysis of magnetosome biomineralization genes of a new genus of South-seeking magnetotactic cocci within the Alphaproteobacteria

    PubMed Central

    Morillo, Viviana; Abreu, Fernanda; Araujo, Ana C.; de Almeida, Luiz G. P.; Enrich-Prast, Alex; Farina, Marcos; de Vasconcelos, Ana T. R.; Bazylinski, Dennis A.; Lins, Ulysses

    2014-01-01

    Although magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats, they are still considered fastidious microorganisms with regard to growth and cultivation with only a relatively low number of axenic cultures available to date. Here, we report the first axenic culture of an MTB isolated in the Southern Hemisphere (Itaipu Lagoon in Rio de Janeiro, Brazil). Cells of this new isolate are coccoid to ovoid in morphology and grow microaerophilically in semi-solid medium containing an oxygen concentration ([O2]) gradient either under chemoorganoheterotrophic or chemolithoautotrophic conditions. Each cell contains a single chain of approximately 10 elongated cuboctahedral magnetite (Fe3O4) magnetosomes. Phylogenetic analysis based on the 16S rRNA gene sequence shows that the coccoid MTB isolated in this study represents a new genus in the Alphaproteobacteria; the name Magnetofaba australis strain IT-1 is proposed. Preliminary genomic data obtained by pyrosequencing shows that M. australis strain IT-1 contains a genomic region with genes involved in biomineralization similar to those found in the most closely related magnetotactic cocci Magnetococcus marinus strain MC-1. However, organization of the magnetosome genes differs from M. marinus. PMID:24616719

  13. Case-Control Study of Telavancin as an Alternative Treatment for Gram-Positive Bloodstream Infections in Patients with Cancer.

    PubMed

    Chaftari, Anne-Marie; Hachem, Ray; Jordan, Mary; Garoge, Kumait; Al Hamal, Zainab; El Zakhem, Aline; Viola, George M; Granwehr, Bruno; Mulanovich, Victor; Gagel, Andrew; Reitzel, Ruth; Yousif, Ammar; Jiang, Ying; Raad, Issam

    2015-10-19

    Gram-positive bacterial infections are an important cause of morbidity and death among cancer patients, despite current therapy. In this case-control study, we evaluated the clinical outcomes and safety of telavancin in cancer patients with uncomplicated Gram-positive bloodstream infections (BSIs). Between March 2011 and May 2013, we enrolled cancer patients with uncomplicated Gram-positive BSIs to receive intravenous telavancin therapy for at least 14 days for Staphylococcus aureus and 7 days for other Gram-positive cocci. Patients with baseline creatinine clearance (CLCR) values of >50 ml/min received 10 mg/kg/day of telavancin, and those with CLCR values between 30 and 49 ml/min received 7.5 mg/kg/day. Patients were compared with a retrospective cohort of 39 historical patients with Gram-positive BSIs, matched for underlying malignancy, infecting organism, and neutropenia status, who had been treated with vancomycin. A total of 78 patients were analyzed, with 39 in each group. The most common pathogen causing BSIs was S. aureus (51%), followed by alpha-hemolytic streptococci (23%), Enterococcus spp. (15%), coagulase-negative staphylococci (8%), and beta-hemolytic streptococci (3%). Sixty-two percent of patients had hematological malignancies, and 38% had solid tumors; 51% of the patients were neutropenic. The overall response rate determined by clinical outcome and microbiological eradication at 72 h following the initiation of therapy, in the absence of relapse, deep-seated infections, and/or infection-related death, was better with telavancin than with vancomycin (86% versus 61%; P = 0.013). Rates of drug-related adverse events were similar in the two groups (telavancin, 31%; vancomycin, 23%; P = 0.79), with similar rates of renal adverse events. Telavancin may provide a useful alternative to standard vancomycin therapy for Gram-positive BSIs in cancer patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT01321879.).

  14. Identification of Aerobic Gram-Positive Bacilli by Use of Vitek MS

    PubMed Central

    Navas, Maria; Pincus, David H.; Wilkey, Kathy; Sercia, Linda; LaSalvia, Margaret; Wilson, Deborah; Procop, Gary W.

    2014-01-01

    The accuracy of Vitek MS mass spectrometric identifications was assessed for 206 clinically significant isolates of aerobic Gram-positive bacilli representing 20 genera and 38 species. The Vitek MS identifications were correct for 85% of the isolates (56.3% to the species level, 28.6% limited to the genus level), with misidentifications occurring for 7.3% of the isolates. PMID:24501030

  15. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments▿ †

    PubMed Central

    Gontang, Erin A.; Fenical, William; Jensen, Paul R.

    2007-01-01

    Major advances in our understanding of marine bacterial diversity have been gained through studies of bacterioplankton, the vast majority of which appear to be gram negative. Less effort has been devoted to studies of bacteria inhabiting marine sediments, yet there is evidence to suggest that gram-positive bacteria comprise a relatively large proportion of these communities. To further expand our understanding of the aerobic gram-positive bacteria present in tropical marine sediments, a culture-dependent approach was applied to sediments collected in the Republic of Palau from the intertidal zone to depths of 500 m. This investigation resulted in the isolation of 1,624 diverse gram-positive bacteria spanning 22 families, including many that appear to represent new taxa. Phylogenetic analysis of 189 representative isolates, based on 16S rRNA gene sequence data, indicated that 124 (65.6%) belonged to the class Actinobacteria while the remaining 65 (34.4%) were members of the class Bacilli. Using a sequence identity value of ≥98%, the 189 isolates grouped into 78 operational taxonomic units, of which 29 (37.2%) are likely to represent new taxa. The high degree of phylogenetic novelty observed during this study highlights the fact that a great deal remains to be learned about the diversity of gram-positive bacteria in marine sediments. PMID:17400789

  16. Diversity of pigmented Gram-positive bacteria associated with marine macroalgae from Antarctica.

    PubMed

    Leiva, Sergio; Alvarado, Pamela; Huang, Ying; Wang, Jian; Garrido, Ignacio

    2015-12-01

    Little is known about the diversity and roles of Gram-positive and pigmented bacteria in Antarctic environments, especially those associated with marine macroorganisms. This work is the first study about the diversity and antimicrobial activity of culturable pigmented Gram-positive bacteria associated with marine Antarctic macroalgae. A total of 31 pigmented Gram-positive strains were isolated from the surface of six species of macroalgae collected in the King George Island, South Shetland Islands. On the basis of 16S rRNA gene sequence similarities ≥99%, 18 phylotypes were defined, which were clustered into 11 genera of Actinobacteria (Agrococcus, Arthrobacter, Brachybacterium, Citricoccus, Kocuria, Labedella, Microbacterium, Micrococcus, Rhodococcus, Salinibacterium and Sanguibacter) and one genus of the Firmicutes (Staphylococcus). It was found that five isolates displayed antimicrobial activity against a set of macroalgae-associated bacteria. The active isolates were phylogenetically related to Agrococcus baldri, Brachybacterium rhamnosum, Citricoccus zhacaiensis and Kocuria palustris. The results indicate that a diverse community of pigmented Gram-positive bacteria is associated with Antartic macroalgae and suggest its potential as a promising source of antimicrobial and pigmented natural compounds.

  17. Evaluation of the Bruker MALDI Biotyper for Identification of Gram-Positive Rods: Development of a Diagnostic Algorithm for the Clinical Laboratory

    PubMed Central

    Bloemberg, Guido V.; Zbinden, Reinhard; Böttger, Erik C.; Hombach, Michael

    2014-01-01

    Reported matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing. PMID:24452159

  18. Genomics of Methylotrophy in Gram-Positive Methylamine-Utilizing Bacteria

    PubMed Central

    McTaggart, Tami L.; Beck, David A. C.; Setboonsarng, Usanisa; Shapiro, Nicole; Woyke, Tanja; Lidstrom, Mary E.; Kalyuzhnaya, Marina G.; Chistoserdova, Ludmila

    2015-01-01

    Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria.

  19. Genomics of Methylotrophy in Gram-Positive Methylamine-Utilizing Bacteria.

    PubMed

    McTaggart, Tami L; Beck, David A C; Setboonsarng, Usanisa; Shapiro, Nicole; Woyke, Tanja; Lidstrom, Mary E; Kalyuzhnaya, Marina G; Chistoserdova, Ludmila

    2015-03-20

    Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria.

  20. Identification of a structural determinant for resistance to β-lactam antibiotics in Gram-positive bacteria

    PubMed Central

    Mouz, N.; Gordon, E.; Di Guilmi, A.-M.; Petit, I.; Pétillot, Y.; Dupont, Y.; Hakenbeck, R.; Vernet, T.; Dideberg, O.

    1998-01-01

    Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to β-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for β-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple β-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the “sensitive” form of PBP2x produces mutants similar, in terms of β-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for β-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of β-lactam antibiotics is lost. PMID:9811812

  1. Gram-negative and Gram-positive bacterial extracellular vesicles.

    PubMed

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria.

  2. Wall Teichoic Acids of Gram-Positive Bacteria

    PubMed Central

    Brown, Stephanie; Santa Maria, John P.; Walker, Suzanne

    2013-01-01

    The peptidoglycan layers of many gram-positive bacteria are densely functionalized with anionic glycopolymers called wall teichoic acids (WTAs). These polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of gram-positive bacterial physiology. Additionally, WTAs are important in pathogenesis and play key roles in antibiotic resistance. We provide an overview of WTA structure and biosynthesis, review recent studies on the biological roles of these polymers, and highlight remaining questions. We also discuss prospects for exploiting WTA biosynthesis as a target for new therapies to overcome resistant infections. PMID:24024634

  3. Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria

    PubMed Central

    Nawrocki, Kathryn L.; Crispell, Emily K.; McBride, Shonna M.

    2014-01-01

    Antimicrobial peptides, or AMPs, play a significant role in many environments as a tool to remove competing organisms. In response, many bacteria have evolved mechanisms to resist these peptides and prevent AMP-mediated killing. The development of AMP resistance mechanisms is driven by direct competition between bacterial species, as well as host and pathogen interactions. Akin to the number of different AMPs found in nature, resistance mechanisms that have evolved are just as varied and may confer broad-range resistance or specific resistance to AMPs. Specific mechanisms of AMP resistance prevent AMP-mediated killing against a single type of AMP, while broad resistance mechanisms often lead to a global change in the bacterial cell surface and protect the bacterium from a large group of AMPs that have similar characteristics. AMP resistance mechanisms can be found in many species of bacteria and can provide a competitive edge against other bacterial species or a host immune response. Gram-positive bacteria are one of the largest AMP producing groups, but characterization of Gram-positive AMP resistance mechanisms lags behind that of Gram-negative species. In this review we present a summary of the AMP resistance mechanisms that have been identified and characterized in Gram-positive bacteria. Understanding the mechanisms of AMP resistance in Gram-positive species can provide guidelines in developing and applying AMPs as therapeutics, and offer insight into the role of resistance in bacterial pathogenesis. PMID:25419466

  4. Conjugative plasmid transfer in gram-positive bacteria.

    PubMed

    Grohmann, Elisabeth; Muth, Günther; Espinosa, Manuel

    2003-06-01

    Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer. PMID:12794193

  5. Which antibiotic for resistant Gram-positives, and why?

    PubMed

    Bradley, John S

    2014-01-01

    Increasing resistance in Gram-positive pathogens, particularly Staphylococcus aureus, and enterococcus, has become a major clinical problem, particularly in the hospital environment, causing significant morbidity and mortality in both healthy hosts and in those with underlying comorbidities. Increased resistance drives the use of empiric therapy with less well-studied and potentially more toxic agents. Resistance mechanisms for currently recommended agents are discussed, with options for therapy of resistant pathogens. For any new agent used, resistance is likely to develop, which underscores the concept that both antibiotics and antimicrobial resistance are ancient, and only by prudent use of antimicrobial agents and effective infection control measures when resistance arises, will effective agents be available to treat Gram-positive pathogens in the future.

  6. Cyclic diguanylate signaling in Gram-positive bacteria.

    PubMed

    Purcell, Erin B; Tamayo, Rita

    2016-09-01

    The nucleotide second messenger 3'-5' cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of the transition between motile and non-motile lifestyles in bacteria, favoring sessility. Most research investigating the functions of c-di-GMP has focused on Gram-negative species, especially pathogens. Recent work in Gram-positive species has revealed that c-di-GMP plays similar roles in Gram-positives, though the precise targets and mechanisms of regulation may differ. The majority of bacterial life exists in a surface-associated state, with motility allowing bacteria to disseminate and colonize new environments. c-di-GMP signaling regulates flagellum biosynthesis and production of adherence factors and appears to be a primary mechanism by which bacteria sense and respond to surfaces. Ultimately, c-di-GMP influences the ability of a bacterium to alter its transcriptional program, physiology and behavior upon surface contact. This review discusses how bacteria are able to sense a surface via flagella and type IV pili, and the role of c-di-GMP in regulating the response to surfaces, with emphasis on studies of Gram-positive bacteria.

  7. Cyclic diguanylate signaling in Gram-positive bacteria.

    PubMed

    Purcell, Erin B; Tamayo, Rita

    2016-09-01

    The nucleotide second messenger 3'-5' cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of the transition between motile and non-motile lifestyles in bacteria, favoring sessility. Most research investigating the functions of c-di-GMP has focused on Gram-negative species, especially pathogens. Recent work in Gram-positive species has revealed that c-di-GMP plays similar roles in Gram-positives, though the precise targets and mechanisms of regulation may differ. The majority of bacterial life exists in a surface-associated state, with motility allowing bacteria to disseminate and colonize new environments. c-di-GMP signaling regulates flagellum biosynthesis and production of adherence factors and appears to be a primary mechanism by which bacteria sense and respond to surfaces. Ultimately, c-di-GMP influences the ability of a bacterium to alter its transcriptional program, physiology and behavior upon surface contact. This review discusses how bacteria are able to sense a surface via flagella and type IV pili, and the role of c-di-GMP in regulating the response to surfaces, with emphasis on studies of Gram-positive bacteria. PMID:27354347

  8. Pilins in gram-positive bacteria: A structural perspective.

    PubMed

    Krishnan, Vengadesan

    2015-07-01

    Pilins or fimbrilins are a class of proteins found in bacterial surface pilus, a hair-like surface appendage. Both the Gram-negative and -positive bacteria produce pilins to assemble pili on their cell-surface for different purposes including adherence, twitching motility, conjugation, immunomodulation, biofilm formation, and electron transfer. Immunogenic properties of the pilins make them attractive vaccine candidates. The polymerized pilins play a key role in the initiation of host adhesion, which is a critical step for bacterial colonization and infection. Because of their key role in adhesion and exposure on the cell surface, targeting the pilins-mediated adhesion (anti-adhesion therapy) is also seen as a promising alternative approach for preventing and treating bacterial infections, one that may overcome their ever-increasing repertoires of resistance mechanisms. Individual pilins interact with each other non-covalently to assemble the pilus fiber with the help of associated proteins like chaperones and Usher in Gram-negative bacteria. In contrast, the pilins in Gram-positive bacteria often connect with each other covalently, with the help of sortases. Certain unique structural features present on the pilins distinguish them from one another across different bacterial strains, and these dictate their cellular targets and functions. While the structure of pilins has been extensively studied in Gram-negative pathogenic bacteria, the pilins in Gram-positive pathogenic bacteria have been in only during the last decade. Recently, the discovery of pilins in non-pathogenic bacteria, such as Lactobacillus rhamnosus GG, has received great attention, though traditionally the attention was on pathogenic bacteria. This review summarizes and discusses the current structural knowledge of pilins in Gram-positive bacteria with emphasis on those pilins which are sortase substrates.

  9. Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

    PubMed

    Al Bulushi, Ismail M; Poole, Susan E; Barlow, Robert; Deeth, Hilton C; Dykes, Gary A

    2010-03-31

    This study identified Gram-positive bacteria in three sub-tropical marine fish species; Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warneri were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body, E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

  10. Current and novel antibiotics against resistant Gram-positive bacteria

    PubMed Central

    Perez, Federico; Salata, Robert A; Bonomo, Robert A

    2008-01-01

    The challenge posed by resistance among Gram-positive bacteria, epitomized by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and vancomycin-intermediate and -resistant S. aureus (VISA and VRSA) is being met by a new generation of antimicrobials. This review focuses on the new β-lactams with activity against MRSA (ceftobiprole and ceftaroline) and on the new glycopeptides (oritavancin, dalbavancin, and telavancin). It will also consider the role of vancomycin in an era of existing alternatives such as linezolid, daptomycin and tigecycline. Finally, compounds in early development are described, such as iclaprim, friulimicin, and retapamulin, among others. PMID:21694878

  11. Acquired inducible antimicrobial resistance in Gram-positive bacteria

    PubMed Central

    Chancey, Scott T; Zähner, Dorothea; Stephens, David S

    2012-01-01

    A major contributor to the emergence of antibiotic resistance in Gram-positive bacterial pathogens is the expansion of acquired, inducible genetic elements. Although acquired, inducible antibiotic resistance is not new, the interest in its molecular basis has been accelerated by the widening distribution and often ‘silent’ spread of the elements responsible, the diagnostic challenges of such resistance and the mounting limitations of available agents to treat Gram-positive infections. Acquired, inducible antibiotic resistance elements belong to the accessory genome of a species and are horizontally acquired by transformation/recombination or through the transfer of mobile DNA elements. The two key, but mechanistically very different, induction mechanisms are: ribosome-sensed induction, characteristic of the macrolide–lincosamide–streptogramin B antibiotics and tetracycline resistance, leading to ribosomal modifications or efflux pump activation; and resistance by cell surface-associated sensing of β-lactams (e.g., oxacillin), glycopeptides (e.g., vancomycin) and the polypeptide bacitracin, leading to drug inactivation or resistance due to cell wall alterations. PMID:22913355

  12. Type IV Pili in Gram-Positive Bacteria

    PubMed Central

    Craig, Lisa

    2013-01-01

    SUMMARY Type IV pili (T4P) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, DNA uptake (competence), and protein secretion and that can act as nanowires carrying electric current. T4P are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type II secretion systems in eubacteria and the flagella of archaea. T4P are found throughout Gram-negative bacterial families and have been studied most extensively in certain model Gram-negative species. Recently, it was discovered that T4P systems are also widespread among Gram-positive species, in particular the clostridia. Since Gram-positive and Gram-negative bacteria have many differences in cell wall architecture and other features, it is remarkable how similar the T4P core proteins are between these organisms, yet there are many key and interesting differences to be found as well. In this review, we compare the two T4P systems and identify and discuss the features they have in common and where they differ to provide a very broad-based view of T4P systems across all eubacterial species. PMID:24006467

  13. Teicoplanin in the treatment of gram-positive-bacterial endocarditis.

    PubMed Central

    Martino, P; Venditti, M; Micozzi, A; Brandimarte, C; Gentile, G; Santini, C; Serra, P

    1989-01-01

    Intravenous teicoplanin has been used to treat 23 cases of gram-positive-bacterial endocarditis, usually with 3 to 7 mg/kg every 12 h on the first day, followed by 3 to 7 mg/kg every 24 h. For some cases (staphylococcal and enterococcal endocarditis), the dosage was 8 to 14.4 mg/kg per day and/or other antibiotics were given. The mean duration was 48.2 days (range, 23 to 130 days). Of 23 patients, 21 (91.3%) had negative cultures or were cured. A total of 18 patients were treated with teicoplanin alone; of these, 4 had surgery, and all (except 2 who relapsed) were cured. Teicoplanin was combined with one or more antibiotics in five cases; in all cases appropriate cultures were negative, but three patients died during therapy or follow-up. Mild renal impairment was seen in two patients; both were receiving teicoplanin in combination with an aminoglycoside. We conclude that intravenous teicoplanin administered once a day at doses of 7 to 14 mg/kg per day is well tolerated, easy to administer, and may represent an efficacious therapy for gram-positive-bacterial endocarditis. PMID:2529815

  14. Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria

    PubMed Central

    2015-01-01

    Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria. PMID:26644434

  15. Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

    PubMed

    Costa, N L; Carlson, H K; Coates, J D; Louro, R O; Paquete, C M

    2015-07-01

    Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

  16. Extraction and purification of lipoteichoic acids from Gram-positive bacteria.

    PubMed

    Coley, J; Duckworth, M; Baddiley, J

    1975-03-01

    Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphlococcus aureus H, Micrococcus 2102, Baccillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.

  17. Catabolite repression and inducer control in Gram-positive bacteria.

    PubMed

    Saier, M H; Chauvaux, S; Cook, G M; Deutscher, J; Paulsen, I T; Reizer, J; Ye, J J

    1996-02-01

    Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria. This protein kinase is not found in enteric bacteria such as E. coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control. In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b). In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc. Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993). In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated. HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins. These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels. Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor. These two proteins together are believed to determine the intensity of catabolite repression. Their relative importance depends on physiological conditions. Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites. CepA possibly binds

  18. New antimicrobial approaches to gram positive respiratory infections.

    PubMed

    Liapikou, Adamantia; Cilloniz, Catia; Mensa, Josep; Torres, Antonio

    2015-06-01

    Nowadays, we face growing resistance among gram-positive and gram-negative pathogens that cause respiratory infection in the hospital and in the community. The spread of penicillin- and macrolide-resistant pneumococci, Community-acquired methicillin-resistant staphylococcus aureus (Ca-MRSA), the emergence of glycopeptide-resistant staphylococci underline the need for underline the need for therapeutic alternatives. A number of new therapeutic agents, with activity against the above Gram (+) respiratory pathogens, as ceftaroline, ceftopibrole, telavancin, tedizolid have become available, either in clinical trials or have been approved for clinical use. Especially, the development of new oral antibiotics, as nemonaxacin, omadacyclin, cethromycin and solithromycin will give a solution to the lack of oral drugs for outpatient treatment. In the future the clinician needs to optimize the use of old and new antibiotics to treat gram (+) respiratory serious infections.

  19. Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

    PubMed

    Wojewoda, Christina M; Sercia, Linda; Navas, Maria; Tuohy, Marion; Wilson, Deborah; Hall, Geraldine S; Procop, Gary W; Richter, Sandra S

    2013-07-01

    Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

  20. A classification system for plasmids from enterococci and other Gram-positive bacteria.

    PubMed

    Jensen, L B; Garcia-Migura, L; Valenzuela, A J S; Løhr, M; Hasman, H; Aarestrup, F M

    2010-01-01

    A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating genes (rep), alignment of these sequences and using a cutoff value of 80% identity on both protein and DNA level, 19 replicon families (rep-families) were defined together with several unique sequences. The prevalence of these rep-families was tested on 79 enterococcal isolates from a collection of isolates of animal and human origin. Difference in prevalence of the designed rep-families were detected with rep(9) being most prevalent in Enterococcus faecalis and rep(2) in Enterococcus faecium. In 33% of the tested E. faecium and 32% of the tested E. faecalis no positive amplicons were detected. Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup.

  1. Reversal of antibiotic resistance in Gram-positive bacteria by the antihistaminic azelastine.

    PubMed

    El-Nakeeb, Moustafa A; Abou-Shleib, Hamida M; Khalil, Amal M; Omar, Hoda G; El-Halfawy, Omar M

    2012-03-01

    Antibiotic resistance represents a serious problem that complicates microbial infection. The use of 'helper compounds' capable of enhancing the antimicrobial activity of antibiotics is being investigated. Azelastine, a new generation antihistaminic, possesses certain antibacterial activity and is capable of inducing alteration in the bacterial membrane permeability. Hence, we hypothesized that it could reverse resistance to antibiotics. Azelastine significantly increased the antibacterial activity of eight antibiotics belonging to five different classes (β-lactams, macrolides, fluoroquinolones, aminoglycosides and tetracyclines) against nine Gram-positive clinical isolates: five Staphylococcus aureus, two Staphylococcus epidermidis and two Enterococcus faecium, seven of which were multi-drug resistant, reversing their resistance to the tested antibiotics. The synergistic effects of azelastine with the studied antibiotics increased with raising the pH from 5 to 8. Antibiotics did not affect the ability of azelastine to alter the permeability of a liposomal artificial membrane model, an effect thought to be critical for the interaction with antibiotics. The findings of this study present azelastine as a potential 'helper compound' that could reverse the resistance of multi-drug resistant Gram-positive clinical isolates to antibiotics. PMID:22339679

  2. Green fluorescent protein-labeled monitoring tool to quantify conjugative plasmid transfer between Gram-positive and Gram-negative bacteria.

    PubMed

    Arends, Karsten; Schiwon, Katarzyna; Sakinc, Türkan; Hübner, Johannes; Grohmann, Elisabeth

    2012-02-01

    On the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positive Enterococcus faecalis and Gram-negative Escherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown from E. faecalis OG1X into the clinical isolate E. faecalis T9.

  3. High-level fluorescence labeling of gram-positive pathogens.

    PubMed

    Aymanns, Simone; Mauerer, Stefanie; van Zandbergen, Ger; Wolz, Christiane; Spellerberg, Barbara

    2011-01-01

    Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

  4. Resistance to bacteriocins produced by Gram-positive bacteria.

    PubMed

    Bastos, Maria do Carmo de Freire; Coelho, Marcus Lívio Varella; Santos, Olinda Cabral da Silva

    2015-04-01

    Bacteriocins are prokaryotic proteins or peptides with antimicrobial activity. Most of them exhibit a broad spectrum of activity, inhibiting micro-organisms belonging to different genera and species, including many bacterial pathogens which cause human, animal or plant infections. Therefore, these substances have potential biotechnological applications in either food preservation or prevention and control of bacterial infectious diseases. However, there is concern that continuous exposure of bacteria to bacteriocins may select cells resistant to them, as observed for conventional antimicrobials. Based on the models already investigated, bacteriocin resistance may be either innate or acquired and seems to be a complex phenomenon, arising at different frequencies (generally from 10(-9) to 10(-2)) and by different mechanisms, even amongst strains of the same bacterial species. In the present review, we discuss the prevalence, development and molecular mechanisms involved in resistance to bacteriocins produced by Gram-positive bacteria. These mechanisms generally involve changes in the bacterial cell envelope, which result in (i) reduction or loss of bacteriocin binding or insertion, (ii) bacteriocin sequestering, (iii) bacteriocin efflux pumping (export) and (iv) bacteriocin degradation, amongst others. Strategies that can be used to overcome this resistance are also addressed.

  5. Multidrug efflux pumps of Gram-positive bacteria.

    PubMed

    Schindler, Bryan D; Kaatz, Glenn W

    2016-07-01

    Gram-positive organisms are responsible for some of the most serious of human infections. Resistance to front-line antimicrobial agents can complicate otherwise curative therapy. These organisms possess multiple drug resistance mechanisms, with drug efflux being a significant contributing factor. Efflux proteins belonging to all five transporter families are involved, and frequently can transport multiple structurally unrelated compounds resulting in a multidrug resistance (MDR) phenotype. In addition to clinically relevant antimicrobial agents, MDR efflux proteins can transport environmental biocides and disinfectants which may allow persistence in the healthcare environment and subsequent acquisition by patients or staff. Intensive research on MDR efflux proteins and the regulation of expression of their genes is ongoing, providing some insight into the mechanisms of multidrug recognition and transport. Inhibitors of many of these proteins have been identified, including drugs currently being used for other indications. Structural modifications guided by structure-activity studies have resulted in the identification of potent compounds. However, lack of broad-spectrum pump inhibition combined with potential toxicity has hampered progress. Further work is required to gain a detailed understanding of the multidrug recognition process, followed by application of this knowledge in the design of safer and more highly potent inhibitors. PMID:27449594

  6. Complete genome sequence of Paenibacillus riograndensis SBR5(T), a Gram-positive diazotrophic rhizobacterium.

    PubMed

    Brito, Luciana Fernandes; Bach, Evelise; Kalinowski, Jörn; Rückert, Christian; Wibberg, Daniel; Passaglia, Luciane M; Wendisch, Volker F

    2015-08-10

    Paenibacillus riograndensis is a Gram-positive rhizobacterium which exhibits plant growth promoting activities. It was isolated from the rhizosphere of wheat grown in the state of Rio Grande do Sul, Brazil. Here we announce the complete genome sequence of P. riograndensis strain SBR5(T). The genome of P. riograndensis SBR5(T) consists of a circular chromosome of 7,893,056bps. The genome was finished and fully annotated, containing 6705 protein coding genes, 87 tRNAs and 27 rRNAs. The knowledge of the complete genome helped to explain why P. riograndensis SBR5(T) can grow with the carbon sources arabinose and mannitol, but not myo-inositol, and to explain physiological features such as biotin auxotrophy and antibiotic resistances. The genome sequence will be valuable for functional genomics and ecological studies as well as for application of P. riograndensis SBR5(T) as plant growth-promoting rhizobacterium.

  7. The ability of electrochemical oxidation with a BDD anode to inactivate Gram-negative and Gram-positive bacteria in low conductivity sulfate medium.

    PubMed

    Bruguera-Casamada, Carmina; Sirés, Ignasi; Prieto, María J; Brillas, Enric; Araujo, Rosa M

    2016-11-01

    The disinfection of 100 mL of synthetic water containing 7 mM Na2SO4 with 10(6) CFU mL(-1) of either Gram-negative or Gram-positive bacteria has been studied by electrochemical oxidation. The electrolytic cell was a stirred tank reactor equipped with a boron-doped diamond (BDD) anode and a stainless steel cathode and the trials were performed at acidic and neutral pH, at 33.3 mA cm(-2) and 25 °C. Reactive oxygen species, pre-eminently hydroxyl radicals, were efficiently produced in both media from water oxidation at the BDD anode and the bacteria concentration was reduced by ≥ 5 log units after 60 min of electrolysis, thus constituting a good chlorine-free disinfection treatment. All the inactivation kinetics were described by a logistic model, with no significant statistical differences between acidic and neutral suspensions. The electrochemical disinfection with BDD was very effective for Gram-negative bacilli like Escherichia coli and Pseudomonas aeruginosa and Gram-positive ones like Bacillus atrophaeus, whereas the Gram-positive cocci Staphylococcus aureus and Enterococcus hirae were more resistant. Thus, the latter organisms are a better choice than E. coli as process indicators. Scanning electron microscopy highlighted a transition from initial cells with standard morphology supported on clean filters to inactivated cells with a highly altered morphology lying on dirty filters with plenty of cellular debris. Larger damage was observed for Gram-negative cells compared to Gram-positive ones. The inactivation effect could then be related to the chemical composition of the outer layers of the cell structure along with the modification of the transmembrane potentials upon current passage. PMID:27567151

  8. The ability of electrochemical oxidation with a BDD anode to inactivate Gram-negative and Gram-positive bacteria in low conductivity sulfate medium.

    PubMed

    Bruguera-Casamada, Carmina; Sirés, Ignasi; Prieto, María J; Brillas, Enric; Araujo, Rosa M

    2016-11-01

    The disinfection of 100 mL of synthetic water containing 7 mM Na2SO4 with 10(6) CFU mL(-1) of either Gram-negative or Gram-positive bacteria has been studied by electrochemical oxidation. The electrolytic cell was a stirred tank reactor equipped with a boron-doped diamond (BDD) anode and a stainless steel cathode and the trials were performed at acidic and neutral pH, at 33.3 mA cm(-2) and 25 °C. Reactive oxygen species, pre-eminently hydroxyl radicals, were efficiently produced in both media from water oxidation at the BDD anode and the bacteria concentration was reduced by ≥ 5 log units after 60 min of electrolysis, thus constituting a good chlorine-free disinfection treatment. All the inactivation kinetics were described by a logistic model, with no significant statistical differences between acidic and neutral suspensions. The electrochemical disinfection with BDD was very effective for Gram-negative bacilli like Escherichia coli and Pseudomonas aeruginosa and Gram-positive ones like Bacillus atrophaeus, whereas the Gram-positive cocci Staphylococcus aureus and Enterococcus hirae were more resistant. Thus, the latter organisms are a better choice than E. coli as process indicators. Scanning electron microscopy highlighted a transition from initial cells with standard morphology supported on clean filters to inactivated cells with a highly altered morphology lying on dirty filters with plenty of cellular debris. Larger damage was observed for Gram-negative cells compared to Gram-positive ones. The inactivation effect could then be related to the chemical composition of the outer layers of the cell structure along with the modification of the transmembrane potentials upon current passage.

  9. Cultivation of aerobic chemoorganotrophic proteobacteria and gram-positive bacteria from a hot spring microbial mat.

    PubMed Central

    Nold, S C; Kopczynski, E D; Ward, D M

    1996-01-01

    The diversity of aerobic chemoorganotrophic bacteria inhabiting the Octopus Spring cyanobacterial mat community (Yellowstone National Park) was examined by using serial-dilution enrichment culture and a variety of enrichment conditions to cultivate the numerically significant microbial populations. The most abundant bacterial populations cultivated from dilutions to extinction were obtained from enrichment flasks which contained 9.0 x 10(2) primary producer (Synechococcus spp.) cells in the inoculum. Two isolates exhibited 16S rRNA nucleotide sequences typical of beta-proteobacteria. One of these isolates contained a 16S rRNA sequence identical to a sequence type previously observed in the mat by molecular retrieval techniques. Both are distantly related to a new sequence directly retrieved from the mat and contributed by a beta-proteobacterial community member. Phenotypically diverse gram-positive isolates genetically similar to Bacillus flavothermus were obtained from a variety of dilutions and enrichment types. These isolates exhibited identical 16S rRNA nucleotide sequences through a variable region of the molecule. Of the three unique sequences observed, only one had been previously retrieved from the mat, illustrating both the inability of the cultivation methods to describe the composition of a microbial community and the limitations of the ability of molecular retrieval techniques to describe populations which may be less abundant in microbial communities. PMID:8899976

  10. C-Terminal WxL Domain Mediates Cell Wall Binding in Enterococcus faecalis and Other Gram-Positive Bacteria▿

    PubMed Central

    Brinster, Sophie; Furlan, Sylviane; Serror, Pascale

    2007-01-01

    Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria. PMID:16963569

  11. Antimicrobials targeted to the replication-specific DNA polymerases of gram-positive bacteria: target potential of dnaE.

    PubMed

    Barnes, Marjorie H; Butler, Michelle M; Wright, George E; Brown, Neal C

    2012-10-01

    DNA polymerases pol IIIC and dnaE [i.e. pol IIIE] are essential for replicative DNA synthesis in low G:C Gram-positive eubacteria. Therefore, they have strong potential as targets for development of Gram-positive-selective antibacterial agents. This work has sought to extend to dnaE the recent discovery of antimicrobial agents based on pol IIIC-specific dGTP analogs. Compound 324C, a member of the same dGTP analog family, was found to be a potent and selective inhibitor of isolated dnaE in vitro. Surprisingly, 324C had no inhibitory effect in either intact Bacillus subtilis cells or in permeabilized cell preparations used to assess replicative DNA synthesis directly. It is proposed that the failure of 324C in the intact cell is a consequence of two major factors: (i) its template-dependent base pairing mechanism, and (ii) a specific subordinate role which dnaE apparently plays to pol IIIC. To generate an effective dnaE-selective inhibitor of replicative DNA synthesis in Gram-positive bacteria, it will likely be necessary to develop a molecule that attacks the enzyme's active site directly, without binding to template DNA.

  12. Uncultivated Magnetotactic Cocci from Yuandadu Park in Beijing, China▿

    PubMed Central

    Lin, Wei; Pan, Yongxin

    2009-01-01

    In the present study, we investigated a group of uncultivated magnetotactic cocci, which was magnetically isolated from a freshwater pond in Beijing, China. Light and transmission electron microscopy showed that these cocci ranged from 1.5 to 2.5 μm and contained two to four chains of magnetite magnetosomes, which sometimes were partially disorganized. Overall, the size of the disorganized magnetosomes was significantly smaller than that arranged in chains. All characterized magnetosome crystals were elongated (shape factor = 0.64) and fall into the single-domain size range (30 to 115 nm). Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the enriched bacteria were a virtually homogeneous population and represented a novel lineage in the Alphaproteobacteria. The closest cultivated relative was magnetotactic coccoid strain MC-1 (88% sequence identity). First-order reversal curve diagrams revealed that these cocci had relatively strong magnetic interactions compared to the single-chain magnetotactic bacteria. Low-temperature magnetic measurements showed that the Verwey transition of them was ∼108 K, confirming magnetite magnetosomes, and the delta ratio δFC/δZFC was >2. Based on the structure, phylogenetic position and magnetic properties, the enriched magnetotactic cocci of Alphaproteobacteria are provisionally named as “Candidatus Magnetococcus yuandaducum.” PMID:19376904

  13. Multistep Resistance Development Studies of Ceftaroline in Gram-Positive and -Negative Bacteria▿

    PubMed Central

    Clark, Catherine; McGhee, Pamela; Appelbaum, Peter C.; Kosowska-Shick, Klaudia

    2011-01-01

    Ceftaroline, the active component of the prodrug ceftaroline fosamil, is a novel broad-spectrum cephalosporin with bactericidal activity against Gram-positive and -negative isolates. This study evaluated the potential for ceftaroline and comparator antibiotics to select for clones of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis with elevated MICs. S. pneumoniae and S. pyogenes isolates in the present study were highly susceptible to ceftaroline (MIC range, 0.004 to 0.25 μg/ml). No streptococcal strains yielded ceftaroline clones with increased MICs (defined as an increase in MIC of >4-fold) after 50 daily passages. Ceftaroline MICs for H. influenzae and M. catarrhalis were 0.06 to 2 μg/ml for four strains and 8 μg/ml for a β-lactamase-positive, efflux-positive H. influenzae with a mutation in L22. One H. influenzae clone with an increased ceftaroline MIC (quinolone-resistant, β-lactamase-positive) was recovered after 20 days. The ceftaroline MIC for this isolate increased 16-fold, from 0.06 to 1 μg/ml. MICs for S. aureus ranged from 0.25 to 1 μg/ml. No S. aureus isolates tested with ceftaroline had clones with increased MIC (>4-fold) after 50 passages. Two E. faecalis isolates tested had ceftaroline MICs increased from 1 to 8 μg/ml after 38 days and from 4 to 32 μg/ml after 41 days, respectively. The parental ceftaroline MIC for the one K. pneumoniae extended-spectrum β-lactamase-negative isolate tested was 0.5 μg/ml and did not change after 50 daily passages. PMID:21343467

  14. Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

    SciTech Connect

    Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.; Preston, J.F.; Aldrich, H.C.

    2003-12-01

    Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in the optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these

  15. Antimicrobial susceptibility of non-enterococcal intrinsic glycopeptide-resistant Gram-positive organisms.

    PubMed

    Vay, Carlos; Cittadini, Roxana; Barberis, Claudia; Hernán Rodríguez, Carlos; Perez Martínez, Herminia; Genero, Fabiana; Famiglietti, Angela

    2007-02-01

    Non-enterococcal Gram-positive bacteria that are intrinsically vancomycin-resistant have been infrequently isolated in association with serious infections. However, well-documented infections have lately been reported with increasing frequency. Because these organisms may be pathogens, we tested the MICs of 19 antimicrobial agents by the agar dilution method for predicting susceptibility. The activity of these antimicrobial agents was assessed against 28 strains (Lactobacillus rhamnosus, 6; Lactobacillus acidophilus, 1; Lactobacillus casei, 1; Lactobacillus fermentum, 2; Lactobacillus brevis, 1; Lactobacillus plantarum, 1; Weissella confusa, 2; Leuconostoc mesenteroides, 7; Leuconostoc lactis, 4; Pediococcus acidilactici, 2; Pediococcus pentosaceus, 1), isolated from clinical specimens in an Argentinian university hospital from 1997 to 2003. The MICs of penicillin for 67% of the Lactobacillus strains and 100% of the Leuconostoc spp. and Pediococcus spp. strains tested were in the 0.25-2 microg/mL range. Erythromycin was the most active antimicrobial overall. Multiresistance was observed in 2 strains (Lactobacillus rhamnosus, 1; Lactobacillus plantarum, 1).

  16. Performance of the Verigene Gram-Positive Blood Culture Assay for Direct Detection of Gram-Positive Organisms and Resistance Markers in a Pediatric Hospital

    PubMed Central

    Mestas, Javier; Polanco, Claudia M.; Felsenstein, Susanna

    2014-01-01

    The performance characteristics of the Verigene Gram-positive blood culture (BC-GP) assay were evaluated in pediatric patients. Concordance of the BC-GP assay was 95.8%, with significant decreases in turnaround time for identification and resistance detection. BC-GP is highly accurate and can be integrated into the routine workflow of the microbiology laboratory. PMID:24131696

  17. Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

    PubMed

    Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

    2011-01-01

    Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

  18. Robenidine Analogues as Gram-Positive Antibacterial Agents.

    PubMed

    Abraham, Rebecca J; Stevens, Andrew J; Young, Kelly A; Russell, Cecilia; Qvist, Anastasia; Khazandi, Manouchehr; Wong, Hui San; Abraham, Sam; Ogunniyi, Abiodun D; Page, Stephen W; O'Handley, Ryan; McCluskey, Adam; Trott, Darren J

    2016-03-10

    Robenidine, 1 (2,2'-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC's of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7-71 μM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1-13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2'-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2'-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC's of 4.2-21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 μL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75. PMID:26765953

  19. Robenidine Analogues as Gram-Positive Antibacterial Agents.

    PubMed

    Abraham, Rebecca J; Stevens, Andrew J; Young, Kelly A; Russell, Cecilia; Qvist, Anastasia; Khazandi, Manouchehr; Wong, Hui San; Abraham, Sam; Ogunniyi, Abiodun D; Page, Stephen W; O'Handley, Ryan; McCluskey, Adam; Trott, Darren J

    2016-03-10

    Robenidine, 1 (2,2'-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC's of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7-71 μM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1-13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2'-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2'-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC's of 4.2-21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 μL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75.

  20. Efficacy of telavancin, a lipoglycopeptide antibiotic, in experimental models of Gram-positive infection.

    PubMed

    Hegde, Sharath S; Janc, James W

    2014-12-01

    Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributing to bactericidal activity against multidrug-resistant Gram-positive pathogens. It has been approved for the treatment of complicated skin and skin structure infections due to susceptible Gram-positive bacteria and hospital-acquired/ventilator-associated bacterial pneumonia due to Staphylococcus aureus when other alternatives are unsuitable. Telavancin has been demonstrated to be efficacious in multiple animal models of soft tissue, cardiac, systemic, lung, bone, brain and device-associated infections involving clinically relevant Gram-positive pathogens, including methicillin-resistant S. aureus, glycopeptide-intermediate S. aureus, heterogeneous vancomycin-intermediate S. aureus and daptomycin non-susceptible methicillin-resistant S. aureus. The AUC0-24h/MIC ratio is the primary pharmacodynamically-linked pharmacokinetic parameter. The preclinical data for telavancin supports further investigative clinical evaluation of its efficacy in additional serious infections caused by susceptible Gram-positive pathogens.

  1. Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria.

    PubMed

    Reichmann, Nathalie T; Cassona, Carolina Picarra; Gründling, Angelika

    2013-09-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with D-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA-D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers D-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for D-alanine incorporation through a process that has been proposed to proceed via a D-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of D-alanine, indicating that LTA has a role, either direct or indirect, in the efficient D-alanine incorporation into WTA in living cells.

  2. Cytokine profile in severe Gram-positive and Gram-negative abdominal sepsis.

    PubMed

    Surbatovic, Maja; Popovic, Nada; Vojvodic, Danilo; Milosevic, Ivan; Acimovic, Gordana; Stojicic, Milan; Veljovic, Milic; Jevdjic, Jasna; Djordjevic, Dragan; Radakovic, Sonja

    2015-06-16

    Sepsis is a principal cause of death in critical care units worldwide and consumes considerable healthcare resources. The aim of our study was to determine whether the early cytokine profile can discriminate between Gram-positive and Gram-negative bacteraemia (GPB and GNB, respectively) and to assess the prognostic value regarding outcome in critically ill patients with severe abdominal sepsis. The outcome measure was hospital mortality. Blood samples were obtained from 165 adult patients with confirmed severe abdominal sepsis. Levels of the proinflammatory mediators TNF-α, IL-8, IL-12 and IFN-γ and the anti-inflammatory mediators IL-1ra, IL-4, IL-10 and TGF-β1 were determined and correlated with the nature of the bacteria isolated from the blood culture and outcome. The cytokine profile in our study indicated that the TNF-α levels were 2-fold, IL-8 were 3.3-fold, IFN-γ were 13-fold, IL-1ra were 1.05-fold, IL-4 were 1.4-fold and IL-10 were 1.83-fold higher in the GNB group compared with the GPB group. The TNF-α levels were 4.7-fold, IL-8 were 4.6-fold, IL-1ra were 1.5-fold and IL-10 were 3.3-fold higher in the non-survivors compared with the survivors.

  3. Use of PNA FISH for blood cultures growing Gram-positive cocci in chains without a concomitant antibiotic stewardship intervention does not improve time to appropriate antibiotic therapy.

    PubMed

    Cosgrove, Sara E; Li, David X; Tamma, Pranita D; Avdic, Edina; Hadhazy, Eric; Wakefield, Teresa; Gherna, Michael; Carroll, Karen C

    2016-09-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a rapid diagnostic assay that can identify certain organisms growing in blood cultures 30-90 min from the time of positive Gram-stain. Existing studies have demonstrated a clinical utility with this assay when antibiotic stewardship programs assist clinicians with interpreting the results. However, the benefit of these rapid assays in the absence of concomitant antibiotic stewardship involvement is unclear. In this randomized study of 220 patients with enterococcal or streptococcal bacteremia, we found that PNA FISH, in the absence of concomitant input from an antibiotic stewardship program, had no impact on time to effective or optimal therapy, length of hospital stay, or in-hospital mortality. Our results suggest that in the absence of guidance from an antibiotic stewardship program, the clinical benefits of rapid diagnostic microbiological tools may be reduced. PMID:27412814

  4. Use of PNA FISH for blood cultures growing Gram-positive cocci in chains without a concomitant antibiotic stewardship intervention does not improve time to appropriate antibiotic therapy.

    PubMed

    Cosgrove, Sara E; Li, David X; Tamma, Pranita D; Avdic, Edina; Hadhazy, Eric; Wakefield, Teresa; Gherna, Michael; Carroll, Karen C

    2016-09-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a rapid diagnostic assay that can identify certain organisms growing in blood cultures 30-90 min from the time of positive Gram-stain. Existing studies have demonstrated a clinical utility with this assay when antibiotic stewardship programs assist clinicians with interpreting the results. However, the benefit of these rapid assays in the absence of concomitant antibiotic stewardship involvement is unclear. In this randomized study of 220 patients with enterococcal or streptococcal bacteremia, we found that PNA FISH, in the absence of concomitant input from an antibiotic stewardship program, had no impact on time to effective or optimal therapy, length of hospital stay, or in-hospital mortality. Our results suggest that in the absence of guidance from an antibiotic stewardship program, the clinical benefits of rapid diagnostic microbiological tools may be reduced.

  5. Rapid detection of Gram-positive organisms by use of the Verigene Gram-positive blood culture nucleic acid test and the BacT/Alert Pediatric FAN system in a multicenter pediatric evaluation.

    PubMed

    Sullivan, K V; Turner, N N; Roundtree, S S; Young, S; Brock-Haag, C A; Lacey, D; Abuzaid, S; Blecker-Shelly, D L; Doern, C D

    2013-11-01

    Assays that expedite the reporting of organism identification and antibiotic susceptibility status in positive blood cultures can fast track interventions that improve clinical outcomes. We evaluated the Verigene Gram-positive blood culture nucleic acid test (BC-GP) in two pediatric hospitals. Positive BacT/Alert Pediatric FAN blood cultures with Gram-positive organisms were tested using the BC-GP in tandem with routine laboratory procedures. To test organisms underrepresented in the clinical blood culture evaluation, blood culture bottles were spiked with diluted organism suspensions at concentrations of 10 to 100 CFU per milliliter. A total of 249 Gram-positive bacterial isolates were recovered from 242 blood cultures. The BC-GP detected Staphylococcus aureus, methicillin-susceptible S. aureus, and methicillin-resistant S. aureus with sensitivities of 100%, 99%, and 100% and specificities of 100%, 100%, and 99.5%, respectively. The BC-GP detected Staphylococcus epidermidis, methicillin-susceptible S. epidermidis, and methicillin-resistant S. epidermidis with sensitivities of 95%, 80%, and 96%, respectively, and 100% specificity. The BC-GP correctly identified 14/15 cases of Enterococcus faecalis and Enterococcus faecium bacteremia and 9 cases of Streptococcus pneumoniae. It misidentified 5/15 clinical blood cultures with Streptococcus mitis/Streptococcus oralis and 1/3 blood cultures spiked with Streptococcus anginosus group as S. pneumoniae. The BC-GP detected a case of Streptococcus pyogenes bacteremia but failed to detect 2/3 clinical blood cultures with Streptococcus agalactiae. BC-GP's rapid accurate detection of Staphylococcus spp., E. faecium, and E. faecalis and its ability to ascertain mecA, vanA, and vanB status may expedite clinical decisions pertaining to optimal antibiotic use. False-positive S. pneumoniae results may warrant reporting of only "Streptococcus spp." when this organism is reported by the BC-GP.

  6. Human cytokine responses induced by Gram-positive cell walls of normal intestinal microbiota

    PubMed Central

    Chen, T; Isomäki, P; Rimpiläinen, M; Toivanen, P

    1999-01-01

    The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a.), Eubacterium limosum(Eu.l.), Lactobacillus casei(L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-γ). Monocytes are the main cell population in PBMC to produce TNF-α and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens. PMID:10540188

  7. Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria.

    PubMed

    Shen, Lirong; Liu, Dandan; Li, Meilu; Jin, Feng; Din, Meihui; Parnell, Laurence D; Lai, Chao-Qiang

    2012-01-01

    The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent. PMID:23056609

  8. Mechanism of Action of Recombinant Acc-Royalisin from Royal Jelly of Asian Honeybee against Gram-Positive Bacteria

    PubMed Central

    Shen, Lirong; Liu, Dandan; Li, Meilu; Jin, Feng; Din, Meihui; Parnell, Laurence D.; Lai, Chao-Qiang

    2012-01-01

    The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent. PMID:23056609

  9. Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria.

    PubMed

    Shen, Lirong; Liu, Dandan; Li, Meilu; Jin, Feng; Din, Meihui; Parnell, Laurence D; Lai, Chao-Qiang

    2012-01-01

    The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.

  10. Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.

    2011-12-01

    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

  11. Thusin, a Novel Two-Component Lantibiotic with Potent Antimicrobial Activity against Several Gram-Positive Pathogens.

    PubMed

    Xin, Bingyue; Zheng, Jinshui; Liu, Hualin; Li, Junhua; Ruan, Lifang; Peng, Donghai; Sajid, Muhammad; Sun, Ming

    2016-01-01

    Due to the rapidly increasing prevalence of multidrug-resistant bacterial strains, the need for new antimicrobial drugs to treat infections has become urgent. Bacteriocins, which are antimicrobial peptides of bacterial origin, are considered potential alternatives to conventional antibiotics and have attracted widespread attention in recent years. Among these bacteriocins, lantibiotics, especially two-component lantibiotics, exhibit potent antimicrobial activity against some clinically relevant Gram-positive pathogens and have potential applications in the pharmaceutical industry. In this study, we characterized a novel two-component lantibiotic termed thusin that consists of Thsα, Thsβ, and Thsβ' (mutation of Thsβ, A14G) and that was isolated from a B. thuringiensis strain BGSC 4BT1. Thsα and Thsβ (or Thsβ') exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells, and they are all highly thermostable (100°C for 30 min) and pH tolerant (pH 2.0 to 9.0). Thusin shows remarkable efficacy against all tested Gram-positive bacteria and greater activities than two known lantibiotics thuricin 4A-4 and ticin A4, and one antibiotic vancomycin against various bacterial pathogens (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus (MRSA), Staphylococcus sciuri, Enterococcus faecalis, and Streptococcus pneumoniae). Moreover, thusin is also able to inhibit the outgrowth of B. cereus spores. The potent antimicrobial activity of thusin against some Gram-positive pathogens indicates that it has potential for the development of new drugs. PMID:27486447

  12. Thusin, a Novel Two-Component Lantibiotic with Potent Antimicrobial Activity against Several Gram-Positive Pathogens

    PubMed Central

    Xin, Bingyue; Zheng, Jinshui; Liu, Hualin; Li, Junhua; Ruan, Lifang; Peng, Donghai; Sajid, Muhammad; Sun, Ming

    2016-01-01

    Due to the rapidly increasing prevalence of multidrug-resistant bacterial strains, the need for new antimicrobial drugs to treat infections has become urgent. Bacteriocins, which are antimicrobial peptides of bacterial origin, are considered potential alternatives to conventional antibiotics and have attracted widespread attention in recent years. Among these bacteriocins, lantibiotics, especially two-component lantibiotics, exhibit potent antimicrobial activity against some clinically relevant Gram-positive pathogens and have potential applications in the pharmaceutical industry. In this study, we characterized a novel two-component lantibiotic termed thusin that consists of Thsα, Thsβ, and Thsβ' (mutation of Thsβ, A14G) and that was isolated from a B. thuringiensis strain BGSC 4BT1. Thsα and Thsβ (or Thsβ') exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells, and they are all highly thermostable (100°C for 30 min) and pH tolerant (pH 2.0 to 9.0). Thusin shows remarkable efficacy against all tested Gram-positive bacteria and greater activities than two known lantibiotics thuricin 4A-4 and ticin A4, and one antibiotic vancomycin against various bacterial pathogens (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus (MRSA), Staphylococcus sciuri, Enterococcus faecalis, and Streptococcus pneumoniae). Moreover, thusin is also able to inhibit the outgrowth of B. cereus spores. The potent antimicrobial activity of thusin against some Gram-positive pathogens indicates that it has potential for the development of new drugs. PMID:27486447

  13. Thusin, a Novel Two-Component Lantibiotic with Potent Antimicrobial Activity against Several Gram-Positive Pathogens.

    PubMed

    Xin, Bingyue; Zheng, Jinshui; Liu, Hualin; Li, Junhua; Ruan, Lifang; Peng, Donghai; Sajid, Muhammad; Sun, Ming

    2016-01-01

    Due to the rapidly increasing prevalence of multidrug-resistant bacterial strains, the need for new antimicrobial drugs to treat infections has become urgent. Bacteriocins, which are antimicrobial peptides of bacterial origin, are considered potential alternatives to conventional antibiotics and have attracted widespread attention in recent years. Among these bacteriocins, lantibiotics, especially two-component lantibiotics, exhibit potent antimicrobial activity against some clinically relevant Gram-positive pathogens and have potential applications in the pharmaceutical industry. In this study, we characterized a novel two-component lantibiotic termed thusin that consists of Thsα, Thsβ, and Thsβ' (mutation of Thsβ, A14G) and that was isolated from a B. thuringiensis strain BGSC 4BT1. Thsα and Thsβ (or Thsβ') exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells, and they are all highly thermostable (100°C for 30 min) and pH tolerant (pH 2.0 to 9.0). Thusin shows remarkable efficacy against all tested Gram-positive bacteria and greater activities than two known lantibiotics thuricin 4A-4 and ticin A4, and one antibiotic vancomycin against various bacterial pathogens (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus (MRSA), Staphylococcus sciuri, Enterococcus faecalis, and Streptococcus pneumoniae). Moreover, thusin is also able to inhibit the outgrowth of B. cereus spores. The potent antimicrobial activity of thusin against some Gram-positive pathogens indicates that it has potential for the development of new drugs.

  14. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

    PubMed Central

    Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

    2012-01-01

    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

  15. Management of gram-positive bacterial infections in patients with cancer.

    PubMed

    Kosmidis, Christos I; Chandrasekar, Pranatharthi H

    2012-01-01

    Bacterial infections, particularly those due to gram-positive bacteria, continue to predominate in patients with cancer. Coagulase-negative and coagulase-positive staphylococci and enterococci remain as common pathogenic microorganisms. Clostridium difficile has emerged as a significant pathogen. Major clinical syndromes include vascular catheter-related infection, febrile neutropenia, diarrhea and colitis. Rising antimicrobial resistance among gram-positive bacteria is of serious concern. The clinical utility of penicillin against streptococci and vancomycin against coagulase-negative and coagulase-positive staphylococci and enterococci may be rapidly diminishing. Liberal empiric use of vancomycin during neutropenic fever needs careful reconsideration. Newer promising anti-gram-positive bacterial drugs with activity against methicillin-resistant staphylococci include daptomycin, linezolid, tigecycline and telavancin. However, toxicity concerns, limited data in immunocompromised populations and high cost prevent the widespread use of these drugs among patients with cancer.

  16. Class D β-lactamases do exist in Gram-positive bacteria

    SciTech Connect

    Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.; Frase, Hilary; Bhattacharya, Monolekha; Smith, Clyde A.; Vakulenko, Sergei B.

    2015-11-09

    Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. In conclusion, these enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

  17. Rose Bengal-decorated silica nanoparticles as photosensitizers for inactivation of gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Guo, Yanyan; Rogelj, Snezna; Zhang, Peng

    2010-02-01

    A new type of photosensitizer, made from Rose Bengal (RB)-decorated silica (SiO2-NH2-RB) nanoparticles, was developed to inactivate gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA), with high efficiency through photodynamic action. The nanoparticles were characterized microscopically and spectroscopically to confirm their structures. The characterization of singlet oxygen generated by RB, both free and immobilized on a nanoparticle surface, was performed in the presence of anthracene-9,10-dipropionic acid. The capability of SiO2-NH2-RB nanoparticles to inactivate bacteria was tested in vitro on both gram-positive and gram-negative bacteria. The results showed that RB-decorated silica nanoparticles can inactivate MRSA and Staphylococcus epidermidis (both gram-positive) very effectively (up to eight-orders-of-magnitude reduction). Photosensitizers of such design should have good potential as antibacterial agents through a photodynamic mechanism.

  18. Class D β-lactamases do exist in Gram-positive bacteria

    PubMed Central

    Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.; Frase, Hilary; Bhattacharya, Monolekha; Smith, Clyde; Vakulenko, Sergei

    2015-01-01

    Production of β-lactamases of the four molecular classes (A, B, C, and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics that have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, they have not been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate binding mode quite different from that of all currently known class A, C, and D β-lactamases. They constitute a novel reservoir of antibiotic resistance enzymes. PMID:26551395

  19. New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

    PubMed

    Gutierrez-Preciado, A; Jensen, R A; Yanofsky, C; Merino, E

    2005-08-01

    The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon. PMID:15953653

  20. Labeling and Selective Inactivation of Gram-Positive Bacteria Employing Bimodal Photoprobes with Dual Readouts.

    PubMed

    Galstyan, Anzhela; Block, Desiree; Niemann, Silke; Grüner, Malte C; Abbruzzetti, Stefania; Oneto, Michele; Daniliuc, Constantin G; Hermann, Sven; Viappiani, Cristiano; Schäfers, Michael; Löffler, Bettina; Strassert, Cristian A; Faust, Andreas

    2016-04-01

    Carbohydrate-conjugated silicon(IV) phthalocyanines with bimodal photoactivity were developed as probes with both fluorescent labeling and photosensitizing capabilities, and the concomitant fluorescent labeling and photoinduced inactivation of Gram-positive and Gram-negative models was explored. The maltohexaose-conjugated photoprobe provides a dual readout to distinguish between both groups of pathogens, as only the Gram-positive species was inactivated, even though both appeared labeled with near-infrared luminescence. Antibiotic resistance did not hinder the phototoxic effect, as even the methicillin-resistant pathogen Staphylococcus aureus (MRSA) was completely photoinactivated. Time-resolved confocal fluorescence microscopy analysis suggests that the photoprobe sticks onto the outer rim of the microorganisms, explaining the resistance of Gram-negative species on the basis of their membrane constitution. The mannose-conjugated photoprobe yields a different readout because it is able to label and to inactivate only the Gram-positive strain.

  1. Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria.

    PubMed

    Nakamura, Keisuke; Ishiyama, Kirika; Sheng, Hong; Ikai, Hiroyo; Kanno, Taro; Niwano, Yoshimi

    2015-09-01

    The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm(2)) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment.

  2. Nucleotide sequence alignment of hdcA from Gram-positive bacteria.

    PubMed

    Diaz, Maria; Ladero, Victor; Redruello, Begoña; Sanchez-Llana, Esther; Del Rio, Beatriz; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2016-03-01

    The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 〈10.2174/157340110791233256〉 [1], Linares et al. 2016 〈http://dx.doi.org/10.1016/j.foodchem.2015.11.013〉〉 [2]). The reaction is catalyzed by a pyruvoyl-dependent histidine decarboxylase (Linares et al. 2011 〈10.1080/10408398.2011.582813〉 [3]), which is encoded by the gene hdcA. In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see 〈http://dx.doi.org/ 10.1016/j.foodcont.2015.11.035〉〉 [4].

  3. Nucleotide sequence alignment of hdcA from Gram-positive bacteria

    PubMed Central

    Diaz, Maria; Ladero, Victor; Redruello, Begoña; Sanchez-Llana, Esther; del Rio, Beatriz; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A.

    2016-01-01

    The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 〈10.2174/157340110791233256〉 [1], Linares et al. 2016 〈http://dx.doi.org/10.1016/j.foodchem.2015.11.013〉〉 [2]). The reaction is catalyzed by a pyruvoyl-dependent histidine decarboxylase (Linares et al. 2011 〈10.1080/10408398.2011.582813〉 [3]), which is encoded by the gene hdcA. In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see 〈http://dx.doi.org/ 10.1016/j.foodcont.2015.11.035〉〉 [4]. PMID:26958625

  4. Class D β-lactamases do exist in Gram-positive bacteria.

    PubMed

    Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K; Frase, Hilary; Bhattacharya, Monolekha; Smith, Clyde A; Vakulenko, Sergei B

    2016-01-01

    Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

  5. The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.

    PubMed

    Arabski, Michał; Konieczna, Iwona; Tusińska, Ewa; Wąsik, Sławomir; Relich, Inga; Zając, Krzysztof; Kamiński, Zbigniew J; Kaca, Wiesław

    2015-01-01

    Lysozyme (1,4-β-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur.

  6. Predictive Factors of Spontaneous Bacterial Peritonitis Caused by Gram-Positive Bacteria in Patients With Cirrhosis.

    PubMed

    Kim, Jung Ho; Jeon, Yong Duk; Jung, In Young; Ahn, Mi Young; Ahn, Hea Won; Ahn, Jin Young; Ku, Nam Su; Han, Sang Hoon; Choi, Jun Yong; Ahn, Sang Hoon; Song, Young Goo; Han, Kwang Hyub; Kim, June Myung

    2016-04-01

    Spontaneous bacterial peritonitis (SBP) in patients with cirrhosis is typically caused by gram-negative bacteria. However, the number of SBP cases due to gram-positive bacteria is steadily increasing. To date, little is known about the predictive factors involved in SBP infections.We performed a retrospective cohort study of patients (>18 years) with SBP due to gram-positive and -negative bacteria who were enrolled from January 2006 to December 2013 at Severance Hospital in Seoul, Korea where the incidences of hepatitis B virus associated chronic liver disease, cirrhosis, and hepatocellular carcinoma are high. Only the 1st SBP episode for each patient within the study period was included in our analysis.We identified 77 patients with cirrhosis and SBP. Of these, 27 patients (35%) had gram-positive bacterial infections and 50 patients (65%) had gram-negative bacterial infections. Our univariate analysis revealed that an early stage of cirrhosis (P = 0.004), lower creatinine level (P = 0.011), lower Sequential Organ Failure Assessment (SOFA) score (P = 0.001), lower Model for End-Stage Liver Disease score (P = 0.005), and use of systemic antibiotics within 30 days before SBP diagnosis (P = 0.03) were significantly associated with gram-positive bacterial infections. Our multivariate analysis indicated that the use of systemic antibiotics within 30 days before SBP diagnosis (odds ratio, 3.94; 95% CI, 1.11-13.96; P = 0.033) and a lower SOFA score (odds ratio, 0.56; 95% CI, 0.37-0.86; P = 0.007) were independent predictive factors of SBP caused by gram-positive bacterial infections in patients with cirrhosis. However, we did not observe a statistically significant difference in the 28-day mortality between the gram-positive and -negative bacterial infection groups (40.7% vs 46.0%, respectively; P = 0.407).In this study, the incidence rate of SBP caused by gram-positive bacteria in patients with cirrhosis was similar to the rates reported

  7. Predictive Factors of Spontaneous Bacterial Peritonitis Caused by Gram-Positive Bacteria in Patients With Cirrhosis

    PubMed Central

    Kim, Jung Ho; Jeon, Yong Duk; Jung, In Young; Ahn, Mi Young; Ahn, Hea Won; Ahn, Jin Young; Ku, Nam Su; Han, Sang Hoon; Choi, Jun Yong; Ahn, Sang Hoon; Song, Young Goo; Han, Kwang Hyub; Kim, June Myung

    2016-01-01

    Abstract Spontaneous bacterial peritonitis (SBP) in patients with cirrhosis is typically caused by gram-negative bacteria. However, the number of SBP cases due to gram-positive bacteria is steadily increasing. To date, little is known about the predictive factors involved in SBP infections. We performed a retrospective cohort study of patients (>18 years) with SBP due to gram-positive and -negative bacteria who were enrolled from January 2006 to December 2013 at Severance Hospital in Seoul, Korea where the incidences of hepatitis B virus associated chronic liver disease, cirrhosis, and hepatocellular carcinoma are high. Only the 1st SBP episode for each patient within the study period was included in our analysis. We identified 77 patients with cirrhosis and SBP. Of these, 27 patients (35%) had gram-positive bacterial infections and 50 patients (65%) had gram-negative bacterial infections. Our univariate analysis revealed that an early stage of cirrhosis (P = 0.004), lower creatinine level (P = 0.011), lower Sequential Organ Failure Assessment (SOFA) score (P = 0.001), lower Model for End-Stage Liver Disease score (P = 0.005), and use of systemic antibiotics within 30 days before SBP diagnosis (P = 0.03) were significantly associated with gram-positive bacterial infections. Our multivariate analysis indicated that the use of systemic antibiotics within 30 days before SBP diagnosis (odds ratio, 3.94; 95% CI, 1.11–13.96; P = 0.033) and a lower SOFA score (odds ratio, 0.56; 95% CI, 0.37–0.86; P = 0.007) were independent predictive factors of SBP caused by gram-positive bacterial infections in patients with cirrhosis. However, we did not observe a statistically significant difference in the 28-day mortality between the gram-positive and -negative bacterial infection groups (40.7% vs 46.0%, respectively; P = 0.407). In this study, the incidence rate of SBP caused by gram-positive bacteria in patients with cirrhosis was similar to the

  8. Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria.

    PubMed

    Woo, Patrick C Y; Teng, Jade L L; Wu, Jeff K L; Leung, Fion P S; Tse, Herman; Fung, Ami M Y; Lau, Susanna K P; Yuen, Kwok-Yung

    2009-08-01

    This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0-2 and 2-13 % of staphylococci, and 0 and 0-10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36-56 and 11-14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S r

  9. Comparison of the Bruker MALDI-TOF mass spectrometry system and conventional phenotypic methods for identification of Gram-positive rods.

    PubMed

    Barberis, Claudia; Almuzara, Marisa; Join-Lambert, Olivier; Ramírez, María Soledad; Famiglietti, Angela; Vay, Carlos

    2014-01-01

    In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥ 1,5 and ≥ 1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥ 1,5 for genus level and ≥ 1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.

  10. Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

    PubMed Central

    Barberis, Claudia; Almuzara, Marisa; Join-Lambert, Olivier; Ramírez, María Soledad; Famiglietti, Angela; Vay, Carlos

    2014-01-01

    In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories. PMID:25184254

  11. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria.

    PubMed

    Jensen, R A; d'Amato, T A; Hochstein, L I

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

  12. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

    NASA Technical Reports Server (NTRS)

    Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

  13. Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting.

    PubMed

    Böhme, Karola; Fernández-No, Inmaculada C; Barros-Velázquez, Jorge; Gallardo, Jose M; Cañas, Benito; Calo-Mata, Pilar

    2011-11-01

    The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram-positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10-30 peak masses. Genus-specific as well as species-specific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web-application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI-TOF MS fingerprinting is applied to Gram-positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.

  14. Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

    PubMed Central

    Poh, Wee Han; Chu, Justin Jang Hann; Loo, Joachim Say Chye; Bazan, Guillermo C.; Hancock, Lynn E.

    2015-01-01

    The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone. PMID:25576607

  15. Oligopolyphenylenevinylene-conjugated oligoelectrolyte membrane insertion molecules selectively disrupt cell envelopes of Gram-positive bacteria.

    PubMed

    Hinks, Jamie; Poh, Wee Han; Chu, Justin Jang Hann; Loo, Joachim Say Chye; Bazan, Guillermo C; Hancock, Lynn E; Wuertz, Stefan

    2015-03-01

    The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone. PMID:25576607

  16. Novel antibiotics targeting respiratory ATP synthesis in Gram-positive pathogenic bacteria.

    PubMed

    Balemans, Wendy; Vranckx, Luc; Lounis, Nacer; Pop, Ovidiu; Guillemont, Jérôme; Vergauwen, Karen; Mol, Selena; Gilissen, Ron; Motte, Magali; Lançois, David; De Bolle, Miguel; Bonroy, Kristien; Lill, Holger; Andries, Koen; Bald, Dirk; Koul, Anil

    2012-08-01

    Emergence of drug-resistant bacteria represents a high, unmet medical need, and discovery of new antibacterials acting on new bacterial targets is strongly needed. ATP synthase has been validated as an antibacterial target in Mycobacterium tuberculosis, where its activity can be specifically blocked by the diarylquinoline TMC207. However, potency of TMC207 is restricted to mycobacteria with little or no effect on the growth of other Gram-positive or Gram-negative bacteria. Here, we identify diarylquinolines with activity against key Gram-positive pathogens, significantly extending the antibacterial spectrum of the diarylquinoline class of drugs. These compounds inhibited growth of Staphylococcus aureus in planktonic state as well as in metabolically resting bacteria grown in a biofilm culture. Furthermore, time-kill experiments showed that the selected hits are rapidly bactericidal. Drug-resistant mutations were mapped to the ATP synthase enzyme, and biochemical analysis as well as drug-target interaction studies reveal ATP synthase as a target for these compounds. Moreover, knockdown of the ATP synthase expression strongly suppressed growth of S. aureus, revealing a crucial role of this target in bacterial growth and metabolism. Our data represent a proof of principle for using the diarylquinoline class of antibacterials in key Gram-positive pathogens. Our results suggest that broadening the antibacterial spectrum for this chemical class is possible without drifting off from the target. Development of the diarylquinolines class may represent a promising strategy for combating Gram-positive pathogens. PMID:22615276

  17. Native and heterologous production of bacteriocins from gram-positive microorganisms.

    PubMed

    Muñoz, Mabel; Jaramillo, Diana; Melendez, Adelina Del Pilar; J Alméciga-Diaz, Carlos; Sánchez, Oscar F

    2011-12-01

    In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.

  18. Distinct localization of the complement C5b-9 complex on Gram-positive bacteria.

    PubMed

    Berends, Evelien T M; Dekkers, Johanna F; Nijland, Reindert; Kuipers, Annemarie; Soppe, Jasper A; van Strijp, Jos A G; Rooijakkers, Suzan H M

    2013-12-01

    The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.

  19. Transformations of the gram-positive honey bee pathogen, Paenibacillus larvae, by electroporation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae—a deadly pathogen of honey bees. The method is substantially different from the only other electroporation method for a Paenibacillus species found in the literature. Using the ty...

  20. Oligopolyphenylenevinylene-conjugated oligoelectrolyte membrane insertion molecules selectively disrupt cell envelopes of Gram-positive bacteria.

    PubMed

    Hinks, Jamie; Poh, Wee Han; Chu, Justin Jang Hann; Loo, Joachim Say Chye; Bazan, Guillermo C; Hancock, Lynn E; Wuertz, Stefan

    2015-03-01

    The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone.

  1. Antimicrobial Growth Promoters Used in Animal Feed: Effects of Less Well Known Antibiotics on Gram-Positive Bacteria

    PubMed Central

    Butaye, Patrick; Devriese, Luc A.; Haesebrouck, Freddy

    2003-01-01

    There are not many data available on antibiotics used solely in animals and almost exclusively for growth promotion. These products include bambermycin, avilamycin, efrotomycin, and the ionophore antibiotics (monensin, salinomycin, narasin, and lasalocid). Information is also scarce for bacitracin used only marginally in human and veterinary medicine and for streptogramin antibiotics. The mechanisms of action of and resistance mechanisms against these antibiotics are described. Special emphasis is given to the prevalence of resistance among gram-positive bacteria isolated from animals and humans. Since no susceptibility breakpoints are available for most of the antibiotics discussed, an alternative approach to the interpretation of MICs is presented. Also, some pharmacokinetic data and information on the influence of these products on the intestinal flora are presented. PMID:12692092

  2. Lipoteichoic Acids, Phosphate-Containing Polymers in the Envelope of Gram-Positive Bacteria

    PubMed Central

    Schneewind, Olaf

    2014-01-01

    Lipoteichoic acids (LTA) are polymers of alternating units of a polyhydroxy alkane, including glycerol and ribitol, and phosphoric acid, joined to form phosphodiester units that are found in the envelope of Gram-positive bacteria. Here we review four different types of LTA that can be distinguished on the basis of their chemical structure and describe recent advances in the biosynthesis pathway for type I LTA, d-alanylated polyglycerol-phosphate linked to di-glucosyl-diacylglycerol. The physiological functions of type I LTA are discussed in the context of inhibitors that block their synthesis and of mutants with discrete synthesis defects. Research on LTA structure and function represents a large frontier that has been investigated in only few Gram-positive bacteria. PMID:24415723

  3. Sulfonylpiperidines as novel, antibacterial inhibitors of Gram-positive thymidylate kinase (TMK).

    PubMed

    Martínez-Botella, Gabriel; Loch, James T; Green, Oluyinka M; Kawatkar, Sameer P; Olivier, Nelson B; Boriack-Sjodin, P Ann; Keating, Thomas A

    2013-01-01

    Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK. Formation of hydrogen bonds with Arg48 in Staphylococcus aureus TMK was key to obtaining excellent enzyme affinity, as verified by protein crystallography. Replacement of a methylene linker in the series by a sulfonamide was accomplished with retention of binding conformation. Further optimization of logD yielded phenol derivative 11, a potent inhibitor of TMK showing excellent MICs against a broad spectrum of Gram-positive bacteria and >10(5) selectivity versus the human TMK homologue. PMID:23206863

  4. Novel Acylphosphate Mimics that Target PlsY, an Essential Acyltransferase in Gram-Positive Bacteria

    PubMed Central

    Grimes, Kimberly D.; Lu, Ying-Jie; Zhang, Yong-Mei; Luna, Vicki A.; Hurdle, Julian G.; Carson, Elizabeth I.; Qi, Jianjun; Kudrimoti, Sucheta; Rock, Charles O.

    2009-01-01

    PlsY is a recently discovered acyltransferase that executes an essential step in membrane phospholipid biosynthesis in Gram-positive bacteria. Using a bioisosteric replacement approach to generate substrate-based inhibitors of PlsY as potential novel antibacterial agents, a series of stabilized acylphosphate mimetics, including acylphosphonates, acyl αα,-difluoromethyl phosphonates, acyl phosphoramides, reverse amide phosphonates, acylsulfamates and acylsulfamides were designed and synthesized. Several acyl phosphonates, phosphoramides and sulfamates were identified as inhibitors of PlsY from Streptococcus pneumoniae and Bacillus anthracis. As anticipated, these inhibitors were competitive inhibitors with respect to the acylphosphate substrate. Antimicrobial testing showed the inhibitors to have generally weak anti Gram-positive activity with the exception of some acyl phosphonates, reverse amide phosphonates, and acylsulfamates that had potent activity against multiple strains of Bacillus anthracis. PMID:19016283

  5. β-Lactam Resistance Mechanisms: Gram-Positive Bacteria and Mycobacterium tuberculosis.

    PubMed

    Fisher, Jed F; Mobashery, Shahriar

    2016-01-01

    The value of the β-lactam antibiotics for the control of bacterial infection has eroded with time. Three Gram-positive human pathogens that were once routinely susceptible to β-lactam chemotherapy-Streptococcus pneumoniae, Enterococcus faecium, and Staphylococcus aureus-now are not. Although a fourth bacterium, the acid-fast (but not Gram-positive-staining) Mycobacterium tuberculosis, has intrinsic resistance to earlier β-lactams, the emergence of strains of this bacterium resistant to virtually all other antibiotics has compelled the evaluation of newer β-lactam combinations as possible contributors to the multidrug chemotherapy required to control tubercular infection. The emerging molecular-level understanding of these resistance mechanisms used by these four bacteria provides the conceptual framework for bringing forward new β-lactams, and new β-lactam strategies, for the future control of their infections. PMID:27091943

  6. Common patterns - unique features: nitrogen metabolism and regulation in Gram-positive bacteria.

    PubMed

    Amon, Johannes; Titgemeyer, Fritz; Burkovski, Andreas

    2010-07-01

    Gram-positive bacteria have developed elaborate mechanisms to control ammonium assimilation, at the levels of both transcription and enzyme activity. In this review, the common and specific mechanisms of nitrogen assimilation and regulation in Gram-positive bacteria are summarized and compared for the genera Bacillus, Clostridium, Streptomyces, Mycobacterium and Corynebacterium, with emphasis on the high G+C genera. Furthermore, the importance of nitrogen metabolism and control for the pathogenic lifestyle and virulence is discussed. In summary, the regulation of nitrogen metabolism in prokaryotes shows an impressive diversity. Virtually every phylum of bacteria evolved its own strategy to react to the changing conditions of nitrogen supply. Not only do the transcription factors differ between the phyla and sometimes even between families, but the genetic targets of a given regulon can also differ between closely related species.

  7. Cyclic-di-GMP signaling in the Gram-positive pathogen Clostridium difficile.

    PubMed

    Bordeleau, Eric; Burrus, Vincent

    2015-11-01

    The anaerobic Gram-positive bacterium Clostridium difficile causes intestinal infections responsible for symptoms ranging from mild diarrhea to fulminant colitis. Like other bacteria, C. difficile needs to sense and integrate environmental signals in order to adapt to changes and thrive in its environment. The second messenger cyclic diguanosine monophosphate (c-di-GMP) was recently recognized as a quasi-ubiquitous phenotype coordinator in bacteria. Mostly known to be involved in the transition from motile to sessile and multicellular behaviors in Gammaproteobacteria, c-di-GMP is now known to regulate many other phenotypes from cell morphogenesis to virulence, in many Gram-negative and a few Gram-positive bacteria. Herein, we review recent advances in our understanding of c-di-GMP signaling in the lifecycle of C. difficile.

  8. Blue green alga mediated synthesis of gold nanoparticles and its antibacterial efficacy against Gram positive organisms.

    PubMed

    Suganya, K S Uma; Govindaraju, K; Kumar, V Ganesh; Dhas, T Stalin; Karthick, V; Singaravelu, G; Elanchezhiyan, M

    2015-02-01

    Biofunctionalized gold nanoparticles (AuNPs) play an important role in design and development of nanomedicine. Synthesis of AuNPs from biogenic materials is environmentally benign and possesses high bacterial inhibition and bactericidal properties. In the present study, blue green alga Spirulina platensis protein mediated synthesis of AuNPs and its antibacterial activity against Gram positive bacteria is discussed. AuNPs were characterized using Ultraviolet-visible (UV-vis) spectroscopy, Fluorescence spectroscopy, Fourier Transform-Infrared (FTIR) spectroscopy, Raman spectroscopy, High Resolution-Transmission Electron Microscopy (HR-TEM) and Energy Dispersive X-ray analysis (EDAX). Stable, well defined AuNPs of smaller and uniform shape with an average size of ~ 5 nm were obtained. The antibacterial efficacy of protein functionalized AuNPs were tested against Gram positive organisms Bacillus subtilis and Staphylococcus aureus. PMID:25492207

  9. When Ribonucleases Come into Play in Pathogens: A Survey of Gram-Positive Bacteria

    PubMed Central

    Jester, Brian C.; Romby, Pascale; Lioliou, Efthimia

    2012-01-01

    It is widely acknowledged that RNA stability plays critical roles in bacterial adaptation and survival in different environments like those encountered when bacteria infect a host. Bacterial ribonucleases acting alone or in concert with regulatory RNAs or RNA binding proteins are the mediators of the regulatory outcome on RNA stability. We will give a current update of what is known about ribonucleases in the model Gram-positive organism Bacillus subtilis and will describe their established roles in virulence in several Gram-positive pathogenic bacteria that are imposing major health concerns worldwide. Implications on bacterial evolution through stabilization/transfer of genetic material (phage or plasmid DNA) as a result of ribonucleases' functions will be covered. The role of ribonucleases in emergence of antibiotic resistance and new concepts in drug design will additionally be discussed. PMID:22550495

  10. Small regulatory RNAs from low-GC Gram-positive bacteria

    PubMed Central

    Brantl, Sabine; Brückner, Reinhold

    2014-01-01

    Small regulatory RNAs (sRNAs) that act by base-pairing were first discovered in so-called accessory DNA elements—plasmids, phages, and transposons—where they control replication, maintenance, and transposition. Since 2001, a huge body of work has been performed to predict and identify sRNAs in a multitude of bacterial genomes. The majority of chromosome-encoded sRNAs have been investigated in E. coli and other Gram-negative bacteria. However, during the past five years an increasing number of sRNAs were found in Gram-positive bacteria. Here, we outline our current knowledge on chromosome-encoded sRNAs from low-GC Gram-positive species that act by base-pairing, i.e., an antisense mechanism. We will focus on sRNAs with known targets and defined regulatory mechanisms with special emphasis on Bacillus subtilis. PMID:24576839

  11. Optimizing identification of clinically relevant Gram-positive organisms by use of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system.

    PubMed

    McElvania Tekippe, Erin; Shuey, Sunni; Winkler, David W; Butler, Meghan A; Burnham, Carey-Ann D

    2013-05-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including "heavy" (H) and "light" (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or "score." We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS.

  12. Synthetic teichoic acid conjugate vaccine against nosocomial Gram-positive bacteria.

    PubMed

    Laverde, Diana; Wobser, Dominique; Romero-Saavedra, Felipe; Hogendorf, Wouter; van der Marel, Gijsbert; Berthold, Martin; Kropec, Andrea; Codee, Jeroen; Huebner, Johannes

    2014-01-01

    Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.

  13. Gram-positive antimicrobial activity of amino acid-based hydrogels.

    PubMed

    Irwansyah, I; Li, Yong-Qiang; Shi, Wenxiong; Qi, Dianpeng; Leow, Wan Ru; Tang, Mark B Y; Li, Shuzhou; Chen, Xiaodong

    2015-01-27

    Antimicrobial hydrogels are prepared based on the co-assembly of commercial Fmoc-phenylalanine and Fmoc-leucine, which act as the hydrogelator and antimicrobial building block, respectively. This co-assembled antimicrobial hydrogel is demonstrated to exhibit selective bactericidal activity for gram-positive bacteria while being biocompatible with normal mammalian cells, showing great potential as an antimicrobial coating for clinical anti-infective applications.

  14. Progress in the Development of Effective Vaccines to Prevent Selected Gram Positive Bacterial Infections

    PubMed Central

    Bronze, Michael S.; Dale, James B.

    2010-01-01

    Infections due to virulent gram positive bacteria, such as Staphylococcus aureus, group B streptococci and group A streptococci remain significant causes of morbidity and mortality despite progress in antimicrobial therapy. Despite significant advances in the understanding of the pathogenesis of infection due to these organisms, there are only limited strategies to prevent infection. In this paper, we review efforts to develop safe and effective vaccines that would prevent infections due to these 3 pathogens. PMID:20697258

  15. Identification of novel aminopiperidine derivatives for antibacterial activity against Gram-positive bacteria.

    PubMed

    Lee, Hee-Yeol; An, Kyung-Mi; Jung, Juyoung; Koo, Je-Min; Kim, Jeong-Geun; Yoon, Jong-Min; Lee, Myong-Jae; Jang, HyeonSoo; Lee, Hong-Sub; Park, Soobong; Kang, Jae-Hoon

    2016-07-01

    We have previously reported amidopiperidine derivatives as a novel peptide deformylase (PDF) inhibitor and evaluated its antibacterial activity against Gram-positive bacteria, but poor pharmacokinetic profiles have resulted in low efficacy in in vivo mouse models. In order to overcome these weaknesses, we newly synthesized aminopiperidine derivatives with remarkable antimicrobial properties and oral bioavailability, and also identified their in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). PMID:27173797

  16. Critical cell wall hole size for lysis in Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  17. The CodY pleiotropic repressor controls virulence in gram-positive pathogens.

    PubMed

    Stenz, Ludwig; Francois, Patrice; Whiteson, Katrine; Wolz, Christiane; Linder, Patrick; Schrenzel, Jacques

    2011-07-01

    CodY is involved in the adaptive response to starvation in at least 30 different low G+C gram-positive bacteria. After dimerization and activation by cofactor binding, CodY binds to a consensus palindromic DNA sequence, leading to the repression of approximately 5% of the genome. CodY represses the transcription of target genes when bound to DNA by competition with the RNA polymerase for promoter binding, or by interference with transcriptional elongation as a roadblock. CodY displays enhanced affinity for its DNA target when bound to GTP and/or branched chain amino acids (BCAA). When nutrients become limiting in the postexponential growth phase, a decrease of intracellular levels of GTP and BCAA causes a deactivation of CodY and decreases its affinity for DNA, leading to the induction of its regulon. CodY-regulated genes trigger adaptation of the bacteria to starvation by highly diverse mechanisms, such as secretion of proteases coupled to expression of amino acid transporters, and promotion of survival strategies like sporulation or biofilm formation. Additionally, in pathogenic bacteria, several virulence factors are regulated by CodY. As a function of their access to nutrients, pathogenic gram-positive bacteria express virulence factors in a codY-dependant manner. This is true for the anthrax toxins of Bacillus anthracis and the haemolysins of Staphylococcus aureus. The purpose of this review is to illustrate CodY-regulated mechanisms on virulence in major gram-positive pathogens.

  18. Class D β-lactamases do exist in Gram-positive bacteria

    DOE PAGES

    Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.; Frase, Hilary; Bhattacharya, Monolekha; Smith, Clyde A.; Vakulenko, Sergei B.

    2015-11-09

    Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinctmore » structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. In conclusion, these enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.« less

  19. Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence.

    PubMed

    Nguyen, Minh Thu; Götz, Friedrich

    2016-09-01

    Since the discovery in 1973 of the first of the bacterial lipoproteins (Lpp) in Escherichia coli, Braun's lipoprotein, the ever-increasing number of publications indicates the importance of these proteins. Bacterial Lpp belong to the class of lipid-anchored proteins that in Gram-negative bacteria are anchored in both the cytoplasmic and outer membranes and in Gram-positive bacteria are anchored only in the cytoplasmic membrane. In contrast to the case for Gram-negative bacteria, in Gram-positive bacteria lipoprotein maturation and processing are not vital. Physiologically, Lpp play an important role in nutrient and ion acquisition, allowing particularly pathogenic species to better survive in the host. Bacterial Lpp are recognized by Toll-like receptor 2 (TLR2) of the innate immune system. The important role of Lpp in Gram-positive bacteria, particularly in the phylum Firmicutes, as key players in the immune response and pathogenicity has emerged only in recent years. In this review, we address the role of Lpp in signaling and modulating the immune response, in inflammation, and in pathogenicity. We also address the potential of Lpp as promising vaccine candidates.

  20. Employing carbon dots modified with vancomycin for assaying Gram-positive bacteria like Staphylococcus aureus.

    PubMed

    Zhong, Dan; Zhuo, Yan; Feng, Yuanjiao; Yang, Xiaoming

    2015-12-15

    By employing attractive performance of fluorescent carbon dots, we herein successfully established an assay for analyzing bacteria firstly. Specifically, carbon dots with blue fluorescence were initially synthesized according to a previous report, and modified with vancomycin on their surfaces. Subsequently, the prepared carbon dots were applied to detect Staphylococcus aureus accompanied with a linear range of 3.18×10(5)-1.59×10(8) cfu/mL as well as a detection limit of 9.40×10(4) cfu/mL. Compared with other regular methods, our method is more rapid and convenient in term of methodology. Meanwhile, the current strategy was applied for detection of other bacteria including Bacillus subtilis, Listeria monocytogenes, Salmonella, Pseudomonas aeruginosa and Escherichia coli, and the modified carbon dots showed obvious affinity with Gram-positive bacteria owing to the ligand-receptor interactions between vancomycin and the cell walls, suggesting its value for detecting Gram-positive bacteria. Additionally, the practicability of this sensing approach was validated by recovery experiments conducted in orange juice, confirming its potential to broaden avenues for detection of Gram-positive bacteria.

  1. Electrochemical classification of gram-negative and gram-positive bacteria.

    PubMed Central

    Matsunaga, T; Nakajima, T

    1985-01-01

    Intestinal bacteria were classified as gram-positive or gram-negative by an electrode system with a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap bacteria. When the potential of the graphite electrode was run in the range of 0 to 1.0 V versus the saturated calomel electrode (SCE), gram-positive bacteria gave peak currents at 0.65 to 0.69 V versus the SCE. The peak potentials of gram-negative bacteria were 0.70 to 0.74 V versus the SCE. Gram-negative bacteria and gram-positive bacteria were also classified based on the ratio of the second peak current to the first peak current when the potential cycle was repeated twice. The numbers of cells on the membrane filter were determined from the peak currents. It was found that the peak currents result from the electrochemical oxidation of coenzyme A in the cells of Escherichia coli and Lactobacillus acidophilus. Images PMID:3931548

  2. High benzene concentrations can favour Gram-positive bacteria in groundwaters from a contaminated aquifer.

    PubMed

    Fahy, Anne; Ball, Andrew S; Lethbridge, Gordon; McGenity, Terry J; Timmis, Kenneth N

    2008-09-01

    Exposure to pollution exerts strong selective pressure on microbial communities, which may affect their potential to adapt to current or future environmental challenges. In this microcosm study, we used DNA fingerprinting based on 16S rRNA genes to document the impact of high concentrations of benzene on two bacterial communities from a benzene-contaminated aquifer situated below a petrochemical plant (SIReN, UK). The two groundwaters harboured distinct aerobic benzene-degrading communities able to metabolize benzene to below detection levels (1 microg L(-1)). A benzene concentration of 100 mg L(-1) caused a major shift from Betaproteobacteria to Actinobacteria, in particular Arthrobacter spp. A similar shift from Betaproteobacteria to Arthrobacter spp. and Rhodococcus erythropolis was observed in minimal medium (MM) inoculated with a third groundwater. These Gram-positive-dominated communities were able to grow on benzene at concentrations up to 600 mg L(-1) in groundwater and up to 1000 mg L(-1) in MM, concentrations that cause significant solvent stress to cellular systems. Therefore, Gram-positive bacteria were better competitors than Gram-negative organisms under experimental conditions of high benzene loads, which suggests that solvent-tolerant Gram-positive bacteria can play a role in the natural attenuation of benzene or the remediation of contaminated sites.

  3. Screening beneficial rhizobacteria from Spartina maritima for phytoremediation of metal polluted salt marshes: comparison of gram-positive and gram-negative strains.

    PubMed

    Paredes-Páliz, Karina I; Caviedes, Miguel A; Doukkali, Bouchra; Mateos-Naranjo, Enrique; Rodríguez-Llorente, Ignacio D; Pajuelo, Eloísa

    2016-10-01

    The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes. PMID:27417328

  4. Screening beneficial rhizobacteria from Spartina maritima for phytoremediation of metal polluted salt marshes: comparison of gram-positive and gram-negative strains.

    PubMed

    Paredes-Páliz, Karina I; Caviedes, Miguel A; Doukkali, Bouchra; Mateos-Naranjo, Enrique; Rodríguez-Llorente, Ignacio D; Pajuelo, Eloísa

    2016-10-01

    The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes.

  5. Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

    PubMed

    Watson, Amanda L; Chiu, Norman H L

    2016-09-01

    Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

  6. Glycerol Monolaurate Inhibits the Effects of Gram Positive Select Agents on Eukaryotic Cells†

    PubMed Central

    Peterson, Marnie L.; Schlievert, Patrick M.

    2008-01-01

    Many exotoxins of gram positive bacteria, such as superantigens (staphylococcal enterotoxins, toxic shock syndrome toxin-1 [TSST-1], and streptococcal pyrogenic exotoxins) and anthrax toxin are bioterrorism agents that cause diseases by immunostimulation or cytotoxicity. Glycerol monolaurate (GML), a fatty acid monoester found naturally in humans, has been reported to prevent synthesis of gram positive bacterial exotoxins. This study explored the ability of GML to inhibit the effects of exotoxins on mammalian cells and prevent rabbit lethality from TSS. GML (≥10 ug/ml) inhibited superantigen (5 ug/ml) immunoproliferation, as determined by inhibition of 3H-thymidine incorporation into DNA of human peripheral blood mononuclear cells (1 × 106 cells/ml) as well as phospholipase Cγ1, suggesting inhibition of signal transduction. The compound (20 ug/ml) prevented superantigen (100 ug/ml) induced cytokine secretion by human vaginal epithelial cells (HVECs) as measured by ELISA. GML (250 ug) inhibited rabbit lethality due to TSST-1 administered vaginally. GML (10 ug/ml) inhibited HVEC and macrophage cytotoxicity by anthrax toxin, prevented erythrocyte lysis by purified hemolysins (staphylococcal α and β) and culture fluids containing streptococcal and Bacillus anthracis hemolysins, and was non-toxic to mammalian cells (up to 100 ug/ml) and rabbits (250 ug). GML stabilized mammalian cell membranes, as erythrocyte lysis was reduced in the presence of hypotonic aqueous solutions (0 to 0.05 M saline) or staphylococcal α and β-hemolysins when erythrocytes were pretreated with GML. GML may be useful in management of gram positive exotoxin illnesses; its action appears to be membrane stabilization with inhibition of signal transduction. PMID:16475828

  7. Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria.

    PubMed

    Evrin, Cécile; Straut, Monica; Slavova-Azmanova, Neli; Bucurenci, Nadia; Onu, Adrian; Assairi, Liliane; Ionescu, Mihaela; Palibroda, Nicolae; Bârzu, Octavian; Gilles, Anne-Marie

    2007-03-01

    In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.

  8. Syndecan-1 Is an in Vivo Suppressor of Gram-positive Toxic Shock*

    PubMed Central

    Hayashida, Kazutaka; Chen, Ye; Bartlett, Allison H.; Park, Pyong Woo

    2008-01-01

    Heparan sulfate proteoglycans bind to and regulate many inflammatory mediators in vitro, suggesting that they serve an important role in influencing inflammatory responses in vivo. Here we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating inflammatory responses in Gram-positive toxic shock, a systemic disease that is a significant cause of morbidity and mortality. Syndecan-1-null and wild-type mice were injected intraperitoneally with staphylococcal enterotoxin B, a pyrogenic superantigen, and their inflammatory responses were assessed. Syndecan-1-null mice showed significantly increased liver injury, vascular permeability, and death in response to staphylococcal enterotoxin B challenge compared with wild-type mice. Although serum levels of systemic IL-2 and IFNγ were similar between the two backgrounds, those of TNFα and IL-6 were significantly increased in syndecan-1-null mice undergoing Gram-positive toxic shock. Furthermore, syndecan-1-null mice challenged with staphylococcal enterotoxin B showed enhanced T cell accumulation in tissues, whereas immunodepletion of T cells protected syndecan-1-null mice from the magnified systemic cytokine storm, inflammatory tissue injury, and death. Importantly, syndecan-1 shedding was induced in wild-type mice injected with staphylococcal enterotoxin B, and the administration of heparan sulfate, but not syndecan-1 core protein, rescued syndecan-1-null mice from lethal toxic shock by suppressing the production of TNFα and IL-6, and attenuating inflammatory tissue injury. Altogether, these data suggest that syndecan-1 shedding is a key endogenous mechanism that protects the host from Gram-positive toxic shock by inhibiting the dysregulation and amplification of the inflammatory response. PMID:18499671

  9. Novel Innate Immune Genes Regulating the Macrophage Response to Gram Positive Bacteria.

    PubMed

    Alper, Scott; Warg, Laura A; De Arras, Lesly; Flatley, Brenna R; Davidson, Elizabeth J; Adams, Jenni; Smith, Keith; Wohlford-Lenane, Christine L; McCray, Paul B; Pedersen, Brent S; Schwartz, David A; Yang, Ivana V

    2016-09-01

    Host variation in Toll-like receptors and other innate immune signaling molecules alters infection susceptibility. However, only a portion of the variability observed in the innate immune response is accounted for by known genes in these pathways. Thus, the identification of additional genes that regulate the response to Gram positive bacteria is warranted. Bone marrow-derived macrophages (BMMs) from 43 inbred mouse strains were stimulated with lipotechoic acid (LTA), a major component of the Gram positive bacterial cell wall. Concentrations of the proinflammatory cytokines IL-6, IL-12, and TNF-α were measured. In silico whole genome association (WGA) mapping was performed using cytokine responses followed by network analysis to prioritize candidate genes. To determine which candidate genes could be responsible for regulating the LTA response, candidate genes were inhibited using RNA interference (RNAi) and were overexpressed in RAW264.7 macrophages. BMMs from Bdkrb1-deficient mice were used to assess the effect of Bdkrb1 gene deletion on the response to LTA, heat-killed Streptococcus pneumoniae, and heat-killed Staphylococcus aureus WGA mapping identified 117 loci: IL-6 analysis yielded 20 loci (average locus size = 0.133 Mb; 18 genes), IL-12 analysis produced 5 loci (0.201 Mb average; 7 genes), and TNF-α analysis yielded 92 loci (0.464 Mb average; 186 genes of which 46 were prioritized by network analysis). The follow-up small interfering RNA screen of 71 target genes identified four genes (Bdkrb1, Blnk, Fbxo17, and Nkx6-1) whose inhibition resulted in significantly reduced cytokine production following LTA stimulation. Overexpression of these four genes resulted in significantly increased cytokine production in response to LTA. Bdkrb1-deficient macrophages were less responsive to LTA and heat-killed S. aureus, validating the genetic and RNAi approach to identify novel regulators of the response to LTA. We have identified four innate immune response genes that

  10. Fighting infections due to multidrug-resistant Gram-positive pathogens.

    PubMed

    Cornaglia, G

    2009-03-01

    Growing bacterial resistance in Gram-positive pathogens means that what were once effective and inexpensive treatments for infections caused by these bacteria are now being seriously questioned, including penicillin and macrolides for use against pneumococcal infections and-in hospitals-oxacillin for use against staphylococcal infections. As a whole, multidrug-resistant (MDR) Gram-positive pathogens are rapidly becoming an urgent and sometimes unmanageable clinical problem. Nevertheless, and despite decades of research into the effects of antibiotics, the actual risk posed to human health by antibiotic resistance has been poorly defined; the lack of reliable data concerning the outcomes resulting from antimicrobial resistance stems, in part, from problems with study designs and the methods used in resistence determination. Surprisingly little is known, too, about the actual effectiveness of the many types of intervention aimed at controlling antibiotic resistance. New antibiotics active against MDR Gram-positive pathogens have been recently introduced into clinical practice, and the antibiotic pipeline contains additional compounds at an advanced stage of development, including new glycopeptides, new anti-methicillin-resistant Staphylococcus aureus (MRSA) beta-lactams, and new diaminopyrimidines. Many novel antimicrobial agents are likely to be niche products, endowed with narrow antibacterial spectra and/or targeted at specific clinical problems. Therefore, an important educational goal will be to change the current, long-lasting attitudes of both physicians and customers towards broad-spectrum and multipurpose compounds. Scientific societies, such as the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), must play a leading role in this process.

  11. Desulfotomaculum spp. and related gram-positive sulfate-reducing bacteria in deep subsurface environments

    PubMed Central

    Aüllo, Thomas; Ranchou-Peyruse, Anthony; Ollivier, Bernard; Magot, Michel

    2013-01-01

    Gram-positive spore-forming sulfate reducers and particularly members of the genus Desulfotomaculum are commonly found in the subsurface biosphere by culture based and molecular approaches. Due to their metabolic versatility and their ability to persist as endospores. Desulfotomaculum spp. are well-adapted for colonizing environments through a slow sedimentation process. Because of their ability to grow autotrophically (H2/CO2) and produce sulfide or acetate, these microorganisms may play key roles in deep lithoautotrophic microbial communities. Available data about Desulfotomaculum spp. and related species from studies carried out from deep freshwater lakes, marine sediments, oligotrophic and organic rich deep geological settings are discussed in this review. PMID:24348471

  12. Telavancin versus Vancomycin for Hospital-Acquired Pneumonia due to Gram-positive Pathogens

    PubMed Central

    Lalani, Tahaniyat; Corey, G. Ralph; Kanafani, Zeina A.; Nannini, Esteban C.; Rocha, Marcelo G.; Rahav, Galia; Niederman, Michael S.; Kollef, Marin H.; Shorr, Andrew F.; Lee, Patrick C.; Lentnek, Arnold L.; Luna, Carlos M.; Fagon, Jean-Yves; Torres, Antoni; Kitt, Michael M.; Genter, Fredric C.; Barriere, Steven L.; Friedland, H. David; Stryjewski, Martin E.

    2011-01-01

    Background. Telavancin is a lipoglycopeptide bactericidal against gram-positive pathogens. Methods. Two methodologically identical, double-blind studies (0015 and 0019) were conducted involving patients with hospital-acquired pneumonia (HAP) due to gram-positive pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA). Patients were randomized 1:1 to telavancin (10 mg/kg every 24 h) or vancomycin (1 g every 12 h) for 7–21 days. The primary end point was clinical response at follow-up/test-of-cure visit. Results. A total of 1503 patients were randomized and received study medication (the all-treated population). In the pooled all-treated population, cure rates with telavancin versus vancomycin were 58.9% versus 59.5% (95% confidence interval [CI] for the difference, –5.6% to 4.3%). In the pooled clinically evaluable population (n = 654), cure rates were 82.4% with telavancin and 80.7% with vancomycin (95% CI for the difference, –4.3% to 7.7%). Treatment with telavancin achieved higher cure rates in patients with monomicrobial S. aureus infection and comparable cure rates in patients with MRSA infection; in patients with mixed gram-positive/gram-negative infections, cure rates were higher in the vancomycin group. Incidence and types of adverse events were comparable between the treatment groups. Mortality rates for telavancin-treated versus vancomycin-treated patients were 21.5% versus 16.6% (95% CI for the difference, –0.7% to 10.6%) for study 0015 and 18.5% versus 20.6% (95% CI for the difference, –7.8% to 3.5%) for study 0019. Increases in serum creatinine level were more common in the telavancin group (16% vs 10%). Conclusions. The primary end point of the studies was met, indicating that telavancin is noninferior to vancomycin on the basis of clinical response in the treatment of HAP due to gram-positive pathogens. PMID:21148517

  13. Comparison of tryptophan biosynthetic operon regulation in different Gram-positive bacterial species.

    PubMed

    Gutiérrez-Preciado, Ana; Yanofsky, Charles; Merino, Enrique

    2007-09-01

    The tryptophan biosynthetic operon has been widely used as a model system for studying transcription regulation. In Bacillus subtilis, the trp operon is primarily regulated by a tryptophan-activated RNA-binding protein, TRAP. Here we show that in many other Gram-positive species the trp operon is regulated differently, by tRNA(Trp) sensing by the RNA-based T-box mechanism, with T-boxes arranged in tandem. Our analyses reveal an apparent relationship between trp operon organization and the specific regulatory mechanism(s) used. PMID:17555843

  14. Zinc, a structural component of adenylate kinases from gram-positive bacteria.

    PubMed Central

    Gilles, A M; Glaser, P; Perrier, V; Meier, A; Longin, R; Sebald, M; Maignan, L; Pistotnik, E; Bârzu, O

    1994-01-01

    The recent finding that Bacillus stearothermophilus adenylate kinase contains a zinc atom coordinated to four cysteines prompted us to investigate the metal-binding properties of the enzyme from various bacteria. We conclude that zinc was present only in adenylate kinase from gram-positive species and that this property is correlated with the presence of three or four Cys residues in the sequence Cys-X2-Cys-X16-Cys-X2-Cys/Asp, in which X stands for different amino acid residues. PMID:8288548

  15. Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials.

    PubMed

    Torres, Sebastian; Pandey, Ashok; Castro, Guillermo R

    2011-01-01

    Organic-solvent-tolerant bacteria are considered extremophiles with different tolerance levels that change among species and strains, but also depend on the inherent toxicity of the solvent. Extensive studies to understand the mechanisms of organic solvent tolerance have been done in Gram-negative bacteria. On the contrary, the information on the solvent tolerance mechanisms in Gram-positive bacteria remains scarce. Possible shared mechanisms among Gram-(-) and Gram-(+) microorganisms include: energy-dependent active efflux pumps that export toxic organic solvents to the external medium; cis-to-trans isomerization of unsaturated membrane fatty acids and modifications in the membrane phospholipid headgroups; formation of vesicles loaded with toxic compounds; and changes in the biosynthesis rate of phospholipids to accelerate repair processes. However, additional physiological responses of Gram-(+) bacteria to organic solvents seem to be specific. The aim of the present work is to review the state of the art of responsible mechanisms for organic solvent tolerance in Gram-positive bacteria, and their industrial and environmental biotechnology potential. PMID:21504787

  16. Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

    PubMed Central

    Weiss, Andy; Shaw, Lindsey N.

    2015-01-01

    The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

  17. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

    PubMed Central

    Pitman, Stephanie; Cho, Kyu Hong

    2015-01-01

    The discovery of small noncoding regulatory RNAs (sRNAs) in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described. PMID:26694351

  18. Fumaric Acid and Slightly Acidic Electrolyzed Water Inactivate Gram Positive and Gram Negative Foodborne Pathogens

    PubMed Central

    Tango, Charles Nkufi; Mansur, Ahmad Rois; Oh, Deog-Hwan

    2015-01-01

    Sanitizing effectiveness of slightly acidic electrolyzed water (SAEW) and fumaric acid (FA) at different dipping temperatures (25–60 °C), times (1–5 min), and concentrations (5–30 ppm for SAEW and 0.125%–0.5% for FA) on pure cultures of two Gram positive pathogens Staphylococcus aureus (SA) and Listeria monocytogenes (LM) and two Gram negative pathogens Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST) was evaluated. FA (0.25%) showed the strongest sanitizing effect, demonstrating complete inactivation of EC, ST, and LM, while SA was reduced by 3.95–5.76 log CFU/mL at 25–60 °C, respectively, after 1 min of treatment. For SAEW, the complete inactivation was obtained when available chlorine concentration was increased to 20 ppm at 40 °C for 3 and 5 min. Moreover, Gram positive pathogens have been shown to resist to all treatment trends more than Gram negative pathogens throughout this experiment. Regardless of the different dipping temperatures, concentrations, and times, FA treatment was more effective than treatment with SAEW for reduction of foodborne pathogens. This study demonstrated that application of FA in food systems may be useful as a method for inactivation of foodborne pathogens.

  19. Optimization of Fluorescent Tools for Cell Biology Studies in Gram-Positive Bacteria

    PubMed Central

    Henriques, Mafalda X.; Gomes, João Paulo; Filipe, Sérgio R.

    2014-01-01

    The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5′ end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. PMID:25464377

  20. Cell to cell communication by autoinducing peptides in gram-positive bacteria.

    PubMed

    Sturme, Mark H J; Kleerebezem, Michiel; Nakayama, Jiro; Akkermans, Antoon D L; Vaugha, Elaine E; de Vos, Willem M

    2002-08-01

    While intercellular communication systems in Gram-negative bacteria are often based on homoserine lactones as signalling molecules, it has been shown that autoinducing peptides are involved in intercellular communication in Gram-positive bacteria. Many of these peptides are exported by dedicated systems, posttranslationally modified in various ways, and finally sensed by other cells via membrane-located receptors that are part of two-component regulatory systems. In this way the expression of a variety of functions including virulence, genetic competence and the production of antimicrobial compounds can be modulated in a co-ordinated and cell density- and growth phase-dependent manner. Occasionally the autoinducing peptide has a dual function, such as in the case of nisin that is both a signalling pheromone involved in quorum sensing and an antimicrobial peptide. Moreover, biochemical, genetic and genomic studies have shown that bacteria may contain multiple quorum sensing systems, underlining the importance of intercellular communication. Finally, in some cases different peptides may be recognised by the same receptor, while also hybrid receptors have been constructed that respond to new peptides or show novel responses. This paper provides an overview of the characteristics of autoinducing peptide-based quorum sensing systems, their application in various gram-positive bacteria, and the discovery of new systems in natural and engineered ecosystems. PMID:12448722

  1. Interfacial charge transfer between CdTe quantum dots and Gram negative vs. Gram positive bacteria.

    SciTech Connect

    Dumas, E.; Gao, C.; Suffern, D.; Bradforth, S. E.; Dimitrejevic, N. M.; Nadeau, J. L.; McGill Univ.; Univ. of Southern California

    2010-01-01

    Oxidative toxicity of semiconductor and metal nanomaterials to cells has been well established. However, it may result from many different mechanisms, some requiring direct cell contact and others resulting from the diffusion of reactive species in solution. Published results are contradictory due to differences in particle preparation, bacterial strain, and experimental conditions. It has been recently found that C{sub 60} nanoparticles can cause direct oxidative damage to bacterial proteins and membranes, including causing a loss of cell membrane potential (depolarization). However, this did not correlate with toxicity. In this study we perform a similar analysis using fluorescent CdTe quantum dots, adapting our tools to make use of the particles fluorescence. We find that two Gram positive strains show direct electron transfer to CdTe, resulting in changes in CdTe fluorescence lifetimes. These two strains also show changes in membrane potential upon nanoparticle binding. Two Gram negative strains do not show these effects - nevertheless, they are over 10-fold more sensitive to CdTe than the Gram positives. We find subtoxic levels of Cd{sup 2+} release from the particles upon irradiation of the particles, but significant production of hydroxyl radicals, suggesting that the latter is a major source of toxicity. These results help establish mechanisms of toxicity and also provide caveats for use of certain reporter dyes with fluorescent nanoparticles which will be of use to anyone performing these assays. The findings also suggest future avenues of inquiry into electron transfer processes between nanomaterials and bacteria.

  2. Regulation of the pts operon in low G+C Gram-positive bacteria.

    PubMed

    Vadeboncoeur, C; Frenette, M; Lortie, L A

    2000-10-01

    The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria. Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species. In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon. In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria. Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins. Their synthesis is not coordinated and is influenced by environmental factors. The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl. Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.

  3. Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.

    PubMed

    Catalão, Maria João; Figueiredo, Joana; Henriques, Mafalda X; Gomes, João Paulo; Filipe, Sérgio R

    2014-01-01

    The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5' end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal.

  4. Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen.

    PubMed Central

    Dahl, T A; Midden, W R; Hartman, P E

    1989-01-01

    Gram-negative and gram-positive bacteria were found to display different sensitivities to pure singlet oxygen generated outside of cells. Killing curves for Salmonella typhimurium and Escherichia coli strains were indicative of multihit killing, whereas curves for Sarcina lutea, Staphylococcus aureus, Streptococcus lactis, and Streptococcus faecalis exhibited single-hit kinetics. The S. typhimurium deep rough strain TA1975, which lacks nearly all of the cell wall lipopolysaccharide coat and manifests concomitant enhancement of penetration by some exogenous substances, responded to singlet oxygen with initially faster inactivation than did the S. typhimurium wild-type strain, although the maximum rates of killing appeared to be quite similar. The structure of the cell wall thus plays an important role in susceptibility to singlet oxygen. The outer membrane-lipopolysaccharide portion of the gram-negative cell wall initially protects the bacteria from extracellular singlet oxygen, although it may also serve as a source for secondary reaction products which accentuate the rates of cell killing. S. typhimurium and E. coli strains lacking the cellular antioxidant, glutathione, showed no difference from strains containing glutathione in response to the toxic effects of singlet oxygen. Strains of Sarcina lutea and Staphylococcus aureus that contained carotenoids, however, were far more resistant to singlet oxygen lethality than were both carotenoidless mutants of the same species and other gram-positive species lacking high levels of protective carotenoids. PMID:2703469

  5. Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

    PubMed Central

    Field, Des; Begley, Maire; O’Connor, Paula M.; Daly, Karen M.; Hugenholtz, Floor; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

    2012-01-01

    Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria. PMID:23056510

  6. Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans.

    PubMed

    Anné, Jozef; Vrancken, Kristof; Van Mellaert, Lieve; Van Impe, Jan; Bernaerts, Kristel

    2014-08-01

    Proteins secreted by Gram-positive bacteria are released into the culture medium with the obvious benefit that they usually retain their native conformation. This property makes these host cells potentially interesting for the production of recombinant proteins, as one can take full profit of established protocols for the purification of active proteins. Several state-of-the-art strategies to increase the yield of the secreted proteins will be discussed, using Streptomyces lividans as an example and compared with approaches used in some other host cells. It will be shown that approaches such as increasing expression and translation levels, choice of secretion pathway and modulation of proteins thereof, avoiding stress responses by changing expression levels of specific (stress) proteins, can be helpful to boost production yield. In addition, the potential of multi-omics approaches as a tool to understand the genetic background and metabolic fluxes in the host cell and to seek for new targets for strain and protein secretion improvement is discussed. It will be shown that S. lividans, along with other Gram-positive host cells, certainly plays a role as a production host for recombinant proteins in an economically viable way. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  7. Teicoplanin or vancomycin in the treatment of gram-positive infections?

    PubMed

    Murphy, S; Pinney, R J

    1995-02-01

    The glycopeptide antibiotics vancomycin and teicoplanin have similar mechanisms of action on bacterial cell wall synthesis. Their spectra of activity are limited to Gram-positive bacteria, with the degree of bactericidal activity depending on the species of micro-organism. Staphylococcus aureus, Staphylococcus epidermis, enterococci and Clostridium difficile are generally sensitive, including methicillin-resistant strains of S. aureus and S. epidermidis. Glycopeptide resistance has recently emerged in staphylococci and enterococci. Vancomycin has a shorter half-life than teicoplanin and requires multiple dosing to maintain adequate serum levels. It can only be given by prolonged intravenous infusion over 1 h. In contrast, the pharmacokinetics of teicoplanin allow for once-daily dosing, either by rapid intravenous infusion or by the intramuscular route. The latter offers reliable absorption for patients with limited venous access and is also of benefit for out-patient therapy. Teicoplanin is a safer drug than vancomycin. It is associated with a lower incidence of nephrotoxicity or ototoxicity. Compared to vancomycin, the availability of the intramuscular route and the absence of a requirement for routine serum monitoring, together with the reduced need to treat drug-related side-effects make teicoplanin more cost-effective. It is as effective as vancomycin for most indications, is safe, easy to administer and an important agent for treating Gram-positive infections. Its role in hospitals is likely to increase if the price of drug acquisition is kept low. PMID:7775615

  8. Synthetic Quorum Sensing and Cell-Cell Communication in Gram-Positive Bacillus megaterium.

    PubMed

    Marchand, Nicholas; Collins, Cynthia H

    2016-07-15

    The components of natural quorum-sensing (QS) systems can be used to engineer synthetic communication systems that regulate gene expression in response to chemical signals. We have used the machinery from the peptide-based agr QS system from Staphylococcus aureus to engineer a synthetic QS system in Bacillus megaterium to enable autoinduction of a target gene at high cell densities. Growth and gene expression from these synthetic QS cells were characterized in both complex and minimal media. We also split the signal production and sensing components between two strains of B. megaterium to produce sender and receiver cells and characterized the resulting communication in liquid media and on semisolid agar. The system described in this work represents the first synthetic QS and cell-cell communication system that has been engineered to function in a Gram-positive host, and it has the potential to enable the generation of dynamic gene regulatory networks in B. megaterium and other Gram-positive organisms. PMID:26203497

  9. Fumaric Acid and Slightly Acidic Electrolyzed Water Inactivate Gram Positive and Gram Negative Foodborne Pathogens

    PubMed Central

    Tango, Charles Nkufi; Mansur, Ahmad Rois; Oh, Deog-Hwan

    2015-01-01

    Sanitizing effectiveness of slightly acidic electrolyzed water (SAEW) and fumaric acid (FA) at different dipping temperatures (25–60 °C), times (1–5 min), and concentrations (5–30 ppm for SAEW and 0.125%–0.5% for FA) on pure cultures of two Gram positive pathogens Staphylococcus aureus (SA) and Listeria monocytogenes (LM) and two Gram negative pathogens Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST) was evaluated. FA (0.25%) showed the strongest sanitizing effect, demonstrating complete inactivation of EC, ST, and LM, while SA was reduced by 3.95–5.76 log CFU/mL at 25–60 °C, respectively, after 1 min of treatment. For SAEW, the complete inactivation was obtained when available chlorine concentration was increased to 20 ppm at 40 °C for 3 and 5 min. Moreover, Gram positive pathogens have been shown to resist to all treatment trends more than Gram negative pathogens throughout this experiment. Regardless of the different dipping temperatures, concentrations, and times, FA treatment was more effective than treatment with SAEW for reduction of foodborne pathogens. This study demonstrated that application of FA in food systems may be useful as a method for inactivation of foodborne pathogens. PMID:27682077

  10. Fumaric Acid and Slightly Acidic Electrolyzed Water Inactivate Gram Positive and Gram Negative Foodborne Pathogens.

    PubMed

    Tango, Charles Nkufi; Mansur, Ahmad Rois; Oh, Deog-Hwan

    2015-02-12

    Sanitizing effectiveness of slightly acidic electrolyzed water (SAEW) and fumaric acid (FA) at different dipping temperatures (25-60 °C), times (1-5 min), and concentrations (5-30 ppm for SAEW and 0.125%-0.5% for FA) on pure cultures of two Gram positive pathogens Staphylococcus aureus (SA) and Listeria monocytogenes (LM) and two Gram negative pathogens Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST) was evaluated. FA (0.25%) showed the strongest sanitizing effect, demonstrating complete inactivation of EC, ST, and LM, while SA was reduced by 3.95-5.76 log CFU/mL at 25-60 °C, respectively, after 1 min of treatment. For SAEW, the complete inactivation was obtained when available chlorine concentration was increased to 20 ppm at 40 °C for 3 and 5 min. Moreover, Gram positive pathogens have been shown to resist to all treatment trends more than Gram negative pathogens throughout this experiment. Regardless of the different dipping temperatures, concentrations, and times, FA treatment was more effective than treatment with SAEW for reduction of foodborne pathogens. This study demonstrated that application of FA in food systems may be useful as a method for inactivation of foodborne pathogens.

  11. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens.

    PubMed

    Pitman, Stephanie; Cho, Kyu Hong

    2015-12-14

    The discovery of small noncoding regulatory RNAs (sRNAs) in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen's survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  12. Adherence of gram-positive and gram-negative bacterial strains to human lung fibroblasts in vitro.

    PubMed

    Martin, D; Mathieu, L G; Lecomte, J; deRepentigny, J

    1986-01-01

    The adherence to eukaryotic cells of Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and the yeast Candida albicans was studied by light microscopy with an in vitro micromethod involving different cell lines. The method is inexpensive, consumes little time and material, and is reproducible. It was used to show that the gram-positive Cowan I strain of S. aureus, which naturally forms protein A on its surface, adheres in much larger numbers to human lung fibroblasts than the protein A-free Wood 46 strain, the strain of S. epidermidis, and the encapsulated Smith strain. The presence of a capsule on the latter strain apparently prevented its attachment to the fibroblasts. Among the gram-negative species studied, a piliated clinical isolate of N. gonorrhoeae, displaying the opaque colonial phenotype, adhered in larger numbers than another isolate lacking pili and displaying the transparent phenotype. E. coli K12 attached slightly to the cell line, whereas P. aeruginosa adhered to it moderately. One strain of C. albicans tested did not attach in any detectable numbers. No clear correlation between bacterial cell surface hydrophobicity, as evaluated by the hexadecane assay, and adherence to eukaryotic cells could be demonstrated for these microorganisms. With our method, bacterial attachment proceeded best at 37 degrees C and did not require more than 1 h of contact with the cell monolayer. The method described revealed differences in the adherence to eukaryotic cells, not only among species, but also between strains of the same species.

  13. Isolation of Bacteroides ureolyticus (B corrodens) from clinical infections.

    PubMed Central

    Duerden, B; Bennet, K W; Faulkner, J

    1982-01-01

    The introduction of an improved anaerobic system resulted in the isolation of Bacteroides ureolyticus (B corrodens) in numbers that suggested a pathogenic role from many more clinical specimens. During a three-year period B ureolyticus was isolated from 103 fairly superficial necrotic or gangrenous lesions all of which showed evidence of active infection. These included 27 perineal or genital infections, 15 perianal abscesses, 15 other soft tissue infections such as pilonidal abscesses and infected sebaceous cysts and 16 ulcers or gangrenous lesions of the lower limb. B ureolyticus was rarely isolated in pure culture but was usually one of the predominant organisms; the other organisms were mostly anaerobes and the combination of B ureolyticus with anaerobic Gram-positive cocci was particularly noticeable. The isolation and identification of B ureolyticus is not difficult but depends upon a reliable anaerobic system and the incubation of primary cultures for at least 72 h. PMID:7068922

  14. Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

    PubMed Central

    Borisova, Marina; Gaupp, Rosmarie; Duckworth, Amanda; Schneider, Alexander; Dalügge, Désirée; Mühleck, Maraike; Deubel, Denise; Unsleber, Sandra; Yu, Wenqi; Muth, Günther; Bischoff, Markus; Götz, Friedrich

    2016-01-01

    ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. PMID:27729505

  15. Clinical experience with linezolid in the treatment of resistant gram-positive infections.

    PubMed Central

    Antony, S. J.; Diaz-Vasquez, E.; Stratton, C.

    2001-01-01

    This study presents our clinical experience with linezolid in 19 patients with serious resistant gram-positive infections enrolled as part of the compassionate study. In this prospective, non-randomized, noncomparative study, 19 patients were enrolled as part of the National Compassionate Study Protocol conducted by Pharmacia-Upjohn. At the time of this writing, these patients had not been published in the literature. All of the patients had to have documented evidence of serious gram-positive infections in normally sterile sites and should have been unable to tolerate available antimicrobial therapy or be unresponsive to available drugs. Clinical characteristics, laboratory values, and pharmacokinetic and pharmacodynamic parameters were obtained. Patients were followed both short-term and long-term after completion of therapy. Nineteen patients were enrolled: 13 females and 6 males. The average age was 63 years. The average length of therapy with linezolid was 22 days. Methicillin-resistant Staphylococcus aureus (MRSA) was treated in eight patients, methicillin-resistant Staphylococcus epidermidis (MRSE) in two patients, vancomycin-resistant Enterococcus faecium (VREF) in eight patients, and coagulase-negative Staphylococcus in two patients. Co-infecting organisms include Enterococcus species colonization in six patients, Pseudomonas species in one patient, Serratia marcenens in one patient, and Candida albicans in one patient. Sterile sites that were infected included bone and joint (wounds and septic joints) in six patients, gastrointestinal system (hepatobiliary, liver abscess, Crohn's) in five patients, genitourinary (kidney and urine) in two patients, blood in five patients, respiratory in one patient, and aortic valve in 1 patient. Linezolid was given at 600 mg IV every 12 hours with a mean length of therapy of 22 days. Surgical drainage was used in combination with linezolid in 11 of the patients. Seventy nine percent of these patients achieved clinical and

  16. [Retrospective analysis of the Gram-positive bacteria-infected cases in the Department of Hematology].

    PubMed

    Jing, Yu; Bo, Jian; Zhao, Yu; Li, Hong-Hua; Wang, Shu-Hong; Huang, Wen-Rong; Wang, Quan-Shun

    2013-10-01

    This study was purposed to evaluate the efficacy and safety of linezolid, vancomycin and teicoplanin for the treatment of patients infected by Gram-positive bacteria in the Department of Hematology by retrospective analysis. The patients with fever in our department from January to December in 2011 were selected for blood culture with Gram-positive bacteria and treated with linezolid, vancomycin or teicoplanin alone.Various parameters were recorded before and after treatment, such as fever time, respiratory symptoms, physical signs, radiographic changes, blood and biochemical routine, and adverse reactions. The efficacy and safety of linezolid, vancomycin and teicoplanin were compared according to the fever abating time, bacterial clearance rate, clinical efficiencies and adverse events. The patients were divided into linezolid group (15 patients), vancomycin group (17 patients) and teicoplanin group (20 patients). The results showed that the mean time of fever abating in linezolid group was (4.43 ± 3.15)d, bacterial clearance rate and clinical efficiency in linezolid group were 55.56% and 86.67%, respectively. The above three data in vancomycin group were (6.83 ± 4.67)d, 54.54% and 76.47% respectively, and were (5.57 ± 4.16)d, 41.67% and 80.00% in teicoplanin group respectively. There was no statistically significant difference between three groups (P > 0.05). There were one case of diarrhea and two cases of thrombocytopenia in the linezolid group, and one case of nausea and two cases of creatinine increase in the vancomycin group. There were three cases of thrombocytopenia in the teicoplanin group. The thrombocytopenia in five cases and the hemogram drop in patients with leukemia after treatment were overlapped, their drug treatment did not stop, but their thrombocytopoiesis recovered to normal-level, thus the drug treatment were considered as no relation with thrombocytopenia. It is concluded that the treatment efficacy between linezolid, vancomycin and

  17. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure

    PubMed Central

    Thangavelu, Bharani; Pavlovsky, Alexander G.; Viola, Ronald

    2014-01-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of l-O-acetyl-homoserine from l-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. PMID:25286936

  18. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure.

    PubMed

    Thangavelu, Bharani; Pavlovsky, Alexander G; Viola, Ronald

    2014-10-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of L-O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA.

  19. Purification Techniques of Bacteriocins from Lactic Acid Bacteria and Other Gram-Positive Bacteria

    NASA Astrophysics Data System (ADS)

    Saavedra, Lucila; Sesma, Fernando

    The search for new antimicrobial peptides produced by lactic acid ­bacteria and other Gram-positive microorganisms has become an interesting field of research in the past decades. The fact that bacteriocins are active against numerous foodborne and human pathogens, are produced by generally regarded as safe (GRAS) microorganisms, and are readily degraded by proteolytic host systems makes them attractive candidates for biotechnological applications. However, before suggesting or choosing a new bacteriocin for future technology developments, it is necessary to elucidate its biochemical structure and its mode of action, which may be carried out once the bacteriocin is purified to homogeneity. This chapter focuses on describing the main strategies used for the purification of numerous bacteriocins.

  20. Selective Inactivation of Resistant Gram-Positive Pathogens with a Light-Driven Hybrid Nanomaterial.

    PubMed

    Grüner, Malte; Tuchscherr, Lorena; Löffler, Bettina; Gonnissen, Dominik; Riehemann, Kristina; Staniford, Mark C; Kynast, Ulrich; Strassert, Cristian A

    2015-09-23

    Herein, we present a straightforward strategy to disperse highly insoluble photosensitizers in aqueous environments, without major synthetic efforts and keeping their photosensitizing abilities unaffected. A layered nanoclay was employed to adsorb and to solubilize a highly efficient yet hydrophobic Si(IV) phthalocyaninate in water. The aggregation of the photoactive dye was correlated with its photophysical properties, particularly with the ability to produce highly cytotoxic singlet oxygen. Moreover, the resulting hybrid nanomaterial is able to selectively photoinactivate Gram-positive pathogens, due to local interactions between the bacterial membranes and the negatively charged nanodiscs. Nanotoxicity assays confirmed its innocuousness toward eukaryotic cells, showing that it constitutes a new class of "phototriggered magic bullet" for the inactivation of pathogens in phototherapy, as well as in the development of coatings for self-disinfecting surfaces. PMID:26360157

  1. Regulating the Intersection of Metabolism and Pathogenesis in Gram-positive Bacteria

    PubMed Central

    RICHARDSON, ANTHONY R.; SOMERVILLE, GREG A.; SONENSHEIN, ABRAHAM L.

    2015-01-01

    Pathogenic bacteria must contend with immune systems that actively restrict the availability of nutrients and cofactors, and create a hostile growth environment. To deal with these hostile environments, pathogenic bacteria have evolved or acquired virulence determinants that aid in the acquisition of nutrients. This connection between pathogenesis and nutrition may explain why regulators of metabolism in nonpathogenic bacteria are used by pathogenic bacteria to regulate both metabolism and virulence. Such coordinated regulation is presumably advantageous because it conserves carbon and energy by aligning synthesis of virulence determinants with the nutritional environment. In Gram-positive bacterial pathogens, at least three metabolite-responsive global regulators, CcpA, CodY, and Rex, have been shown to coordinate the expression of metabolism and virulence genes. In this chapter, we discuss how environmental challenges alter metabolism, the regulators that respond to this altered metabolism, and how these regulators influence the host-pathogen interaction. PMID:26185086

  2. Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH

    PubMed Central

    Cotter, Paul D.; Hill, Colin

    2003-01-01

    Gram-positive bacteria possess a myriad of acid resistance systems that can help them to overcome the challenge posed by different acidic environments. In this review the most common mechanisms are described: i.e., the use of proton pumps, the protection or repair of macromolecules, cell membrane changes, production of alkali, induction of pathways by transcriptional regulators, alteration of metabolism, and the role of cell density and cell signaling. We also discuss the reponses of Listeria monocytogenes, Rhodococcus, Mycobacterium, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, oral streptococci, and lactic acid bacteria to acidic environments and outline ways in which this knowledge has been or may be used to either aid or prevent bacterial survival in low-pH environments. PMID:12966143

  3. Buffering Capacity and Membrane H+ Conductance of Neutrophilic and Alkalophilic Gram-Positive Bacteria

    PubMed Central

    Rius, Núria; Lorén, José G.

    1998-01-01

    Buffering capacity and membrane H+ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H+ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and l-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells. PMID:9546171

  4. Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae.

    PubMed

    Olaya-Abril, Alfonso; Gómez-Gascón, Lidia; Jiménez-Munguía, Irene; Obando, Ignacio; Rodríguez-Ortega, Manuel J

    2012-06-27

    Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.

  5. Characterization of lectinlike surface components on Capnocytophaga ochracea ATCC 33596 that mediate coaggregation with gram-positive oral bacteria.

    PubMed Central

    Weiss, E I; London, J; Kolenbrander, P E; Kagermeier, A S; Andersen, R N

    1987-01-01

    The interactions between Capnocytophaga ochracea ATCC 33596 and Streptococcus sanguis H1, Actinomyces naeslundii PK984, or Actinomyces israelii PK16 are dependent on specific recognitions between heat-sensitive adhesins on C. ochracea and heat-stable structures (probably carbohydrate-containing receptors) on the surfaces of these gram-positive coaggregation partners. The coaggregation of C. ochracea with each of these three organisms was inhibited by L-rhamnose and D-fucose and to a lesser extent by beta-methyl-galactoside. The reaction with S. sanguis was the most sensitive, while the coaggregation with A. israelii was the least sensitive and was only partially inhibited by each of the sugars that were considered to be effective inhibitors. A more effective inhibition of the coaggregation between C. ochracea and A. israelii was achieved by adding a combination of the 6-deoxysugars and N-acetylneuraminic acid. To further characterize the coaggregations, naturally occurring coaggregation-defective (Cog-) mutants of C. ochracea were obtained from several different selections. Three phenotypically distinct groups of mutants were were isolated. Type 1 mutants failed to coaggregate with S. sanguis only. Type 2 mutants lost ability to interact with both S. sanguis and A. naeslundii. Type 3 mutants failed to coaggregate with all three coaggregation partners. Characterization of the Cog- mutants by sugar inhibition studies made it possible to distinguish three classes of adhesin activity. PMID:3570460

  6. Synthesis and evaluation of isatin-β-thiosemicarbazones as novel agents against antibiotic-resistant Gram-positive bacterial species.

    PubMed

    Zhang, Xu-Meng; Guo, Hui; Li, Zai-Shun; Song, Fu-Hang; Wang, Wei-Min; Dai, Huan-Qin; Zhang, Li-Xin; Wang, Jian-Guo

    2015-08-28

    Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have caused an increasing mortality rate, which means that antibiotic resistance is becoming an important health issue. In the course to screen new agents for resistant bacteria, we identified that a series of isatin-β-thiosemicarbazones (IBTs) could inhibit the growth of MRSA and VRE. This was the first time that the "familiar" IBT compounds exhibited significant anti Gram-positive pathogen activity. Against a clinical isolated MRSA strain, 20 of the 51 synthesized compounds showed minimum inhibitory concentration (MIC) data of 0.78 mg/L and another 12 novel compounds had MICs of 0.39 mg/L. Moreover, these compounds also inhibited Enterococcus faecalis and VRE at similar levels, indicating that IBTs might have different mode of action compared with vancomycin. For these IBTs, comparative field analysis (CoMFA) models were further established to understand the structure-activity relationships in order to design new compounds from steric and electrostatic contributions. This work has suggested that IBTs can be considered as potential lead compounds to discover antibacterial inhibitors to combat drug resistance.

  7. Synthesis and evaluation of isatin-β-thiosemicarbazones as novel agents against antibiotic-resistant Gram-positive bacterial species.

    PubMed

    Zhang, Xu-Meng; Guo, Hui; Li, Zai-Shun; Song, Fu-Hang; Wang, Wei-Min; Dai, Huan-Qin; Zhang, Li-Xin; Wang, Jian-Guo

    2015-08-28

    Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have caused an increasing mortality rate, which means that antibiotic resistance is becoming an important health issue. In the course to screen new agents for resistant bacteria, we identified that a series of isatin-β-thiosemicarbazones (IBTs) could inhibit the growth of MRSA and VRE. This was the first time that the "familiar" IBT compounds exhibited significant anti Gram-positive pathogen activity. Against a clinical isolated MRSA strain, 20 of the 51 synthesized compounds showed minimum inhibitory concentration (MIC) data of 0.78 mg/L and another 12 novel compounds had MICs of 0.39 mg/L. Moreover, these compounds also inhibited Enterococcus faecalis and VRE at similar levels, indicating that IBTs might have different mode of action compared with vancomycin. For these IBTs, comparative field analysis (CoMFA) models were further established to understand the structure-activity relationships in order to design new compounds from steric and electrostatic contributions. This work has suggested that IBTs can be considered as potential lead compounds to discover antibacterial inhibitors to combat drug resistance. PMID:26185006

  8. Microcins from Enterobacteria: On the Edge Between Gram-Positive Bacteriocins and Colicins

    NASA Astrophysics Data System (ADS)

    Rebuffat, Sylvie

    Most bacteria and archaea produce gene-encoded antimicrobial peptides/proteins called bacteriocins, which are secreted by the producing bacteria to compete against other microorganisms in a given niche. They are considered important mediators of intra- and interspecies interactions and therefore a factor in ­maintaining the microbial diversity and stability. They are ribosomally synthesized, and most of them are produced as inactive precursor proteins, which in some cases are further enzymatically modified. Bacteriocins generally exert potent antibacterial activities directed against bacterial species closely related to the producing bacteria. Bacteriocins are abundant and diverse in Gram-negative and Gram-positive bacteria. This chapter focuses on colicins and microcins from enterobacteria (mainly Escherichia coli) and on bacteriocins from lactic acid bacteria (LAB). Microcins are the lower-molecular-mass bacteriocins produced by Gram-negative bacteria with a repertoire of only 14 representatives. They form a very restricted family of bacteriocins, compared to the huge family of LAB bacteriocins that is constituted of several hundreds of peptides, with which microcins share common characteristics. Nevertheless, microcins also show similarities, particularly in their uptake mechanisms, with the higher-molecular-mass colicins, also produced by E. coli strains. On the edge between LAB bacteriocins and colicins, microcins appear to combine highly efficient strategies developed by both Gram-positive and Gram-negative bacteria at different levels, including uptake, translocation, killing of target cells, and immunity of the producing bacteria, making them important actors of bacterial competitions and fascinating models for novel concepts toward antimicrobial strategies and against resistance mechanisms.

  9. Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

    PubMed Central

    Ji, Yuetong; He, Zhili; Pu, Yunting; Zhou, Jizhong; Xu, Jian

    2010-01-01

    Background Thermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes. Methodology/Principal Findings Here we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic. Conclusions/Significance In this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically. PMID:20838444

  10. Influence of bentonite particles on representative gram negative and gram positive bacterial deposition in porous media.

    PubMed

    Yang, Haiyan; Tong, Meiping; Kim, Hyunjung

    2012-11-01

    The significance of clay particles on the transport and deposition kinetics of bacteria in irregular quartz sand was examined by direct comparison of both breakthrough curves and retained profiles with clay particles in bacteria suspension versus those without clay particles. Two representative cell types, Gram-negative strain E. coli DH5α and Gram-positive strain Bacillus subtilis were utilized to systematically determine the influence of clay particles (bentonite) on cell transport behavior. Packed column experiments for both cell types were conducted in both NaCl (5 and 25 mM ionic strengths) and CaCl(2) (5 mM ionic strength) solutions at pH 6.0. The breakthrough plateaus with bentonite in solutions (30 mg L(-1) and 50 mg L(-1)) were lower than those without bentonite for both cell types under all examined conditions, indicating that bentonite in solutions decreased cell transport in porous media regardless of cell types (Gram-negative or Gram-positive) and solution chemistry (ionic strength and ion valence). The enhanced cell deposition with bentonite particles was mainly observed at segments near to column inlet, retained profiles for both cell types with bentonite particles were therefore steeper relative to those without bentonite. The increased cell deposition with bentonite observed in NaCl solutions was attributed to the codeposition of bacteria with bentonite particles whereas, in addition to codeposition of bacteria with bentonite, the bacteria-bentonite-bacteria cluster formed in suspensions also contributed to the increased deposition of bacteria with bentonite in CaCl(2) solution. PMID:22970735

  11. In Vitro Activity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens

    PubMed Central

    Huband, Michael D.; Hackel, Meredith; de Jonge, Boudewijn L. M.; Sahm, Daniel F.; Bradford, Patricia A.

    2015-01-01

    AZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed the in vitro activity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter against Staphylococcus aureus (n = 11,680), coagulase-negative staphylococci (n = 1,923), streptococci (n = 4,380), and Moraxella catarrhalis (n = 145), 0.5 mg/liter against Staphylococcus lugdunensis (n = 120) and Haemophilus influenzae (n = 352), 1 mg/liter against Enterococcus faecalis (n = 1,241), and 2 mg/liter against Haemophilus parainfluenzae (n = 70). The activity against Enterococcus faecium was more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, β-lactamase-producing Haemophilus spp., and M. catarrhalis. Based on these in vitro findings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species. PMID:26195518

  12. In Vitro Activity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens.

    PubMed

    Biedenbach, Douglas J; Huband, Michael D; Hackel, Meredith; de Jonge, Boudewijn L M; Sahm, Daniel F; Bradford, Patricia A

    2015-10-01

    AZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed the in vitro activity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter against Staphylococcus aureus (n = 11,680), coagulase-negative staphylococci (n = 1,923), streptococci (n = 4,380), and Moraxella catarrhalis (n = 145), 0.5 mg/liter against Staphylococcus lugdunensis (n = 120) and Haemophilus influenzae (n = 352), 1 mg/liter against Enterococcus faecalis (n = 1,241), and 2 mg/liter against Haemophilus parainfluenzae (n = 70). The activity against Enterococcus faecium was more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, β-lactamase-producing Haemophilus spp., and M. catarrhalis. Based on these in vitro findings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species.

  13. Species identification and susceptibility to 17 antibiotics of coagulase-negative staphylococci isolated from clinical specimens.

    PubMed Central

    Marsik, F J; Brake, S

    1982-01-01

    A total of 299 isolates of gram-positive, catalase-positive, coagulase-negative cocci were isolated from a variety of specimens collected from patients at a large university hospital, and 281 (94%) were identified as staphylococci by established methods. Using the scheme of Kloos and Schleifer, we determined the species of the coagulase-negative staphylococci. Staphylococcus epidermidis was the cause of all bacteremias and the most commonly isolated species from bone, joint, and wound infections. Staphylococcus haemolyticus was the second most common isolate from wound infections, and Staphylococcus saprophyticus was the most commonly isolated species from urinary tract infections. Antibiograms to 17 antimicrobial agents were performed by a microdilution technique, and the results revealed that S. epidermidis was resistant to a water spectrum of antimicrobial agents than the other species of staphylococci were. PMID:7068839

  14. Sequence-Based Characterization of Tn5801-Like Genomic Islands in Tetracycline-Resistant Staphylococcus pseudintermedius and Other Gram-positive Bacteria from Humans and Animals

    PubMed Central

    de Vries, Lisbeth E.; Hasman, Henrik; Jurado Rabadán, Sonia; Agersø, Yvonne

    2016-01-01

    Antibiotic resistance in pathogens is often associated with mobile genetic elements, such as genomic islands (GI) including integrative and conjugative elements (ICEs). These can transfer resistance genes within and between bacteria from humans and/or animals. The aim of this study was to investigate whether Tn5801-like GIs carrying the tetracycline resistance gene, tet(M), are common in Staphylococcus pseudintermedius from pets, and to do an overall sequences-based characterization of Tn5801-like GIs detected in Gram-positive bacteria from humans and animals. A total of 27 tetracycline-resistant S. pseudintermedius isolates from Danish pets (1998–2005) were screened for tet(M) by PCR. Selected isolates (13) were screened for GI- or ICE-specific genes (intTn5801 or xisTn916) and their tet(M) gene was sequenced (Sanger-method). Long-range PCR mappings and whole-genome-sequencing (Illumina) were performed for selected S. pseudintermedius-isolates (seven and three isolates, respectively) as well as for human S. aureus isolates (seven and one isolates, respectively) and one porcine Enterococcus faecium isolate known to carry Tn5801-like GIs. All 27 S. pseudintermedius were positive for tet(M). Out of 13 selected isolates, seven contained Tn5801-like GIs and six contained Tn916-like ICEs. Two different Tn5801-like GI types were detected among S. pseudintermedius (Tn5801 and GI6287) - both showed high similarity compared to GenBank sequences from human pathogens. Two distinct Tn5801-like GI types were detected among the porcine E. faecium and human S. aureus isolates (Tn6014 and GI6288). Tn5801-like GIs were detected in GenBank-sequences from Gram-positive bacteria of human, animal or food origin worldwide. Known Tn5801-like GIs were divided into seven types. The results showed that Tn5801-like GIs appear to be relatively common in tetracycline-resistant S. pseudintermedius in Denmark. Almost identical Tn5801-like GIs were identified in different Gram-positive species

  15. Deoxyribonucleic acid base composition and biochemical properties of certain coagulase-negative enterotoxigenic cocci.

    PubMed

    Lotter, L P; Genigeorgis, C A

    1975-02-01

    Eight coagulase-negative, enterotoxigenic strains of cocci and one weakly coagulase-positive strain isolated from a number of different sources, including cases of food poisoning incidents, were evaluated for their relationship to Staphylococcus aureus on the basis of deoxyribonucleic acid (DNA) buoyant density and physiological studies. One strain of cocci produced enterotoxins A and C, two strains produced types B and C, four strains produced only type C, and one strain only type D. The enterotoxin produced by one strain of cocci was serologically untypable. None of the test organisms produced detectable amounts of enterotoxin in broth cultures. The test strains of cocci exhibited the following profile: all produced catalase; all grew anaerobically and fermented glucse; five were sensitive to lysostaphin; the percentage of guanine plus cytosine content of their DNA varied from 32.7 to 37.6; five produced acid from mannitol both aerobically and anaerobically; two formed delta-hemolysin; five produced phosphatase and acetoin; and all produced heat-stable nuclease. None of the organisms exhibited typical characteristics of S. aureus, S. epidermidis, or S. saprophyticus. On the basis of the present data and data reported elsewhere, these organisms should be considered as variants or mutants of S. aureus.

  16. Transport Capabilities of Eleven Gram-positive Bacteria: Comparative Genomic Analyses

    PubMed Central

    Lorca, Graciela L.; Barabote, Ravi D.; Zlotopolski, Vladimir; Tran, Can; Winnen, Brit; Hvorup, Rikki N.; Stonestrom, Aaron J.; Nguyen, Elizabeth; Huang, Li-Wen; Kim, David S.; Saier, Milton H.

    2007-01-01

    The genomes of eleven Gram-positive bacteria that are important for human health and the food industry, nine low G+C lactic acid bacteria and two high G+C Gram-positive organisms, were analyzed for their complement of genes encoding transport proteins. Thirteen to eighteen percent of their genes encode transport proteins, larger percentages than observed for most other bacteria. All of these bacteria possess channel proteins, some of which probably function to relieve osmotic stress. Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugar uptake porters, those specific for oligosaccharides and glycosides often outnumber those for free sugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumps with Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all other types. An exception to this generalization is Streptococcus thermophilus with 54% of its drug efflux pumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumps that may function in intercellular signalling, and macromolecular efflux pumps, many of predictable specificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electron carriers. The one exception is Brevibacterium linens, which in addition to these carriers, also has transporters of several families not represented in the other ten bacteria examined. Comparisons with the genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possess distinctive proportions of recognized transporter types (e.g., more porters specific for glycosides than reducing sugars). Some homologues of transporters identified had previously been identified only in Gram-negative bacteria or in eukaryotes

  17. Results of the surveillance of Tedizolid activity and resistance program: in vitro susceptibility of gram-positive pathogens collected in 2011 and 2012 from the United States and Europe.

    PubMed

    Sahm, Daniel F; Deane, Jennifer; Bien, Paul A; Locke, Jeffrey B; Zuill, Douglas E; Shaw, Karen J; Bartizal, Ken F

    2015-02-01

    The in vitro activity and spectrum of tedizolid and comparators were analyzed against 6884 Gram-positive clinical isolates collected from multiple US and European sites as part of the Surveillance of Tedizolid Activity and Resistance Program in 2011 and 2012. Organisms included 4499 Staphylococcus aureus, 537 coagulase-negative staphylococci (CoNS), 873 enterococci, and 975 β-hemolytic streptococci. The MIC values that inhibited 90% of the isolates within each group (MIC90) were 0.25 μg/mL for Staphylococcus epidermidis and β-hemolytic streptococci and 0.5 μg/mL for S. aureus, other CoNS, and enterococci. Of 16 isolates with elevated tedizolid or linezolid MIC values (intermediate or resistant isolates), 10 had mutations in the genes encoding 23S rRNA (primarily G2576T), 5 had mutations in the genes encoding ribosomal proteins L3 or L4, and 5 carried the cfr multidrug resistance gene. Overall, tedizolid showed excellent activity against Gram-positive bacteria and was at least 4-fold more potent than linezolid against wild-type and linezolid-resistant isolates. Given the low overall frequency of isolates that would be resistant to tedizolid at the proposed break point of 0.5 μg/mL (0.19%) and potent activity against contemporary US and European isolates, tedizolid has the potential to serve as a valuable therapeutic option in the treatment of infections caused by Gram-positive pathogens.

  18. Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

    PubMed Central

    Burnham, Carey-Ann D.; Bythrow, Maureen; Garner, Omai B.; Ginocchio, Christine C.; Jennemann, Rebecca; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Sercia, Linda; Westblade, Lars F.; Ferraro, Mary Jane; Branda, John A.

    2013-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting. PMID:23658261

  19. Gramicidin A Mutants with Antibiotic Activity against Both Gram-Positive and Gram-Negative Bacteria.

    PubMed

    Zerfas, Breanna L; Joo, Yechaan; Gao, Jianmin

    2016-03-17

    Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic-resistant bacteria. One promising example of this is gramicidin A (gA). In its wild-type sequence, gA is active by permeating the plasma membrane of Gram-positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa. PMID:26918268

  20. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains.

    PubMed

    Smits, T H; Röthlisberger, M; Witholt, B; van Beilen, J B

    1999-08-01

    We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C6-C11) or long-chain n-alkanes (C12-C16), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n-alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases. PMID:11207749

  1. Fate study of water-borne gram positive vegetative bacterial cells with Raman microscopy

    NASA Astrophysics Data System (ADS)

    Guicheteau, Jason; Tripathi, Ashish; Minter, Jennifer; Wilcox, Phillip; Christesen, Steven

    2010-04-01

    We present an initial bacterial fate study of Gram positive vegetative cells suspended in water and stored at ambient room temperature via Raman spectroscopy monitoring. Two types of cells were considered for this study: vegetative cells of Bacillus cereus, Bacillus thuringiensis which contain the polyhydroxybutyric acid (PHBA) as an energy storage compound and Bacillus subtlilis cells which do not. The cells were cultured specifically for this project. Immediately following the culturing phase, the bacteria were extracted, cleaned and at the onset of the study were suspended in de-ionized water and stored at room temperature. Aliquots of suspensions were deposited onto aluminum slides at different times and allowed to dry for Raman analysis. Spectra from multiple regions of each dried spot and each deposit time were acquired along with the bright-field and fluorescence images. Results were examined to investigate the effect of suspension time on the spectral signatures as well as the fate behavior of the three types of cells investigated. The cells were monitored daily for over a 14 period during which time the onset of starvation induced sporulation was observed.

  2. Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria.

    PubMed

    Vaid, A; Bishop, A H

    1999-10-01

    Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA. The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell. Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls. A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only. Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei. Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L. lactis and M. phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.

  3. Antibacterial properties of biosurfactants against selected Gram-positive and -negative bacteria.

    PubMed

    Díaz De Rienzo, Mayri A; Stevenson, Paul; Marchant, Roger; Banat, Ibrahim M

    2016-01-01

    The antibacterial properties and ability to disrupt biofilms of biosurfactants (rhamnolipids, sophorolipids) and sodium dodecyl sulphate (SDS) in the presence and absence of selected organic acids were investigated. Pseudomonas aeruginosa PAO1 was inhibited by sophorolipids and SDS at concentrations >5% v/v, and the growth of Escherichia coli NCTC 10418 was also inhibited by sophorolipids and SDS at concentrations >5% and 0.1% v/v, respectively. Bacillus subtilis NCTC 10400 was inhibited by rhamnolipids, sophorolipids and SDS at concentrations >0.5% v/v of all three; the same effect was observed with Staphylococcus aureus ATCC 9144. The ability to attach to surfaces and biofilm formation of P. aeruginosa PAO1, E. coli NCTC 10418 and B. subtilis NCTC 10400 was inhibited by sophorolipids (1% v/v) in the presence of caprylic acid (0.8% v/v). In the case of S. aureus ATCC 9144, the best results were obtained using caprylic acid on its own. It was concluded that sophorolipids are promising compounds for the inhibition/disruption of biofilms formed by Gram-positive and Gram-negative microorganisms and this activity can be enhanced by the presence of booster compounds such as caprylic acid. PMID:26598715

  4. Effect of betamethasone in combination with antibiotics on gram positive and gram negative bacteria.

    PubMed

    Artini, M; Papa, R; Cellini, A; Tilotta, M; Barbato, G; Koverech, A; Selan, L

    2014-01-01

    Betamethasone is an anti-inflammatory steroid drug used in cases of anaphylactic and allergic reactions, of Alzheimer and Addison diseases and in soft tissue injuries. It modulates gene expression for anti-inflammatory activity suppressing the immune system response. This latter effect might decrease the effectiveness of immune system response against microbial infections. Corticosteroids, in fact, mask some symptoms of infection and during their use superimposed infections may occur. Thus, the use of glucocorticoids in patients with sepsis remains extremely controversial. In this study we analyzed the in vitro effect of a commercial formulation of betamethasone (Bentelan) on several Gram positive and Gram negative bacteria of clinical relevance. It was found to be an inhibitor of the growth of most of the strains examined. Also the effect of betamethasone in combination with some classes of antibiotics was evaluated. Antibiotic-steroid combination therapy is, in such cases, superior to antibiotic-alone treatment to impair bacterial growths. Such effect was essentially not at all observable on Staphylococcus aureus or Coagulase Negative Staphylococci (CoNS).

  5. Comparative analysis of gene expression among low G+C gram-positive genomes.

    PubMed

    Karlin, Samuel; Theriot, Julie; Mrázek, Jan

    2004-04-20

    We present a comparative analysis of predicted highly expressed (PHX) genes in the low G+C Gram-positive genomes of Bacillus subtilis, Bacillus halodurans, Listeria monocytogenes, Listeria innocua, Lactococcus lactis, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Clostridium acetobutylicum, and Clostridium perfringens. Most enzymes acting in glycolysis and fermentation pathways are PHX in these genomes, but not those involved in the TCA cycle and respiration, suggesting that these organisms have predominantly adapted to grow rapidly in an anaerobic environment. Only B. subtilis and B. halodurans have several TCA cycle PHX genes, whereas the TCA pathway is entirely missing from the metabolic repertoire of the two Streptococcus species and is incomplete in Listeria, Lactococcus, and Clostridium. Pyruvate-formate lyase, an enzyme critical in mixed acid fermentation, is among the highest PHX genes in all these genomes except for C. acetobutylicum (not PHX), and B. subtilis, and B. halodurans (missing). Pyruvate-formate lyase is also prominently PHX in enteric gamma-proteobacteria, but not in other prokaryotes. Phosphotransferase system genes are generally PHX with selection of different substrates in different genomes. The various substrate specificities among phosphotransferase systems in different genomes apparently reflect on differences in habitat, lifestyle, and nutrient sources.

  6. Inactivation of Gram-positive biofilms by low-temperature plasma jet at atmospheric pressure

    NASA Astrophysics Data System (ADS)

    Marchal, F.; Robert, H.; Merbahi, N.; Fontagné-Faucher, C.; Yousfi, M.; Romain, C. E.; Eichwald, O.; Rondel, C.; Gabriel, B.

    2012-08-01

    This work is devoted to the evaluation of the efficiency of a new low-temperature plasma jet driven in ambient air by a dc-corona discharge to inactivate adherent cells and biofilms of Gram-positive bacteria. The selected microorganisms were lactic acid bacteria, a Weissella confusa strain which has the particularity to excrete a polysaccharide polymer (dextran) when sucrose is present. Both adherent cells and biofilms were treated with the low-temperature plasma jet for different exposure times. The antimicrobial efficiency of the plasma was tested against adherent cells and 48 h-old biofilms grown with or without sucrose. Bacterial survival was estimated using both colony-forming unit counts and fluorescence-based assays for bacterial cell viability. The experiments show the ability of the low-temperature plasma jet at atmospheric pressure to inactivate the bacteria. An increased resistance of bacteria embedded within biofilms is clearly observed. The resistance is also significantly higher with biofilm in the presence of sucrose, which indicates that dextran could play a protective role.

  7. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

    PubMed Central

    Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

    2016-01-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

  8. Biofilms affecting progression of mild steel corrosion by Gram positive Bacillus sp.

    PubMed

    Lin, Johnson; Madida, Bafana B

    2015-10-01

    The biodeterioration of metals have detrimental effects on the environment with economic implications. The deterioration of metals is of great concern to industry. In this study, mild steel coupons which were immersed in a medium containing Gram-positive Bacillus spp. and different nutrient sources were compared with the control in sterile deionized water. The weight loss of the coupons in the presence of Bacillus spp. alone was lower than the control and was further reduced when additional carbon sources, especially fructose, were added. The level of metal corrosion was significantly increased in the presence of nitrate with or without bacteria. There was a significant strong correlation between the weight loss and biofilm level (r =  0.64; p < 0.05). The addition of nitrate and Bacillus spp. produced more biofilms on the coupons and resulted in greater weight loss compared to that with Bacillus spp. only under the same conditions. However, Bacillus spp. enriched with carbon sources formed less biofilms and results in lower weight loss compared to that with Bacillus spp. only. The production of biofilm by Bacillus spp. influences the level of metal corrosion under different environmental conditions, thereby, supporting the development of a preventive strategy against corrosion.

  9. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

    PubMed

    Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

    2016-07-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications.

  10. Linezolid in late-chronic prosthetic joint infection caused by gram-positive bacteria.

    PubMed

    Cobo, Javier; Lora-Tamayo, Jaime; Euba, Gorane; Jover-Sáenz, Alfredo; Palomino, Julián; del Toro, Ma Dolores; Rodríguez-Pardo, Dolors; Riera, Melchor; Ariza, Javier

    2013-05-01

    Linezolid may be an interesting alternative for prosthetic joint infection (PJI) due to its bioavailability and its antimicrobial spectrum. However, experience in this setting is scarce. The aim of the study was to assess linezolid's clinical and microbiological efficacy, and also its tolerance. This was a prospective, multicenter, open-label, non-comparative study of 25 patients with late-chronic PJI caused by Gram-positive bacteria managed with a two-step exchange procedure plus 6 weeks of linezolid. Twenty-two (88%) patients tolerated linezolid without major adverse effects, although a global decrease in the platelet count was observed. Three patients were withdrawn because of major toxicity, which reversed after linezolid stoppage. Among patients who completed treatment, 19 (86%) demonstrated clinical and microbiological cure. Two patients presented with clinical and microbiological failure, and one showed clinical cure and microbiological failure. In conclusion, linezolid showed good results in chronic PJI managed with a two-step exchange procedure. Tolerance seems acceptable, though close surveillance is required.

  11. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    PubMed

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  12. Antibacterial properties of biosurfactants against selected Gram-positive and -negative bacteria.

    PubMed

    Díaz De Rienzo, Mayri A; Stevenson, Paul; Marchant, Roger; Banat, Ibrahim M

    2016-01-01

    The antibacterial properties and ability to disrupt biofilms of biosurfactants (rhamnolipids, sophorolipids) and sodium dodecyl sulphate (SDS) in the presence and absence of selected organic acids were investigated. Pseudomonas aeruginosa PAO1 was inhibited by sophorolipids and SDS at concentrations >5% v/v, and the growth of Escherichia coli NCTC 10418 was also inhibited by sophorolipids and SDS at concentrations >5% and 0.1% v/v, respectively. Bacillus subtilis NCTC 10400 was inhibited by rhamnolipids, sophorolipids and SDS at concentrations >0.5% v/v of all three; the same effect was observed with Staphylococcus aureus ATCC 9144. The ability to attach to surfaces and biofilm formation of P. aeruginosa PAO1, E. coli NCTC 10418 and B. subtilis NCTC 10400 was inhibited by sophorolipids (1% v/v) in the presence of caprylic acid (0.8% v/v). In the case of S. aureus ATCC 9144, the best results were obtained using caprylic acid on its own. It was concluded that sophorolipids are promising compounds for the inhibition/disruption of biofilms formed by Gram-positive and Gram-negative microorganisms and this activity can be enhanced by the presence of booster compounds such as caprylic acid.

  13. Gramicidin A Mutants with Antibiotic Activity against Both Gram-Positive and Gram-Negative Bacteria.

    PubMed

    Zerfas, Breanna L; Joo, Yechaan; Gao, Jianmin

    2016-03-17

    Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic-resistant bacteria. One promising example of this is gramicidin A (gA). In its wild-type sequence, gA is active by permeating the plasma membrane of Gram-positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa.

  14. Stuck in the Middle: Fibronectin-Binding Proteins in Gram-Positive Bacteria

    PubMed Central

    Hymes, Jeffrey P.; Klaenhammer, Todd R.

    2016-01-01

    Fibronectin is a multidomain glycoprotein found ubiquitously in human body fluids and extracellular matrices of a variety of cell types from all human tissues and organs, including intestinal epithelial cells. Fibronectin plays a major role in the regulation of cell migration, tissue repair, and cell adhesion. Importantly, fibronectin also serves as a common target for bacterial adhesins in the gastrointestinal tract. Fibronectin-binding proteins (FnBPs) have been identified and characterized in a wide variety of host-associated bacteria. Single bacterial species can contain multiple, diverse FnBPs. In pathogens, some FnBPs contribute to virulence via host cell attachment, invasion, and interference with signaling pathways. Although FnBPs in commensal and probiotic strains are not sufficient to confer virulence, they are essential for attachment to their ecological niches. Here we describe the interaction between human fibronectin and bacterial adhesins by highlighting the FnBPs of Gram-positive pathogens and commensals. We provide an overview of the occurrence and diversity of FnBPs with a focus on the model pathogenic organisms in which FnBPs are most characterized. Continued investigation of FnBPs is needed to fully understand their divergence and specificity in both pathogens and commensals. PMID:27713740

  15. Tribolium castaneum defensins are primarily active against Gram-positive bacteria.

    PubMed

    Tonk, Miray; Knorr, Eileen; Cabezas-Cruz, Alejandro; Valdés, James J; Kollewe, Christian; Vilcinskas, Andreas

    2015-11-01

    The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi, but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis. None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum, mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species. PMID:26522790

  16. Fasting hypochlorhydria with gram positive gastric flora is highly prevalent in healthy old people.

    PubMed Central

    Husebye, E; Skar, V; Høverstad, T; Melby, K

    1992-01-01

    Fifteen healthy old people mean age 84 years (range 80-91 years), were examined to assess the effect of advanced age on the microecology of the upper gastrointestinal tract. Twelve of 15 (80%) were hypochlorhydric with pH 6.6 (0.3) (mean (SEM) and a mean bacterial count of 10(8) colony forming units (CFU) per ml (range 10(5)-10(10)) in fasting gastric aspirate. Normochlorhydric subjects had low counts (< or = 10(1) CFU/ml). The microbial flora was dominated by viridans streptococci, coagulase negative staphylococci, and Haemophilus sp. Only one subject harboured significant concentrations of Gram negative bacilli with Escherichia coli (10(4-5) CFU/ml) and Klebsiella (10(4-5)). Strict anaerobes were not found. The total concentration of short chain fatty acids in gastric aspirate was 10.6 (2.9) mmol/l (mean (SEM). Absence of significant, intraluminal fermentation of xylose to CO2 was shown by the 14C-d Xylose breath test, and ambulatory manometry showed preserved fasting motility pattern of the small intestine. Serum immunoglobulins were normal. Advanced age is accompanied by fasting hypochlorhydria and colonisation with mainly Gram positive flora in the upper gut. Other factors than old age and fasting hypochlorhydria are required for colonisation with Gram negative bacilli. PMID:1446855

  17. Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci.

    PubMed

    Boda, Sunil Kumar; Broda, Janine; Schiefer, Frank; Weber-Heynemann, Josefine; Hoss, Mareike; Simon, Ulrich; Basu, Bikramjit; Jahnen-Dechent, Willi

    2015-07-01

    The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 × 10(-6) m [Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections. PMID:25712910

  18. Multivalent Artificial Opsonin for the Recognition and Phagocytosis of Gram-Positive Bacteria by Human Phagocytes

    PubMed Central

    Katzenmeyer, Kristy N.; Bryers, James D.

    2011-01-01

    Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body’s natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly(L-lysine)-graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin’s inherent ability to bind to D-Ala-D-Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL-g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of S. epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys-D-Ala-D-Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, E. faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms. PMID:21388677

  19. Lipoglycopeptide Antibacterial Agents in Gram-Positive Infections: A Comparative Review.

    PubMed

    Van Bambeke, Françoise

    2015-12-01

    Oritavancin, telavancin, and dalbavancin are recently marketed lipoglycopeptides that exhibit remarkable differences to conventional molecules. While dalbavancin inhibits the late stages of peptidoglycan synthesis by mainly impairing transglycosylase activity, oritavancin and telavancin anchor in the bacterial membrane by the lipophilic side chain linked to their disaccharidic moiety, disrupting membrane integrity and causing bacteriolysis. Oritavancin keeps activity against vancomycin-resistant enterocococci, being a stronger inhibitor of transpeptidase than of transglycosylase activity. These molecules have potent activity against Gram-positive organisms, most notably staphylococci (including methicillin-resistant Staphylococcus aureus and to some extent vancomycin-intermediate S. aureus), streptococci (including multidrug-resistant pneumococci), and Clostridia. All agents are indicated for the treatment of acute bacterial skin and skin structure infections, and telavancin, for hospital-acquired and ventilator-associated bacterial pneumonia. While telavancin is administered daily at 10 mg/kg, the remarkably long half-lives of oritavancin and dalbavancin allow for infrequent dosing (single dose of 1200 mg for oritavancin and 1000 mg at day 1 followed by 500 mg at day 8 for dalbavancin), which could be exploited in the future for outpatient therapy. Among possible safety issues evidenced during clinical development were an increased risk of developing osteomyelitis with oritavancin; taste disturbance, nephrotoxicity, and risk of corrected QT interval prolongation (especially in the presence of at-risk co-medications) with telavancin; and elevation of hepatic enzymes with dalbavancin. Interference with coagulation tests has been reported with oritavancin and telavancin. These drugs proved non-inferior to conventional treatments in clinical trials but their advantages may be better evidenced upon future evaluation in more severe infections.

  20. Antioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible mushrooms.

    PubMed

    Ahmad, Nisar; Mahmood, Fazal; Khalil, Shahid Akbar; Zamir, Roshan; Fazal, Hina; Abbasi, Bilal Haider

    2014-10-01

    Edible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the present study, the antioxidant activity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial potential in EMs (Pleurotus ostreatus, Morchella esculenta, P. ostreatus (Black), P. ostreatus (Yellow) and Pleurotus sajor-caju) were investigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) was observed in Morchella esculenta at a maximum concentration. Similarly, the dose-dependent concentrations (200, 400, 600, 800 and 1000 µg) were also used for other four EMs. Pleurotus ostreatus exhibited 36.13% activity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-caju exhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigated for its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillus subtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison with standard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active against all microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus and A. tumifaciens. P. sajor-caju showed best activities (12.5 mm) against B. subtilis, B. atrophaeus and K. pneumonia. P. ostreatus (Y) showed best activities against P. aeroginosa (21.83 mm), B. atrophaeus (20 mm) and C. albicans (21 mm). P. ostreatus (B) exhibited best activities against C. albicans (16 mm) and slightly lower activities against all other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against all microorganisms tested except S. typhi.

  1. Improving linezolid use decreases the incidence of resistance among Gram-positive microorganisms.

    PubMed

    Ramírez, Elena; Gómez-Gil, Rosa; Borobia, Alberto M; Moreno, Francisco; Zegarra, Claudia; Muñoz, Raúl; Reutero, Zaida; de Montreuil, Carolina; González, Diana; Hernández, Sonsoles; Herrero, Alicia; Gutiérrez, Avelino; Frías, Jesús

    2013-02-01

    Surveillance studies have shown the emergence of infections with linezolid-resistant bacteria. The relationship between appropriate linezolid use and the spread of linezolid resistance among Gram-positive microorganisms in a single tertiary referral centre was evaluated. In an initial observational study, a prospective prescription-indication study was conducted on intensive care areas and haematology, neurosurgery, vascular surgery and nephrology wards during 2009. An intervention through follow-up feedback on audit results from May-June 2010 was then conducted. From July-December 2010, a second drug-use study of linezolid was conducted, with the same objectives and methodology. To assess the antimicrobial pressure of linezolid, an ecological study was conducted from 2006-2010 in the same hospital wards. Indications for linezolid in the initial study were considered suitable in 38.5% of cases, whilst in the second study the rate was 51.2% (33% increase). Linezolid consumption fell by 57% in the second half of 2010. A significant correlation was found between its inadequate use (DDD/1000 patient-days) and the incidence of linezolid-resistant strains/1000 patient-days (r=0.93; P=6.9e-024); 85% of the variability in the incidence of linezolid resistance was predicted by its inadequate use. Its partial correlations were significant for Enterococcus faecium (r=0.407; P=0.049), Staphylococcus epidermidis (r=0.874; P=2.3e-008) and Staphylococcus haemolyticus (r=0.406; P=0.049) but not Staphylococcus aureus (r=0.051; P=0.704). A relationship was found between appropriate linezolid use and the incidence of linezolid-resistant strains of E. faecium, S. epidermidis and S. haemolyticus. PMID:23276501

  2. Dustborne and airborne gram-positive and gram-negative bacteria in high versus low ERMI homes

    EPA Science Inventory

    The study aimed at investigating Gram-positive and Gram-negative bacteria in moldy and non-moldy homes, as defined by the home's Environmental Relative Moldiness Index (ERMI) value. The ERMI values were determined from floor dust samples in 2010 and 2011 and homes were classified...

  3. The ability of "low G + C gram-positive" ruminal bacteria to resist monensin and counteract potassium depletion.

    PubMed

    Callaway, T R; Adams, K A; Russell, J B

    1999-10-01

    Gram-negative ruminal bacteria with an outer membrane are generally more resistant to the feed additive, monensin, than Gram-positive species, but some bacteria can adapt and increase their resistance. 16S rRNA sequencing indicates that a variety of ruminal bacteria are found in the "low G + C Gram-positive group," but some of these bacteria are monensin resistant and were previously described as Gram-negative species (e.g., Selenomonas ruminantium and Megasphaera elsdenii). The activity of monensin can be assayed by its ability to cause potassium loss, and results indicated that the amount of monensin needed to catalyze half maximal potassium depletion (K(d)) from low G + C gram-positive ruminal bacteria varied by as much as 130-fold. The K(d) values for Butyrivibrio fibrisolvens 49, Streptococcus bovis JB1, Clostridium aminophilum F, S. ruminantium HD4, and M. elsdenii B159 were 10, 65, 100, 1020, and 1330 nM monensin, respectively. B. fibrisolvens was very sensitive to monensin, and it did not adapt. S. bovis and C. aminophilum cultures that were transferred repeatedly with sub-lethal doses of monensin had higher K(d) values than unadapted cultures, but the K(d) was always less than 800 nM. S. ruminantium and M. elsdenii cells were highly resistant (K(d) > 1000 nM), and this resistance could be explained by the ability of these low G + C Gram-positive bacteria to synthesize outer membranes.

  4. Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle

    PubMed Central

    Dubuffet, Aurore; Zanchi, Caroline; Boutet, Gwendoline; Moreau, Jérôme; Teixeira, Maria; Moret, Yannick

    2015-01-01

    In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called “Trans-generational immune priming” (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations. PMID:26430786

  5. Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle.

    PubMed

    Dubuffet, Aurore; Zanchi, Caroline; Boutet, Gwendoline; Moreau, Jérôme; Teixeira, Maria; Moret, Yannick

    2015-10-01

    In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called "Trans-generational immune priming" (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations.

  6. Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle.

    PubMed

    Dubuffet, Aurore; Zanchi, Caroline; Boutet, Gwendoline; Moreau, Jérôme; Teixeira, Maria; Moret, Yannick

    2015-10-01

    In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called "Trans-generational immune priming" (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations. PMID:26430786

  7. Daptomycin: an evidence-based review of its role in the treatment of Gram-positive infections.

    PubMed

    Gonzalez-Ruiz, Armando; Seaton, R Andrew; Hamed, Kamal

    2016-01-01

    Infections caused by Gram-positive pathogens remain a major public health burden and are associated with high morbidity and mortality. Increasing rates of infection with Gram-positive bacteria and the emergence of resistance to commonly used antibiotics have led to the need for novel antibiotics. Daptomycin, a cyclic lipopeptide with rapid bactericidal activity against a wide range of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, has been shown to be effective and has a good safety profile for the approved indications of complicated skin and soft tissue infections (4 mg/kg/day), right-sided infective endocarditis caused by S. aureus, and bacteremia associated with complicated skin and soft tissue infections or right-sided infective endocarditis (6 mg/kg/day). Based on its pharmacokinetic profile and concentration-dependent bactericidal activity, high-dose (>6 mg/kg/day) daptomycin is considered an important treatment option in the management of various difficult-to-treat Gram-positive infections. Although daptomycin resistance has been documented, it remains uncommon despite the increasing use of daptomycin. To enhance activity and to minimize resistance, daptomycin in combination with other antibiotics has also been explored and found to be beneficial in certain severe infections. The availability of daptomycin via a 2-minute intravenous bolus facilitates its outpatient administration, providing an opportunity to reduce risk of health care-associated infections, improve patient satisfaction, and minimize health care costs. Daptomycin, not currently approved for use in the pediatric population, has been shown to be widely used for treating Gram-positive infections in children.

  8. Daptomycin: an evidence-based review of its role in the treatment of Gram-positive infections

    PubMed Central

    Gonzalez-Ruiz, Armando; Seaton, R Andrew; Hamed, Kamal

    2016-01-01

    Infections caused by Gram-positive pathogens remain a major public health burden and are associated with high morbidity and mortality. Increasing rates of infection with Gram-positive bacteria and the emergence of resistance to commonly used antibiotics have led to the need for novel antibiotics. Daptomycin, a cyclic lipopeptide with rapid bactericidal activity against a wide range of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, has been shown to be effective and has a good safety profile for the approved indications of complicated skin and soft tissue infections (4 mg/kg/day), right-sided infective endocarditis caused by S. aureus, and bacteremia associated with complicated skin and soft tissue infections or right-sided infective endocarditis (6 mg/kg/day). Based on its pharmacokinetic profile and concentration-dependent bactericidal activity, high-dose (>6 mg/kg/day) daptomycin is considered an important treatment option in the management of various difficult-to-treat Gram-positive infections. Although daptomycin resistance has been documented, it remains uncommon despite the increasing use of daptomycin. To enhance activity and to minimize resistance, daptomycin in combination with other antibiotics has also been explored and found to be beneficial in certain severe infections. The availability of daptomycin via a 2-minute intravenous bolus facilitates its outpatient administration, providing an opportunity to reduce risk of health care-associated infections, improve patient satisfaction, and minimize health care costs. Daptomycin, not currently approved for use in the pediatric population, has been shown to be widely used for treating Gram-positive infections in children. PMID:27143941

  9. N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge

    PubMed Central

    Lee, Ji Young; Linge, Helena M.; Ochani, Kanta; Lin, Ke; Miller, Edmund J.

    2016-01-01

    Background The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition. Methods Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN. Results Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1–10 nM. Conclusions TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an

  10. Genome-wide gene order distances support clustering the gram-positive bacteria

    PubMed Central

    House, Christopher H.; Pellegrini, Matteo; Fitz-Gibbon, Sorel T.

    2015-01-01

    Initially using 143 genomes, we developed a method for calculating the pair-wise distance between prokaryotic genomes using a Monte Carlo method to estimate the conservation of gene order. The method was based on repeatedly selecting five or six non-adjacent random orthologs from each of two genomes and determining if the chosen orthologs were in the same order. The raw distances were then corrected for gene order convergence using an adaptation of the Jukes-Cantor model, as well as using the common distance correction D′ = −ln(1-D). First, we compared the distances found via the order of six orthologs to distances found based on ortholog gene content and small subunit rRNA sequences. The Jukes-Cantor gene order distances are reasonably well correlated with the divergence of rRNA (R2 = 0.24), especially at rRNA Jukes-Cantor distances of less than 0.2 (R2 = 0.52). Gene content is only weakly correlated with rRNA divergence (R2 = 0.04) over all distances, however, it is especially strongly correlated at rRNA Jukes-Cantor distances of less than 0.1 (R2 = 0.67). This initial work suggests that gene order may be useful in conjunction with other methods to help understand the relatedness of genomes. Using the gene order distances in 143 genomes, the relations of prokaryotes were studied using neighbor joining and agreement subtrees. We then repeated our study of the relations of prokaryotes using gene order in 172 complete genomes better representing a wider-diversity of prokaryotes. Consistently, our trees show the Actinobacteria as a sister group to the bulk of the Firmicutes. In fact, the robustness of gene order support was found to be considerably greater for uniting these two phyla than for uniting any of the proteobacterial classes together. The results are supportive of the idea that Actinobacteria and Firmicutes are closely related, which in turn implies a single origin for the gram-positive cell. PMID:25653643

  11. Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

    PubMed Central

    Farfour, E.; Leto, J.; Barritault, M.; Barberis, C.; Meyer, J.; Dauphin, B.; Le Guern, A.-S.; Leflèche, A.; Badell, E.; Guiso, N.; Leclercq, A.; Le Monnier, A.; Lecuit, M.; Rodriguez-Nava, V.; Bergeron, E.; Raymond, J.; Vimont, S.; Bille, E.; Carbonnelle, E.; Guet-Revillet, H.; Lécuyer, H.; Beretti, J.-L.; Vay, C.; Berche, P.; Ferroni, A.; Nassif, X.

    2012-01-01

    Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry. PMID:22692743

  12. Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader rhodococcus sp. strain RHA1

    SciTech Connect

    Masai, Eiji; Hatta, Takashi; Kimbara, Kazuhide

    1995-06-01

    Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB-48 (equivalent to Aroclor 1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30-65%). FIn Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Djoi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments. The bphA1 and bphC insertion mutants lost the ability to grow on biphenyl, demonstrating that the cloned bph genes are essential for biphenyl catabolism in this strain. 31 refs., 5 figs., 2 tabs.

  13. In vitro assessment of Ag2O nanoparticles toxicity against Gram-positive and Gram-negative bacteria.

    PubMed

    Negi, Harshita; Rathinavelu Saravanan, Palaniyandi; Agarwal, Tithi; Ghulam Haider Zaidi, Mohd; Goel, Reeta

    2013-01-01

    In view of antibiotic resistance among pathogens, the present study is to address the toxicity of Ag2O nanoparticles against the Gram-positive and Gram-negative bacteria through in vitro assays. The preliminary screening by agar diffusion assay confirms the antibacterial activity of Ag2O nanoparticles against all the test bacteria. Comparative antibacterial activity of Ag2O nanoparticles and respective antibiotics reveals their broad range of activity and lower inhibitory dose against the used bacterial strains. Further, they can inhibit E. coli with an effective dose of 0.036 mg/ml within 1 h of exposure time as determined by luciferin based ATP assay. Moreover, the Ag2O nanoparticles exhibit higher antibacterial efficacy against Gram-negative bacteria than Gram-positive bacteria, as revealed by their MIC & MBC values. Therefore, Ag2O nanoparticles pave the way for a new generation of antibacterial agents against the emerging multidrug resistant pathogens.

  14. Disinfection of gram-negative and gram-positive bacteria using DynaJets® hydrodynamic cavitating jets.

    PubMed

    Loraine, Gregory; Chahine, Georges; Hsiao, Chao-Tsung; Choi, Jin-Keun; Aley, Patrick

    2012-05-01

    Cavitating jet technologies (DynaJets®) were investigated as a means of disinfection of gram-negative Escherichia coli, Klebsiellapneumoniae, Pseudomonas syringae, and Pseudomonas aeruginosa, and gram-positive Bacillus subtilis. The hydrodynamic cavitating jets were found to be very effective in reducing the concentrations of all of these species. In general, the observed rates of disinfection of gram-negative species were higher than for gram-positive species. However, different gram-negative species also showed significant differences (P. syringae 6-log(10) reduction, P. aeruginosa 2-log(10) reduction) under the same conditions. Disinfection of E. coli repeatedly showed five orders of magnitude reduction in concentration within 45-60-min at low nozzle pressure (2.1 bar). Optimization of nozzle design and operating pressures increased disinfection rates per input energy by several orders of magnitude. The power efficiencies of the hydrodynamic cavitating jets were found to be 10-100 times greater than comparable ultrasonic systems.

  15. In Vitro Activity of Ozenoxacin against Quinolone-Susceptible and Quinolone-Resistant Gram-Positive Bacteria

    PubMed Central

    López, Y.; Tato, M.; Espinal, P.; Garcia-Alonso, F.; Gargallo-Viola, D.; Cantón, R.

    2013-01-01

    In vitro activity of ozenoxacin, a novel nonfluorinated topical (L. D. Saravolatz and J. Leggett, Clin. Infect. Dis. 37:1210–1215, 2003) quinolone, was compared with the activities of other quinolones against well-characterized quinolone-susceptible and quinolone-resistant Gram-positive bacteria. Ozenoxacin was 3-fold to 321-fold more active than other quinolones. Ozenoxacin could represent a first-in-class nonfluorinated quinolone for the topical treatment of a broad range of dermatological infections. PMID:24080666

  16. Comparative in vitro activities of L-695,256, a novel carbapenem, against gram-positive bacteria.

    PubMed Central

    Malanoski, G; Collins, L; Eliopoulos, C T; Moellering, R C; Eliopoulos, G M

    1995-01-01

    The in vitro activity of a prototype 2-aryl carbapenem, L-695,256, against gram-positive bacteria was examined. All streptococci and oxacillin-susceptible and -resistant staphylococci were inhibited at concentrations of < or = 0.125, < or = 0.125, and 4 micrograms/ml, respectively. The activity of L-695,256 was superior to that of imipenem against other organisms intrinsically resistant to beta-lactams. PMID:7786011

  17. Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of Gram-negative and Gram-positive shock

    PubMed Central

    Murch, Oliver; Collin, Marika; Sepodes, Bruno; Foster, Simon J; Mota-Filipe, Helder; Thiemermann, Christoph

    2006-01-01

    Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA). Rats were subjected to (i) endotoxaemia (LPS 6 mg kg–1 i.v.) and treated with sLPC (1–100 mg kg−1), (ii) endotoxaemia and treated with nLPC (10 mg kg−1) or (iii) Gram-positive shock (PepG 10 mg kg–1 and LTA 3 mg kg–1 i.v.) and treated with sLPC (10 mg kg−1). Endotoxaemia or Gram-positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury. Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1–10 mg kg−1) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg−1) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by Gram-positive shock. nLPC was found to be protective in endotoxaemic animals. The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin-1β (IL-1β). In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL-1β levels caused by Gram-negative or Gram-positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies. PMID:16751791

  18. Efficacy of linezolid on gram-positive bacterial infection in elderly patients and the risk factors associated with thrombocytopenia

    PubMed Central

    Bi, Li-qing; Zhou, Jing; Huang, Ming; Zhou, Su-ming

    2013-01-01

    Objective : Linezolid is active against drug-resistant gram-positive bacteria. However, the efficacy and safety of linezolid in the treatment of the elderly have not been well characterized. The purpose of this study was to evaluate the efficacy of linezolid in the treatment of the elderly with gram-positive bacterial infection and to investigate the risk factors associated with the development of thrombocytopenia in these patients. Methodology: This was a retrospective analysis of 50 elderly patients who were treated with intravenous linezolid for gram-positive bacterial infection. Clinical data and bacteriological responses were assessed. Risk factors associated with thrombocytopenia in elderly patients were analyzed. Results: The overall clinical cure rate of linezolid was 74%, and the bacteriological eradication rate was 69%. Thrombocytopenia occurred in 24 patients, and thrombocytopenia was associated with both the duration of treatment (P = 0.005) and the baseline platelet count (P = 0.042). Based on a logistic regression analysis, the baseline platelet count <200×109/L (OR = 0.244; 95% CI = 0.068- 0.874; P = 0.030) was identified as the only significant risk factor for linezolid-associated thrombocytopenia in elderly patients. The mean platelet count decreased significantly from the 7th day of treatment, and decreased to the lowest value 1-2 days after the end of therapy. Conclusions : Linezolid is effective and safe for the elderly with gram-positive bacterial infections. Adverse effects such as thrombocytopenia are of greater concern. Platelet counts should be monitored in patients who are treated with linezolid and that measures should be taken in advance to avoid hemorrhagic tendencies. PMID:24353639

  19. Relevance of GC content to the conservation of DNA polymerase III/mismatch repair system in Gram-positive bacteria

    PubMed Central

    Akashi, Motohiro; Yoshikawa, Hirofumi

    2013-01-01

    The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3′-5′exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3′-5′ exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3′-5′ exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria. PMID:24062730

  20. Wide distribution of virulence genes among Enterococcus faecium and Enterococcus faecalis clinical isolates.

    PubMed

    Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasanthakumari; Sadeghifard, Nourkhoda; Ramli, Ramliza; Hamat, Rukman Awang

    2014-01-01

    Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5(°)C and 65(°)C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.

  1. RNA-mediated regulation in Gram-positive pathogens: an overview punctuated with examples from the group A Streptococcus

    PubMed Central

    Miller, Eric W.; Cao, Tram N.; Pflughoeft, Kathryn J.; Sumby, Paul

    2014-01-01

    RNA-based mechanisms of regulation represent a ubiquitous class of regulators that are associated with diverse processes including nutrient sensing, stress response, modulation of horizontal gene transfer, and virulence factor expression. While better studied in Gram-negative bacteria, the literature is replete with examples of the importance of RNA-mediated regulatory mechanisms to the virulence and fitness of Gram-positives. Regulatory RNAs are classified as cis-acting, e.g. riboswitches, which modulate the transcription, translation, or stability of co-transcribed RNA, or trans-acting, e.g. small regulatory RNAs, which target separate mRNAs or proteins. The group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive bacterial pathogen from which several regulatory RNA mechanisms have been characterized. The study of RNA-mediated regulation in GAS has uncovered novel concepts with respect to how small regulatory RNAs may positively regulate target mRNA stability, and to how CRISPR RNAs are processed from longer precursors. This review provides an overview of RNA-mediated regulation in Gram-positive bacteria, and is highlighted with specific examples from GAS research. The key roles that these systems play in regulating bacterial virulence are discussed and future perspectives outlined. PMID:25091277

  2. Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

    NASA Technical Reports Server (NTRS)

    Gehron, M. J.; Davis, J. D.; Smith, G. A.; White, D. C.

    1984-01-01

    Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydrofluoric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.

  3. Evolution of sensitivity to fosfomycin in bacteria isolated in 1973, 1974 and 1975 in the Servicio de Microbiologia y Epidemiologia of the 'Clinica Puerta de Hierro', Madrid.

    PubMed

    Dámaso, D; Moreno-López, M; Martínez-Beltrán, J

    1977-01-01

    The bacteriostatic activity of fosfomycin was studied in vitro against 1,243 clinical isolations of gram-positive cocci and 4,086 isolations of gram-negative bacilli that were obtained in 1973, 1974 and in the period from January to May of 1975. MIC was determined by the agar diffusion method, quantifying it by means of the standard curve that was worked out with the strain of E. coli NCTC 10,418. A slight increase in resistance was observed in the gram-positive cocci: 64 mug/ml were inhibitory for 63% of the 249 isolations obtained in 1973, 59.1% of the 716 isolations obtained in 1974, and 57.5% of the 278 isolations from 1975. A slight loss of sensitivity was also observed in the gram-negative bacilli: the aforementioned concentration of fosfomycin inhibited 36% of the 742 isolations from 1973, 33.6% of the 2,387 isolations from 1974 and 32.6% of the 957 isolations from 1975. 933 g of this antibiotic were consumed in our hospital in 1973, 4,203 g in 1974 and 957 g in 1975. The consumption rate per patient per year was 0.15, 0.72 and 0.20 g, respectively. In conclusion, although no change was observed in the sensitivity of some bacterial strains to fosfomycin, the overall study indicates a slight decrease in the sensitivity, although it does not apparently have any relationship to the consumption of fosfomycin in our hospital.

  4. [Infections caused by multi-resistant Gram-positive bacteria (Staphylococcus aureus and Enterococcus spp.)].

    PubMed

    Cantón, Rafael; Ruiz-Garbajosa, Patricia

    2013-10-01

    Methicillin -resistant Staphylocccus aureus (MRSA) and multirresistant entorococci are still problematic in nosocomial infections and new challenges have emerged for their containment. MRSA has increased the multiresistant profile; it has been described vancomycin and linezolid resistant isolates and isolates with decreased daptomycin susceptibility. Moreover, new clones (ST398) have emerged, initially associated with piggeries, and new mec variants (mecC) with livestock origin that escape to the detection with current molecular methods based on mecA gene have been detected. In enterococci, linzeolid resistant isolates and isolates with deceased susceptibility to daptomycin have been described. Moreover, ampicillin resistant Enterococcus faecium due to β-lactamase production has been recently found in Europe. Control of MRSA isolates and multiresistant enteroccocci should combined antibiotic stewardship strategies and epidemiological measures, including detection of colonized patients in order to reduce colonization pressure and their transmission.

  5. Linezolid-resistant clinical isolates of enterococci and Staphylococcus cohnii from a multicentre study in China: molecular epidemiology and resistance mechanisms.

    PubMed

    Chen, Hongbin; Wu, Weiyuan; Ni, Ming; Liu, Yingmei; Zhang, Jixia; Xia, Fei; He, Wenqiang; Wang, Qi; Wang, Zhanwei; Cao, Bin; Wang, Hui

    2013-10-01

    Genetic characterisation of linezolid-resistant Gram-positive cocci in a multicentre study in China has not been reported previously. To study the mechanism underlying the resistance of linezolid-resistant isolates, nine Enterococcus faecalis, one Enterococcus faecium and three Staphylococcus cohnii isolates with various levels of resistance were collected from five hospitals across China in 2009-2012. The nine E. faecalis isolates were classified into seven sequence types, indicating that these linezolid-resistant E. faecalis isolates were polyclonal. Enterococci isolates had reduced susceptibility to linezolid (MICs of 4-8 mg/L) and had mutation of ribosomal protein L3, with three also having mutation of L4, but without the multidrug resistance gene cfr or the 23S rRNA mutation G2576T. The three S. cohnii isolates were highly resistant to linezolid (MICs of 64 mg/L to >256 mg/L), harboured the cfr gene and had the 23S rRNA mutation G2576T. Southern blotting indicated that the cfr gene of these three isolates resided on different plasmids (pHK01, pRM01 and pRA01). In plasmid pHK01, IS21-558 and the cfr gene were integrated into transposon Tn558. In plasmids pRM01 and pRA01, the cfr gene was flanked by two copies of an IS256-like insertion sequence, indicating that the transferable form of linezolid resistance is conferred by the cfr gene. In conclusion, the emergence of linezolid-resistant Gram-positive cocci in different regions of China is of concern. The cfr gene and the 23S rRNA mutation contribute to high-level linezolid resistance in S. cohnii, and the L3 and L4 mutations are associated with low-level linezolid resistance in enterococci.

  6. Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

    PubMed

    Ben Bacha, Abir; Abid, Islem

    2013-03-01

    The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

  7. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.

    PubMed

    Corrigan, Rebecca M; Bellows, Lauren E; Wood, Alison; Gründling, Angelika

    2016-03-22

    The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases--RsgA, RbgA, Era, HflX, and ObgE--as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

  8. Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

    PubMed Central

    Navarre, William Wiley; Schneewind, Olaf

    1999-01-01

    The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

  9. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

    PubMed Central

    Corrigan, Rebecca M.; Bellows, Lauren E.; Wood, Alison

    2016-01-01

    The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria. PMID:26951678

  10. Design, synthesis, and antibacterial activities of neomycin-lipid conjugates: polycationic lipids with potent gram-positive activity.

    PubMed

    Bera, Smritilekha; Zhanel, George G; Schweizer, Frank

    2008-10-01

    Aminoglycoside antibiotics and cationic detergents constitute two classes of clinically important drugs and antiseptics. Their bacteriological and clinical efficacy, however, has decreased recently due to antibiotic resistance. We have synthesized aminoglycoside-lipid conjugates in which the aminoglycoside neomycin forms the cationic headgroup of a polycationic detergent. Our results show that neomycin-C16 and neomycin-C20 conjugates exhibit strong Gram-positive activity but reduced Gram-negative activity. The MIC of neomycin-C16 (C20) conjugates against methicillin-resistant Staphylococcus aureus (MRSA) is comparable to clinically used antiseptics.

  11. Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria I. Partition systems.

    PubMed

    Dmowski, Michał; Jagura-Burdzy, Grazyna

    2013-01-01

    Low copy number plasmids cannot rely on the random segregation during bacterial cell division. To be stably maintained in the population they evolved two types of mechanisms (i) partition systems (PAR) that actively separate replicated plasmid molecules to the daughter cells and (ii) toxin-andidote systems (TA) that act after cell division to kill plasmid-less cells. Our knowledge of partition systems has been based mainly on analysis of plasmids from Gram-negative bacteria. Now, numerous partition systems of plasmids from Gram-positive bacteria have also been characterized and make significant contribution to our understanding of these mechanisms.

  12. Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

    PubMed

    Nakonieczna, A; Cooper, C J; Gryko, R

    2015-09-01

    Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

  13. Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum.

    PubMed Central

    Janssen, P J; Jones, W A; Jones, D T; Woods, D R

    1988-01-01

    The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum glnA gene was determined. The upstream region of the glnA gene contained two putative extended promoter consensus sequences (p1 and p2), characteristic of gram-positive bacteria. A third putative extended gram-positive promoter consensus sequence (p3), oriented towards the glnA gene, was detected downstream of the structural gene. The sequences containing the proposed promoter regions p1 and p2 or p3 were shown to have promoter activity by subcloning into promoter probe vectors. The complete amino acid sequence (444 residues) of the C. acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms. To determine whether the putative promoter p3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C. acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking p3 and various downstream sequences. Deletion of the putative promoter p3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions. PMID:2891680

  14. First Multitarget Chemo-Bioinformatic Model To Enable the Discovery of Antibacterial Peptides against Multiple Gram-Positive Pathogens.

    PubMed

    Speck-Planche, Alejandro; Kleandrova, Valeria V; Ruso, Juan M; Cordeiro, M N D S

    2016-03-28

    Antimicrobial peptides (AMPs) have emerged as promising therapeutic alternatives to fight against the diverse infections caused by different pathogenic microorganisms. In this context, theoretical approaches in bioinformatics have paved the way toward the creation of several in silico models capable of predicting antimicrobial activities of peptides. All current models have several significant handicaps, which prevent the efficient search for highly active AMPs. Here, we introduce the first multitarget (mt) chemo-bioinformatic model devoted to performing alignment-free prediction of antibacterial activity of peptides against multiple Gram-positive bacterial strains. The model was constructed from a data set containing 2488 cases of AMPs sequences assayed against at least 1 out of 50 Gram-positive bacterial strains. This mt-chemo-bioinformatic model displayed percentages of correct classification higher than 90.00% in both training and prediction (test) sets. For the first time, two computational approaches derived from basic concepts in genetics and molecular biology were applied, allowing the calculations of the relative contributions of any amino acid (in a defined position) to the antibacterial activity of an AMP and depending on the bacterial strain used in the biological assay. The present mt-chemo-bioinformatic model constitutes a powerful tool to enable the discovery of potent and versatile AMPs.

  15. Neither Single nor a Combination of Routine Laboratory Parameters can Discriminate between Gram-positive and Gram-negative Bacteremia

    PubMed Central

    Ratzinger, Franz; Dedeyan, Michel; Rammerstorfer, Matthias; Perkmann, Thomas; Burgmann, Heinz; Makristathis, Athanasios; Dorffner, Georg; Loetsch, Felix; Blacky, Alexander; Ramharter, Michael

    2015-01-01

    Adequate early empiric antibiotic therapy is pivotal for the outcome of patients with bloodstream infections. In clinical practice the use of surrogate laboratory parameters is frequently proposed to predict underlying bacterial pathogens; however there is no clear evidence for this assumption. In this study, we investigated the discriminatory capacity of predictive models consisting of routinely available laboratory parameters to predict the presence of Gram-positive or Gram-negative bacteremia. Major machine learning algorithms were screened for their capacity to maximize the area under the receiver operating characteristic curve (ROC-AUC) for discriminating between Gram-positive and Gram-negative cases. Data from 23,765 patients with clinically suspected bacteremia were screened and 1,180 bacteremic patients were included in the study. A relative predominance of Gram-negative bacteremia (54.0%), which was more pronounced in females (59.1%), was observed. The final model achieved 0.675 ROC-AUC resulting in 44.57% sensitivity and 79.75% specificity. Various parameters presented a significant difference between both genders. In gender-specific models, the discriminatory potency was slightly improved. The results of this study do not support the use of surrogate laboratory parameters for predicting classes of causative pathogens. In this patient cohort, gender-specific differences in various laboratory parameters were observed, indicating differences in the host response between genders. PMID:26522966

  16. CRIg Functions as a Macrophage Pattern Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

    PubMed

    Zeng, Zhutian; Surewaard, Bas G J; Wong, Connie H Y; Geoghegan, Joan A; Jenne, Craig N; Kubes, Paul

    2016-07-13

    Kupffer cells (KCs), the vast pool of intravascular macrophages in the liver, help to clear blood-borne pathogens. The mechanisms by which KCs capture circulating pathogens remain unknown. Here we use intra-vital imaging of mice infected with Staphylococcus aureus to directly visualize the dynamic process of bacterial capture in the liver. Circulating S. aureus were captured by KCs in a manner dependent on the macrophage complement receptor CRIg, but the process was independent of complement. CRIg bound Staphylococcus aureus specifically through recognition of lipoteichoic acid (LTA), but not cell-wall-anchored surface proteins or peptidoglycan. Blocking the recognition between CRIg and LTA in vivo diminished the bacterial capture in liver and led to systemic bacterial dissemination. All tested Gram-positive, but not Gram-negative, bacteria bound CRIg in a complement-independent manner. These findings reveal a pattern recognition role for CRIg in the direct capture of circulating Gram-positive bacteria from the bloodstream. PMID:27345697

  17. Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria.

    PubMed

    Qazi, S.N.A.; Rees, C.E.D.; Mellits, K.H.; Hill, P.J.

    2001-02-01

    The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the Pxyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the Pxyn promoter was replaced with the PxylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3+ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.

  18. Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Biswas, Bipul; Rogers, Kimberly; McLaughlin, Fredrick; Daniels, Dwayne; Yadav, Anand

    2013-01-01

    Aim. To determine the antimicrobial potential of guava (Psidium guajava) leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis) and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50  μ L leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties. PMID:24223039

  19. Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Biswas, Bipul; Rogers, Kimberly; McLaughlin, Fredrick; Daniels, Dwayne; Yadav, Anand

    2013-01-01

    Aim. To determine the antimicrobial potential of guava (Psidium guajava) leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis) and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50  μ L leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties.

  20. Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria

    PubMed Central

    Biswas, Bipul; Rogers, Kimberly; McLaughlin, Fredrick; Yadav, Anand

    2013-01-01

    Aim. To determine the antimicrobial potential of guava (Psidium guajava) leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis) and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50 μL leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties. PMID:24223039

  1. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

    PubMed

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Iñigo; Novick, Richard P; Christie, Gail E; Penadés, José R

    2013-08-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

  2. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

    PubMed Central

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

    2013-01-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  3. Neither Single nor a Combination of Routine Laboratory Parameters can Discriminate between Gram-positive and Gram-negative Bacteremia.

    PubMed

    Ratzinger, Franz; Dedeyan, Michel; Rammerstorfer, Matthias; Perkmann, Thomas; Burgmann, Heinz; Makristathis, Athanasios; Dorffner, Georg; Loetsch, Felix; Blacky, Alexander; Ramharter, Michael

    2015-11-02

    Adequate early empiric antibiotic therapy is pivotal for the outcome of patients with bloodstream infections. In clinical practice the use of surrogate laboratory parameters is frequently proposed to predict underlying bacterial pathogens; however there is no clear evidence for this assumption. In this study, we investigated the discriminatory capacity of predictive models consisting of routinely available laboratory parameters to predict the presence of Gram-positive or Gram-negative bacteremia. Major machine learning algorithms were screened for their capacity to maximize the area under the receiver operating characteristic curve (ROC-AUC) for discriminating between Gram-positive and Gram-negative cases. Data from 23,765 patients with clinically suspected bacteremia were screened and 1,180 bacteremic patients were included in the study. A relative predominance of Gram-negative bacteremia (54.0%), which was more pronounced in females (59.1%), was observed. The final model achieved 0.675 ROC-AUC resulting in 44.57% sensitivity and 79.75% specificity. Various parameters presented a significant difference between both genders. In gender-specific models, the discriminatory potency was slightly improved. The results of this study do not support the use of surrogate laboratory parameters for predicting classes of causative pathogens. In this patient cohort, gender-specific differences in various laboratory parameters were observed, indicating differences in the host response between genders.

  4. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

    PubMed

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Iñigo; Novick, Richard P; Christie, Gail E; Penadés, José R

    2013-08-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  5. Quantitative proteomic view associated with resistance to clinically important antibiotics in Gram-positive bacteria: a systematic review

    PubMed Central

    Lee, Chang-Ro; Lee, Jung Hun; Park, Kwang Seung; Jeong, Byeong Chul; Lee, Sang Hee

    2015-01-01

    The increase of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) poses a worldwide and serious health threat. Although new antibiotics, such as daptomycin and linezolid, have been developed for the treatment of infections of Gram-positive pathogens, the emergence of daptomycin-resistant and linezolid-resistant strains during therapy has now increased clinical treatment failures. In the past few years, studies using quantitative proteomic methods have provided a considerable progress in understanding antibiotic resistance mechanisms. In this review, to understand the resistance mechanisms to four clinically important antibiotics (methicillin, vancomycin, linezolid, and daptomycin) used in the treatment of Gram-positive pathogens, we summarize recent advances in studies on resistance mechanisms using quantitative proteomic methods, and also examine proteins playing an important role in the bacterial mechanisms of resistance to the four antibiotics. Proteomic researches can identify proteins whose expression levels are changed in the resistance mechanism to only one antibiotic, such as LiaH in daptomycin resistance and PrsA in vancomycin resistance, and many proteins simultaneously involved in resistance mechanisms to various antibiotics. Most of resistance-related proteins, which are simultaneously associated with resistance mechanisms to several antibiotics, play important roles in regulating bacterial envelope biogenesis, or compensating for the fitness cost of antibiotic resistance. Therefore, proteomic data confirm that antibiotic resistance requires the fitness cost and the bacterial envelope is an important factor in antibiotic resistance. PMID:26322035

  6. Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria.

    PubMed

    Grau-Campistany, Ariadna; Manresa, Ángeles; Pujol, Montserrat; Rabanal, Francesc; Cajal, Yolanda

    2016-02-01

    Resistance to all known antibiotics is a growing concern worldwide, and has renewed the interest in antimicrobial peptides, a structurally diverse class of amphipathic molecules that essentially act on the bacterial membrane. Propelled by the antimicrobial potential of this compound class, we have designed three new lipopeptides derived from polymyxin B, sp-34, sp-96 and sp-100, with potent antimicrobial activity against both Gram positive and Gram negative bacteria. The three peptides bind with high affinity to lipopolysaccharide as demonstrated by monolayer penetration and dansyl-displacement. The interaction with the cytoplasmic membrane has been elucidated by biophysical experiments with model membranes of POPG or POPE/POPG (6:4), mimicking the Gram positive and Gram negative bacterial membrane. Trp-based fluorescence experiments including steady-state, quenching, anisotropy and FRET, reveal selectivity for anionic phospholipids and deep insertion into the membrane. All three lipopeptides induce membrane fusion and leakage from anionic vesicles, a process that is favored by the presence of POPE. The molecules bind to zwitterionic POPC vesicles, a model of the eukaryotic membrane, but in a different way, with lower affinity, less penetration into the bilayer and no fusion or permeabilization of the membrane. Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.

  7. The filamentous bacterial morphotype 'Nostocoida limicola' I contains at least two previously described genera in the low G+C gram positive bacteria.

    PubMed

    Liu, J R; Burrell, P; Seviour, E M; Soddell, J A; Blackall, L L; Seviour, R J

    2000-12-01

    Isolates of eight bacterial filaments fitting the published morphological description of 'Nostocoida limicola' I were obtained from the mixed liquor of four different Australian and one Czech Republic activated sludge plants by micromanipulation. On the basis of their near complete (Ben 200 and Ben 201), or partial (Ben 77, Ben 78, Ben 202, Ben 203, Ben 204 and Ben 205) 16S rRNA gene sequences, six of these isolates were 99.3-100% similar to Lactosphaera pasteurii and Trichococcus flocculiformis, a bulking filament only reported previously in Germany. The other two (Ben 203 and Ben 204) were 99.9% similar to Streptococcus suis. Hence, all are in the low mol % G+C gram-positive bacteria division of the Bacteria. On this evidence 'N. limicola' I is phylogenetically unrelated to 'Nostocoida limicola' II, which is now known to be in the Actinobacteria, even though these two filamentous bacteria appearing in activated sludge systems have been considered to be closely related to each other historically.

  8. Targeting agr- and agr-Like Quorum Sensing Systems for Development of Common Therapeutics to Treat Multiple Gram-Positive Bacterial Infections

    PubMed Central

    Gray, Brian; Hall, Pamela; Gresham, Hattie

    2013-01-01

    Invasive infection by the Gram-positive pathogen Staphylococcus aureus is controlled by a four gene operon, agr that encodes a quorum sensing system for the regulation of virulence. While agr has been well studied in S. aureus, the contribution of agr homologues and analogues in other Gram-positive pathogens is just beginning to be understood. Intriguingly, other significant human pathogens, including Clostridium perfringens, Listeria monocytogenes, and Enterococcus faecalis contain agr or analogues linked to virulence. Moreover, other significant human Gram-positive pathogens use peptide based quorum sensing systems to establish or maintain infection. The potential for commonality in aspects of these signaling systems across different species raises the prospect of identifying therapeutics that could target multiple pathogens. Here, we review the status of research into these agr homologues, analogues, and other peptide based quorum sensing systems in Gram-positive pathogens as well as the potential for identifying common pathways and signaling mechanisms for therapeutic discovery. PMID:23598501

  9. Metabolism of 4-chloro-2-nitrophenol in a Gram-positive bacterium, Exiguobacterium sp. PMA

    PubMed Central

    2012-01-01

    Background Chloronitrophenols (CNPs) are widely used in the synthesis of dyes, drugs and pesticides, and constitute a major group of environmental pollutants. 4-Chloro-2-nitrophenol (4C2NP) is an isomer of CNPs that has been detected in various industrial effluents. A number of physicochemical methods have been used for treatment of wastewater containing 4C2NP. These methods are not as effective as microbial degradation, however. Results A 4C2NP-degrading bacterium, Exiguobacterium sp. PMA, which uses 4C2NP as the sole carbon and energy source was isolated from a chemically-contaminated site in India. Exiguobacterium sp. PMA degraded 4C2NP with the release of stoichiometeric amounts of chloride and ammonium ions. The effects of different substrate concentrations and various inoculum sizes on degradation of 4C2NP were investigated. Exiguobacterium sp. PMA degraded 4C2NP up to a concentration of 0.6 mM. High performance liquid chromatography and gas chromatography–mass spectrometry identified 4-chloro-2-aminophenol (4C2AP) and 2-aminophenol (2AP) as possible metabolites of the 4C2NP degradation pathway. The crude extract of 4C2NP-induced PMA cells contained enzymatic activity for 4C2NP reductase and 4C2AP dehalogenase, suggesting the involvement of these enzymes in the degradation of 4C2NP. Microcosm studies using sterile and non-sterile soils spiked with 4C2NP were carried out to monitor the bioremediation potential of Exiguobacterium sp. PMA. The bioremediation of 4C2NP by Exiguobacterium sp. PMA was faster in non-sterilized soil than sterilized soil. Conclusions Our studies indicate that Exiguobacterium sp. PMA may be useful for the bioremediation of 4C2NP-contaminated sites. This is the first report of (i) the formation of 2AP in the 4C2NP degradation pathway by any bacterium and (iii) the bioremediation of 4C2NP by any bacterium. PMID:23171039

  10. LiF Reduces MICs of Antibiotics against Clinical Isolates of Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    Syed, H. C.; Ravaoarinoro, M.

    2012-01-01

    Antibiotic resistance is an ever-growing problem yet the development of new antibiotics has slowed to a trickle, giving rise to the use of combination therapy to eradicate infections. The purpose of this study was to evaluate the combined inhibitory effect of lithium fluoride (LiF) and commonly used antimicrobials on the growth of the following bacteria: Enterococcus faecalis, Staphyloccoccus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, and Streptococcus pneumoniae. The in vitro activities of ceftazidime, sulfamethoxazole-trimethoprim, streptomycin, erythromycin, amoxicillin, and ciprofloxacin, doxycycline, alone or combined with LiF were performed by microdilution method. MICs were determined visually following 18–20 h of incubation at 37°C. We observed reduced MICs of antibiotics associated with LiF ranging from two-fold to sixteen-fold. The strongest decreases of MICs observed were for streptomycin and erythromycin associated with LiF against Acinetobacter baumannii and Streptococcus pneumoniae. An eight-fold reduction was recorded for streptomycin against S. pneumoniae whereas an eight-fold and a sixteen-fold reduction were obtained for erythromycin against A. baumannii and S. pneumoniae. This suggests that LiF exhibits a synergistic effect with a wide range of antibiotics and is indicative of its potential as an adjuvant in antibiotic therapy. PMID:22518143

  11. Distinct Mechanisms Underlie Boosted Polysaccharide-Specific IgG Responses Following Secondary Challenge with Intact Gram-Negative versus Gram-Positive Extracellular Bacteria.

    PubMed

    Kar, Swagata; Arjunaraja, Swadhinya; Akkoyunlu, Mustafa; Pier, Gerald B; Snapper, Clifford M

    2016-06-01

    Priming of mice with intact, heat-killed cells of Gram-negative Neisseria meningitidis, capsular serogroup C (MenC) or Gram-positive group B Streptococcus, capsular type III (GBS-III) bacteria resulted in augmented serum polysaccharide (PS)-specific IgG titers following booster immunization. Induction of memory required CD4(+) T cells during primary immunization. We determined whether PS-specific memory for IgG production was contained within the B cell and/or T cell populations, and whether augmented IgG responses following booster immunization were also dependent on CD4(+) T cells. Adoptive transfer of purified B cells from MenC- or GBS-III-primed, but not naive mice resulted in augmented PS-specific IgG responses following booster immunization. Similar responses were observed when cotransferred CD4(+) T cells were from primed or naive mice. Similarly, primary immunization with unencapsulated MenC or GBS-III, to potentially prime CD4(+) T cells, failed to enhance PS-specific IgG responses following booster immunization with their encapsulated isogenic partners. Furthermore, in contrast to GBS-III, depletion of CD4(+) T cells during secondary immunization with MenC or another Gram-negative bacteria, Acinetobacter baumannii, did not inhibit augmented PS-specific IgG booster responses of mice primed with heat-killed cells. Also, in contrast with GBS-III, booster immunization of MenC-primed mice with isolated MenC-PS, a TI Ag, or a conjugate of MenC-PS and tetanus toxoid elicited an augmented PS-specific IgG response similar to booster immunization with intact MenC. These data demonstrate that memory for augmented PS-specific IgG booster responses to Gram-negative and Gram-positive bacteria is contained solely within the B cell compartment, with a differential requirement for CD4(+) T cells for augmented IgG responses following booster immunization. PMID:27183619

  12. Distinct Mechanisms Underlie Boosted Polysaccharide-Specific IgG Responses Following Secondary Challenge with Intact Gram-Negative versus Gram-Positive Extracellular Bacteria.

    PubMed

    Kar, Swagata; Arjunaraja, Swadhinya; Akkoyunlu, Mustafa; Pier, Gerald B; Snapper, Clifford M

    2016-06-01

    Priming of mice with intact, heat-killed cells of Gram-negative Neisseria meningitidis, capsular serogroup C (MenC) or Gram-positive group B Streptococcus, capsular type III (GBS-III) bacteria resulted in augmented serum polysaccharide (PS)-specific IgG titers following booster immunization. Induction of memory required CD4(+) T cells during primary immunization. We determined whether PS-specific memory for IgG production was contained within the B cell and/or T cell populations, and whether augmented IgG responses following booster immunization were also dependent on CD4(+) T cells. Adoptive transfer of purified B cells from MenC- or GBS-III-primed, but not naive mice resulted in augmented PS-specific IgG responses following booster immunization. Similar responses were observed when cotransferred CD4(+) T cells were from primed or naive mice. Similarly, primary immunization with unencapsulated MenC or GBS-III, to potentially prime CD4(+) T cells, failed to enhance PS-specific IgG responses following booster immunization with their encapsulated isogenic partners. Furthermore, in contrast to GBS-III, depletion of CD4(+) T cells during secondary immunization with MenC or another Gram-negative bacteria, Acinetobacter baumannii, did not inhibit augmented PS-specific IgG booster responses of mice primed with heat-killed cells. Also, in contrast with GBS-III, booster immunization of MenC-primed mice with isolated MenC-PS, a TI Ag, or a conjugate of MenC-PS and tetanus toxoid elicited an augmented PS-specific IgG response similar to booster immunization with intact MenC. These data demonstrate that memory for augmented PS-specific IgG booster responses to Gram-negative and Gram-positive bacteria is contained solely within the B cell compartment, with a differential requirement for CD4(+) T cells for augmented IgG responses following booster immunization.

  13. Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Zheng, Amy; Panja, Subrata; Woodson, Sarah A

    2016-06-01

    The Sm-protein Hfq facilitates interactions between small non-coding RNA (sRNA) and target mRNAs. In enteric Gram-negative bacteria, Hfq is required for sRNA regulation, and hfq deletion results in stress intolerance and reduced virulence. By contrast, the role of Hfq in Gram-positive is less established and varies among species. The RNA binding and RNA annealing activity of Hfq from Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, and Staphylococcus aureus were compared using minimal RNAs and fluorescence spectroscopy. The results show that RNA annealing activity increases with the number of arginines in a semi-conserved patch on the rim of the Hfq hexamer and correlates with the previously reported requirement for Hfq in sRNA regulation. Thus, the amino acid sequence of the arginine patch can predict the chaperone function of Hfq in sRNA regulation in different organisms. PMID:27049793

  14. Highly active modulators of indole signaling alter pathogenic behaviors in Gram-negative and Gram-positive bacteria.

    PubMed

    Minvielle, Marine J; Eguren, Kristen; Melander, Christian

    2013-12-16

    Indole is a universal signal that regulates various bacterial behaviors, such as biofilm formation and antibiotic resistance. To generate mechanistic probes of indole signaling and control indole-mediated pathogenic phenotypes in both Gram-positive and Gram-negative bacteria, we have investigated the use of desformylflustrabromine (dFBr) derivatives to generate highly active indole mimetics. We have developed non-microbicidal dFBr derivatives that are 27-2000 times more active than indole in modulating biofilm formation, motility, acid resistance, and antibiotic resistance. The activity of these analogues parallels indole, because they are dependent on temperature, the enzyme tryptophanase TnaA, and the transcriptional regulator SdiA. This investigation demonstrates that molecules based on the dFBr scaffold can alter pathogenic behaviors by mimicking indole-signaling pathways.

  15. Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis

    PubMed Central

    Jana, Ninkovic; Vidhu, Anand; Raini, Dutta; Zhang, Li; Saluja, Anuj; Meng, Jingjing; Lisa, Koodie; Santanu, Banerjee; Sabita, Roy

    2016-01-01

    Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (−) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (−) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (−) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers. PMID:26891899

  16. Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria.

    PubMed

    Brzozowska, Iwona; Brzozowska, Kinga; Zielenkiewicz, Urszula

    2012-07-01

    Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.

  17. Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter

    PubMed Central

    Nandi, Sobhan; Maurer, John J.; Hofacre, Charles; Summers, Anne O.

    2004-01-01

    Reversing the spread of antibiotic multiresistant bacteria is hampered by ignorance of the natural history of resistance genes, the mobile elements carrying them, and the bacterial hosts harboring them. Using traditional cultivation and cultivation-independent molecular techniques, we quantified antibiotic resistance genes and mobile elements called integrons in poultry house litter from commercial poultry farms. Unexpectedly, the major reservoir for Class 1 integrons in poultry litter is not their previously identified hosts, Gram-negative Enterobacteriaceae such as Escherichia coli. Rather, integrons and associated resistance genes abound in several genera of Gram-positive bacteria that constitute >85% of the litter community compared with Enterobacteriaceae that comprise <2% of this ecosystem. This finding warrants reexamination of our assumptions about the persistence and spread of antibiotic resistance genes. PMID:15107498

  18. Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis.

    PubMed

    Ninkovic, Jana; Jana, Ninkovic; Anand, Vidhu; Vidhu, Anand; Dutta, Raini; Raini, Dutta; Zhang, Li; Saluja, Anuj; Meng, Jingjing; Koodie, Lisa; Lisa, Koodie; Banerjee, Santanu; Santanu, Banerjee; Roy, Sabita; Sabita, Roy

    2016-02-19

    Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (-) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (-) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (-) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers.

  19. Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Zheng, Amy; Panja, Subrata; Woodson, Sarah A

    2016-06-01

    The Sm-protein Hfq facilitates interactions between small non-coding RNA (sRNA) and target mRNAs. In enteric Gram-negative bacteria, Hfq is required for sRNA regulation, and hfq deletion results in stress intolerance and reduced virulence. By contrast, the role of Hfq in Gram-positive is less established and varies among species. The RNA binding and RNA annealing activity of Hfq from Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, and Staphylococcus aureus were compared using minimal RNAs and fluorescence spectroscopy. The results show that RNA annealing activity increases with the number of arginines in a semi-conserved patch on the rim of the Hfq hexamer and correlates with the previously reported requirement for Hfq in sRNA regulation. Thus, the amino acid sequence of the arginine patch can predict the chaperone function of Hfq in sRNA regulation in different organisms.

  20. Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

    PubMed

    Niwa, Takashi; Kawamura, Yoshiaki; Katagiri, Yoshihiro; Ezaki, Takayuki

    2005-05-01

    The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols.

  1. Armadillidin: a novel glycine-rich antibacterial peptide directed against gram-positive bacteria in the woodlouse Armadillidium vulgare (Terrestrial Isopod, Crustacean).

    PubMed

    Herbinière, Juline; Braquart-Varnier, Christine; Grève, Pierre; Strub, Jean-Marc; Frère, Jacques; Van Dorsselaer, Alain; Martin, Gilbert

    2005-01-01

    We report the isolation and the characterization of a novel antibacterial peptide from hemocytes of the woodlouse Armadillidium vulgare, naturally infected or uninfected by Wolbachia, an intracellular Gram-negative bacterium. This molecule displays antibacterial activity against Gram-positive bacteria despite its composition which classes it into the glycine-rich antibacterial peptide family, usually directed against fungi and Gram-negative bacteria. The complete sequence was determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a hemocyte library. The mature peptide (53 residues) has a 5259 Da molecular mass and is post-translationally modified by a C-terminal amidation. This peptide is characterized by a high level of glycine (47%) and a fivefold repeated motif GGGFH(R/S). As no evident sequence homology to other hitherto described antibacterial peptides has been found out, this antibacterial peptide was named armadillidin. Armadillidin is constitutively expressed in hemocytes and appears to be specific of A. vulgare. PMID:15752546

  2. Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

    PubMed

    Niwa, Takashi; Kawamura, Yoshiaki; Katagiri, Yoshihiro; Ezaki, Takayuki

    2005-05-01

    The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols. PMID:15722152

  3. Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR.

    PubMed

    Carroll, N M; Jaeger, E E; Choudhury, S; Dunlop, A A; Matheson, M M; Adamson, P; Okhravi, N; Lightman, S

    2000-05-01

    A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.

  4. Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus.

    PubMed

    Morita, Shuu; Tagai, Chihiro; Shiraishi, Takayuki; Miyaji, Kazuyuki; Iwamuro, Shawichi

    2013-10-01

    We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.

  5. Nanoemulsion Therapy for Burn Wounds Is Effective as a Topical Antimicrobial Against Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Dolgachev, Vladislav A; Ciotti, Susan M; Eisma, Rone; Gracon, Stephen; Wilkinson, J Erby; Baker, James R; Hemmila, Mark R

    2016-01-01

    The aim of this study is to investigate the antimicrobial efficacy of two different nanoemulsion (NE) formulations against Gram-positive and Gram-negative bacteria in an in vivo rodent scald burn model. Male Sprague-Dawley rats were anesthetized and received a partial-thickness scald burn. Eight hours after burn injury, the wound was inoculated with 1 × 10(8) colony-forming units of Pseudomonas aeruginosa or Staphylococcus aureus. Treatment groups consisted of two different NE formulations (NB-201 and NB-402), NE vehicle, or saline. Topical application of the treatment was performed at 16 and 24 hours after burn injury. Animals were killed 32 hours after burn injury, and skin samples were obtained for quantitative wound culture and determination of dermal inflammation markers. In a separate set of experiments, burn wound progression was measured histologically after 72 hours of treatment. Both NE formulations (NB-201 and NB-402) significantly reduced burn wound infections with either P. aeruginosa or S. aureus and decreased median bacterial counts at least three logs when compared with animals with saline applications (p < .0001). NB-201 and NB-402 also decreased dermal neutrophil recruitment and sequestration into the wound as measured by myeloperoxidase (MPO) assay and histopathology (p < .05). In addition, there was a decrease in the proinflammatory dermal cytokines (interleukin 1-beta [IL-1β], IL-6, and tumor necrosis factor alpha [TNF-α]) and the neutrophil chemoattractants CXCL1 and CXCL2. Using histologic examination, it was found that both NB-201 and NB-402 appeared to suppress burn wound progression 72 hours after injury. Topically applied NB-201 and NB-402 are effective in decreasing Gram-positive and Gram-negative bacteria growth in burn wounds, reducing inflammation, and abrogating burn wound progression.

  6. Differential sensitivity of aerobic gram-positive and gram-negative microorganisms to 2,4,6-trinitrotoluene (TNT) leads to dissimilar growth and TNT transformation: Results of soil and pure culture studies

    SciTech Connect

    Fuller, M.E.; Manning, J.F. Jr.

    1996-07-30

    The effects of 2,4,6-trinitrotoluene (TNT) on indigenous soil populations and pure bacterial cultures were examined. The number of colony-forming units (CFU) appearing when TNT-contaminated soil was spread on 0.3% molasses plates decreased by 50% when the agar was amended with 67 {mu}g TNT mL{sup -1}, whereas a 99% reduction was observed when uncontaminated soil was plated. Furthermore, TNT-contaminated soil harbored a greater number of organisms able to grow on plates amended with greater than 10 {mu}g TNT mL{sup -1}. The percentage of gram-positive isolates was markedly less in TNT-contaminated soil (7%; 2 of 30) than in uncontaminated soil (61%; 20 of 33). Pseudomonas aeruginosa, Pseudomonas corrugate, Pseudomonasfluorescens and Alcaligenes xylosoxidans made up the majority of the gram-negative isolates from TNT-contaminated soil. Gram-positive isolates from both soils demonstrated marked growth inhibition when greater than 8-16 {mu}g TNT mL{sup -1} was present in the culture media. Most pure cultures of known aerobic gram-negative organisms readily degraded TNT and evidenced net consumption of reduced metabolites. However, pure cultures of aerobic gram-positive bacteria were sensitive to relatively low concentrations of TNT as indicated by the 50% reduction in growth and TNT transformation which was observed at approximately 10 {mu}g TNT mL{sup -1}. Most non-sporeforming gram-positive organisms incubated in molasses media amended with 80 {mu}g TNT mL{sup -1} or greater became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 {mu}g TNT mL{sup -1}, indicating that TNT sensitivity is likely linked to cell growth. These results indicate that gram-negative organisms are most likely responsible for any TNT transformation in contaminated soil, due to their relative insensitivity to high TNT concentrations and their ability to transform TNT.

  7. Induction of nitric oxide production by polyosides from the cell walls of Streptococcus mutans OMZ 175, a gram-positive bacterium, in the rat aorta.

    PubMed Central

    Martin, V; Kleschyov, A L; Klein, J P; Beretz, A

    1997-01-01

    The cardiovascular dysfunctions associated with septic shock induced by gram-negative or gram-positive bacteria (gram-positive or gram-negative septic shock) are comparable. In gram-negative septic shock, lipopolysaccharide (LPS) induces nitric oxide (NO) synthase, which contributes to the vascular hypotension and hyporeactivity to vasoconstrictors. The role of NO in gram-positive septic shock and the nature of the bacterial wall components responsible for the vascular effects of gram-positive bacteria are not well known. This study investigated the vascular effects of cell wall serotype polyosides, rhamnose glucose polymers (RGPs), from Streptococcus mutans, in comparison with lipoteichoic acid (LTA) from Staphylococcus aureus, on the induction of NO synthase activity in the rat aorta. We show that 10 microg of both RGPs and LTA per ml induced hyporeactivity to noradrenaline, L-arginine-induced relaxation, increases of 2.2- and 7.8-fold, respectively, of cyclic GMP production, and increases of 7- and 12-fold in nitrite release. All of these effects appeared after several hours of incubation and were inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Electron paramagnetic resonance spin trapping experiments demonstrated directly that RGPs and LTA induced NO overproduction (four- to eightfold, respectively) in rat aortic rings; this production was inhibited by L-NAME and prevented by dexamethasone. These results demonstrate directly the induction of NO production in vascular tissue by LTA and show that another, chemically different component of gram-positive bacteria can also have these properties. This result suggests that different components of the gram-positive bacterial wall could be implicated in the genesis of cardiovascular dysfunctions observed in gram-positive septic shock. PMID:9169734

  8. Phoenix 100 versus Vitek 2 in the identification of gram-positive and gram-negative bacteria: a comprehensive meta-analysis.

    PubMed

    Chatzigeorgiou, Kalliopi-Stavroula; Sergentanis, Theodoros N; Tsiodras, Sotirios; Hamodrakas, Stavros J; Bagos, Pantelis G

    2011-09-01

    Phoenix 100 and Vitek 2 (operating with the current colorimetric cards) are commonly used in hospital laboratories for rapid identification of microorganisms. The present meta-analysis aims to evaluate and compare their performance on Gram-positive and Gram-negative bacteria. The MEDLINE database was searched up to October 2010 for the retrieval of relevant articles. Pooled correct identification rates were derived from random-effects models, using the arcsine transformation. Separate analyses were conducted at the genus and species levels; subanalyses and meta-regression were undertaken to reveal meaningful system- and study-related modifiers. A total of 29 (6,635 isolates) and 19 (4,363 isolates) articles were eligible for Phoenix and colorimetric Vitek 2, respectively. No significant differences were observed between Phoenix and Vitek 2 either at the genus (97.70% versus 97.59%, P = 0.919) or the species (92.51% versus 88.77%, P = 0.149) level. Studies conducted with conventional comparator methods tended to report significantly better results compared to those using molecular reference techniques. Speciation of Staphylococcus aureus was significantly more accurate in comparison to coagulase-negative staphylococci by both Phoenix (99.78% versus 88.42%, P < 0.00001) and Vitek 2 (98.22% versus 91.89%, P = 0.043). Vitek 2 also reached higher correct identification rates for Gram-negative fermenters versus nonfermenters at the genus (99.60% versus 95.90%, P = 0.004) and the species (97.42% versus 84.85%, P = 0.003) level. In conclusion, the accuracy of both systems seems modified by underlying sample- and comparator method-related parameters. Future simultaneous assessment of the instruments against molecular comparator procedures may facilitate interpretation of the current observations.

  9. Quantifying Glosair™ 400 efficacy for surface disinfection of American Type Culture Collection strains and micro-organisms recently isolated from intensive care unit patients.

    PubMed

    Herruzo, R; Vizcaíno, M J; Herruzo, I

    2014-07-01

    Microbial contamination of hospital surfaces may be a source of infection for hospitalized patients. We evaluated the efficacy of Glosair™ 400 against two American Type Culture Collection strains and 18 clinical isolates, placed on glass germ-carriers. Carriers were left to air-dry for 60 min and then exposed to a cycle before detection of any surviving micro-organisms. Antibiotic-susceptible Gram-negative bacilli were less susceptible (although not significantly) to this technique than resistant Gram-negative bacilli or Gram-positive cocci and yeasts (3, 3.4 and 4.6 log10 reduction, respectively). In conclusion, in areas that had not been cleaned, aerosolized hydrogen peroxide obtained >3 log10 mean destruction of patients' micro-organisms.

  10. Efficacy of the New Lantibiotic NAI-107 in Experimental Infections Induced by Multidrug-Resistant Gram-Positive Pathogens▿

    PubMed Central

    Jabés, Daniela; Brunati, Cristina; Candiani, GianPaolo; Riva, Simona; Romanó, Gabriella; Donadio, Stefano

    2011-01-01

    NAI-107 is a novel lantibiotic active against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus (GISA), and vancomycin-resistant enterococci (VRE). The aim of this study was to evaluate the in vivo efficacy of NAI-107 in animal models of severe infection. In acute lethal infections induced with a penicillin-intermediate Streptococcus pneumoniae strain in immunocompetent mice, or with MRSA, GISA, and VRE strains in neutropenic mice, the 50% effective dose (ED50) values of NAI-107 were comparable or lower than those of reference compounds, irrespective of the strain and immune status (0.51 to 14.2 mg/kg of body weight for intravenous [i.v.] NAI-107, 5.1 to 22.4 for oral linezolid, and 22.4 for subcutaneous [s.c.] vancomycin). In the granuloma pouch model induced in rats with a MRSA strain, intravenous NAI-107 showed a dose-proportional bactericidal activity that, at a single 40-mg/kg dose, compared with 2 20-mg/kg doses at a 12-h or 24-h interval, caused a 3-log10-CFU/ml reduction of viable MRSA in exudates that persisted for more than 72 h. Rat endocarditis was induced with a MRSA strain and treated for five consecutive days. In a first experiment, using 5, 10, or 20 mg/kg/day, and in a second experiment, when 10 mg/kg at 12-h intervals was compared to 20 mg/kg/day, intravenous NAI-107 was effective in reducing the bacterial load in heart vegetations in a dose-proportional manner. Trough plasma levels, as determined on days 2 and 5, were several times higher than the NAI-107 minimal bactericidal concentration (MBC). NAI-107 binding to rat and human serum ranges between 93% and 98.6%. The rapid bactericidal activity of NAI-107 observed in vitro was thus confirmed by the efficacy in several models of experimental infection induced by Gram-positive pathogens, supporting further investigation of the compound. PMID:21220527

  11. Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles - a retrospective chart review.

    PubMed

    Mushtaq, Ammara; Chen, Derrick J; Strand, Gregory J; Dylla, Brenda L; Cole, Nicolynn C; Mandrekar, Jayawant; Patel, Robin

    2016-07-01

    With the advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), most Gram-positive rods (GPRs) are readily identified; however, their clinical relevance in blood cultures remains unclear. Herein, we assessed the clinical significance of GPRs isolated from blood and identified in the era of MALDI-TOF MS. A retrospective chart review of patients presenting to the Mayo Clinic, Rochester, MN, from January 1, 2013, to October 13, 2015, was performed. Any episode of a positive blood culture for a GPR was included. We assessed the number of bottles positive for a given isolate, time to positivity of blood cultures, patient age, medical history, interpretation of culture results by the healthcare team and whether infectious diseases consultation was obtained. We also evaluated the susceptibility profiles of a larger collection of GPRs tested in the clinical microbiology laboratory of the Mayo Clinic, Rochester, MN from January 1, 2013, to October 31, 2015. There were a total of 246 GPRs isolated from the blood of 181 patients during the study period. 56% (n = 101) were deemed contaminants by the healthcare team and were not treated; 33% (n = 59) were clinically determined to represent true bacteremia and were treated; and 8% (n = 14) were considered of uncertain significance, with patients prescribed treatment regardless. Patient characteristics associated with an isolate being treated on univariate analysis included younger age (P = 0.02), identification to the species level (P = 0.02), higher number of positive blood culture sets (P < 0.0001), lower time to positivity (P < 0.0001), immunosuppression (P = 0.03), and recommendation made by an infectious disease consultant (P = 0.0005). On multivariable analysis, infectious diseases consultation (P = 0.03), higher number of positive blood culture sets (P = 0.0005) and lower time to positivity (P = 0.03) were associated with an isolate being treated. 100, 83, 48 and 34% of GPRs

  12. Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles - a retrospective chart review.

    PubMed

    Mushtaq, Ammara; Chen, Derrick J; Strand, Gregory J; Dylla, Brenda L; Cole, Nicolynn C; Mandrekar, Jayawant; Patel, Robin

    2016-07-01

    With the advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), most Gram-positive rods (GPRs) are readily identified; however, their clinical relevance in blood cultures remains unclear. Herein, we assessed the clinical significance of GPRs isolated from blood and identified in the era of MALDI-TOF MS. A retrospective chart review of patients presenting to the Mayo Clinic, Rochester, MN, from January 1, 2013, to October 13, 2015, was performed. Any episode of a positive blood culture for a GPR was included. We assessed the number of bottles positive for a given isolate, time to positivity of blood cultures, patient age, medical history, interpretation of culture results by the healthcare team and whether infectious diseases consultation was obtained. We also evaluated the susceptibility profiles of a larger collection of GPRs tested in the clinical microbiology laboratory of the Mayo Clinic, Rochester, MN from January 1, 2013, to October 31, 2015. There were a total of 246 GPRs isolated from the blood of 181 patients during the study period. 56% (n = 101) were deemed contaminants by the healthcare team and were not treated; 33% (n = 59) were clinically determined to represent true bacteremia and were treated; and 8% (n = 14) were considered of uncertain significance, with patients prescribed treatment regardless. Patient characteristics associated with an isolate being treated on univariate analysis included younger age (P = 0.02), identification to the species level (P = 0.02), higher number of positive blood culture sets (P < 0.0001), lower time to positivity (P < 0.0001), immunosuppression (P = 0.03), and recommendation made by an infectious disease consultant (P = 0.0005). On multivariable analysis, infectious diseases consultation (P = 0.03), higher number of positive blood culture sets (P = 0.0005) and lower time to positivity (P = 0.03) were associated with an isolate being treated. 100, 83, 48 and 34% of GPRs

  13. Dynamic NETosis is Carried Out by Live Neutrophils in Human and Mouse Bacterial Abscesses and During Severe Gram-Positive Infection

    PubMed Central

    Yipp, Bryan G.; Petri, Björn; Salina, Davide; Jenne, Craig N.; Scott, Brittney N. V.; Zbytnuik, Lori D.; Pittman, Keir; Asaduzzaman, Muhammad; Wu, Kaiyu; Meijndert, H. Christopher; Malawista, Stephen E.; de Boisfleury Chevance, Anne; Zhang, Kunyan; Conly, John; Kubes, Paul

    2013-01-01

    Neutrophil extracellular traps (NETs) are released, as neutrophils die in vitro, in a process requiring hours, leaving a temporal gap for invasive microbes to exploit. Functional neutrophils undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live PMN in vivo rapidly releasing NETs, which prevented bacterial dissemination. NETosis occurred during crawling thereby casting large areas of NETs. NET-releasing PMN developed diffuse decondensed nuclei ultimately becoming devoid of DNA. Cells with abnormal nuclei displayed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A combined requirement of Tlr2 and complement mediated opsonization tightly regulated NET release. Additionally live human PMN developed decondensed nuclei and formed NETS in vivo and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection, non-cell death NETosis occurs in vivo during Gram-positive infection in mice and humans. PMID:22922410

  14. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    NASA Astrophysics Data System (ADS)

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-07-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

  15. Antibacterial Activity of Stenotrophomonas maltophilia Endolysin P28 against both Gram-positive and Gram-negative Bacteria.

    PubMed

    Dong, Hongling; Zhu, Chaoyang; Chen, Jingyi; Ye, Xing; Huang, Yu-Ping

    2015-01-01

    Maltocin P28 is a phage-tail like bacteriocin produced by Stenotrophomonas maltophilia P28. The ORF8 of maltocin P28 gene cluster is predicted to encode an endolysin and we name it endolysin P28. Sequence analysis revealed that it contains the lysozyme_like superfamily conserved domain. Endolysin P28 has the four consensus motifs as that of Escherichia coli phage lambda gpR. In this study, endolysin P28 was expressed in E. coli BL21 (DE3) and purified with a C-terminal oligo-histidine tag. The antibacterial activity of endolysin P28 increased as the temperature rose from 25 to 45°C. Thermostability assays showed that endolysin P28 was stable up to 50°C, while its residual activity was reduced by 55% after treatment at 70°C for 30 min. Acidity and high salinity could enhance its antibacterial activity. Endolysin P28 exhibited a broad antibacterial activity against 14 out of 16 tested Gram-positive and Gram-negative bacteria besides S. maltophilia. Moreover, it could effectively lyse intact Gram-negative bacteria in the absence of ethylenediaminetetraacetic acid as an outer membrane permeabilizer. Therefore, the characteristics of endolysin P28 make it a potential therapeutic agent against multi-drug-resistant pathogens. PMID:26635765

  16. Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

    PubMed Central

    Wright, David P; Ulijasz, Andrew T

    2014-01-01

    Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

  17. Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer.

    PubMed

    Fernández-López, Cris; Bravo, Alicia; Ruiz-Cruz, Sofía; Solano-Collado, Virtu; Garsin, Danielle A; Lorenzo-Díaz, Fabián; Espinosa, Manuel

    2014-10-01

    Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmids are more frequently found in nature. In this sense, replication and mobilization can be considered important mechanisms influencing plasmid promiscuity. Here we review the currently available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed.

  18. Inhibition of various gram-positive and gram-negative bacteria growth on selenium nanoparticle coated paper towels.

    PubMed

    Wang, Qi; Larese-Casanova, Philip; Webster, Thomas J

    2015-01-01

    There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns. PMID:25926733

  19. Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

    PubMed

    San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

    2015-10-01

    This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain.

  20. Prediction of surface exposed proteins in Streptococcus pyogenes, with a potential application to other Gram-positive bacteria.

    PubMed

    Barinov, Aleksandr; Loux, Valentin; Hammani, Amal; Nicolas, Pierre; Langella, Philippe; Ehrlich, Dusko; Maguin, Emmanuelle; van de Guchte, Maarten

    2009-01-01

    The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria-host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host-environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane-associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram-positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.

  1. Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium.

    PubMed

    Hahn, Veronika; Sünwoldt, Katharina; Mikolasch, Annett; Schauer, Frieder

    2013-02-01

    Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself. PMID:22828982

  2. A zebrafish intelectin ortholog agglutinates both Gram-negative and Gram-positive bacteria with binding capacity to bacterial polysaccharide.

    PubMed

    Chen, Lei; Yan, Jie; Sun, Weiping; Zhang, Yan; Sui, Chao; Qi, Jing; Du, Yijun; Feng, Lijun

    2016-08-01

    Intelectins are glycan-binding lectins found in various species including cephalochordates, urochordates, fish, amphibians and mammals. But their detailed functions are not well studied in zebrafish which is a good model to study native immunity. In this study, we cloned a zebrafish intelectin ortholog, zebrafish intelectin 2 (zITLN2), which contains a conserved fibrinogen-related domain (FReD) in the N-terminus and the unique intelectin domain in the C-terminus. We examined the tissue distribution of zITLN2 in adult zebrafish and found that zITLN2 was expressed in various organs with the highest level in intestine. Like amphioxus intelectins, zITLN2 expression was upregulated in adult zebrafish infected with Staphylococcus aureus with the highest expression level at 12 h after challenge. Recombinant zITLN2 protein expressed in E. coli was able to agglutinate both Gram-negative and Gram-positive bacteria to similar degrees in a calcium-dependent manner. Furthermore, recombinant zITLN2 bound lipopolysaccharide (LPS) and peptidoglycan (PGN) comparably. Our work on zITLN2 provided further information to understand functions of this new family of lectins and the innate immunity in vertebrates. PMID:27329687

  3. Inhibition of various gram-positive and gram-negative bacteria growth on selenium nanoparticle coated paper towels.

    PubMed

    Wang, Qi; Larese-Casanova, Philip; Webster, Thomas J

    2015-01-01

    There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns.

  4. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    PubMed Central

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-01-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis. PMID:27457620

  5. Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer

    PubMed Central

    Fernández-López, Cris; Bravo, Alicia; Ruiz-Cruz, Sofía; Solano-Collado, Virtu; Garsin, Danielle A.; Lorenzo-Díaz, Fabián; Espinosa, Manuel

    2014-01-01

    Chapter summary Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmidsare more frequently found in nature. In this sense, replication and mobilization can be considered as important mechanisms influencing plasmid promiscuity. Here we review the present available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed. PMID:25606350

  6. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity.

    PubMed

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-01-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis. PMID:27457620

  7. Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis

    PubMed Central

    San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

    2015-01-01

    This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. PMID:26185055

  8. What are archaebacteria: life's third domain or monoderm prokaryotes related to gram-positive bacteria? A new proposal for the classification of prokaryotic organisms.

    PubMed

    Gupta, R S

    1998-08-01

    The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria. A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/ protein sequences. To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low-G+C Gram-positive bacteria are phylogenetically distinct from the high-G+C Gram-positive group and that the diderm prokaryotes (i.e. Gram-negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also

  9. Study of the comparative activity of piperacillin/tazobactam with currently available antibiotics against 8206 aerobic isolates

    PubMed Central

    Forward, Kevin R; Low, Donald E; Laverdiere, Michel; Rennie, Robert; Simor, Andrew E; Franks, Patricia A

    1997-01-01

    OBJECTIVES: To compare the activity of piperacillin-tazobactam with piperacillin and other parenterally administered antibiotics against aerobic Gram-negative bacilli and Gram-positive cocci isolated from across Canada, and to determine the prevalence of resistance mediated by extended-spectrum cephalosporinases. METHODS: Sixty-one laboratories participated. Disk diffusion testing was performed in accordance with methods outlined by the National Committee for Clinical Laboratory Standards. Susceptibilities were performed on 8206 strains. Escherichia coli and Klebsiella pneumoniae with reduced susceptibilities to third-generation cephalosporins were screened for extended-spectrum beta-lactamases (ESBLs). RESULTS: Piperacillin-tazobactam was active against 92% of the strains, piperacillin against 81% and ticarcillin-clavulanic acid against 88%. Few differences were observed in the relative susceptibility of strains from teaching or community hospitals, from different anatomic sites or from different regions of the country. Aerobic Gram-negative bacilli tested tended to be more susceptible to all the agents than was recently reported in a similar American study. Only 43% of Enterococcus faecium were susceptible to ampicillin and 42% to piperacillin piperacillin with and without tazobactam. Only two enterococcal strains were resistant to vancomycin, and 19 had intermediate zone sizes. Of the 10 strains of E coli and eight strains of K pneumoniae with reduced susceptibility to extended spectrum cephalosporins, only one demonstrated typical ESBL activity. CONCLUSIONS: Canadian aerobic Gram-positive cocci and Gram-negative bacilli remain highly susceptible to many currently available antibiotics. The findings confirm a broad spectrum of activity of piperacillin-tazobactam and indicate that the pattern of susceptibility is quite uniform from different body sites, in both teaching and community hospitals, and across the country. PMID:22514487

  10. Adsorption and Fenton regeneration of SBA-15 for di-(2-ethylhexyl) phthalate leached from PVC sheets by Gram-positive strains LHM1 and LHM2

    NASA Astrophysics Data System (ADS)

    Hwang, S.; Latorre, I.; Caban, M.; Soto, B.; Montalvo-Rodríguez, R.; Hernández-Maldonado, A.

    2012-12-01

    Bioleaching of Di-(2-ethylhexyl) phthalate (DEHP) from PVC sheets was studied with newly isolated, Gram-positive strains LHM1 and LHM2 capable of growing on DEHP as the sole carbon source. According to 16S rRNA gene analysis, strains LHM1 and LHM2 were closely related (more than 97% similarity) to Chryseomicrobium imtechense MW 10(T) and Lysinibacillus fusiformis NBRC 15717(T), respectively. The biodeteriorated PVC sheets by the strains LHM1 and LHM2 had thicker biofilm development. Despite their metabolic capability of degrading DEHP as the sole carbon source, the strains LHM1 and LHM2 did not metabolize all DEHP leached out of the PVC sheets. Thermogravimetric analysis (TGA) showed that the biodeterioration by strains LHM1 and LHM2 resulted in less amount of and weakly bonded DEHP present in PVC sheets, in comparison to the virgin PVC sheet. Therefore, PVC biodeterioration by strains LHM1 and LHM2 might play an important role in stability of PVC sheets and fate and effect of leached DEHP on the environmental receptors. In response to this, an advanced adsorption with SBA-15 was assessed as a potential alternative DEHP remediation with arsenic as a co-contaminant. SBA-15 had an excellent arsenic adsorption showing >90% arsenic removal when arsenic was present as a singular contaminant. Adsorption effectiveness was irrelevant to the solid/liquid (S/L) ratio. However, when arsenic was present together with DEHP, arsenic adsorption to bare SBA-15 was reduced by 10 - 40%, with lesser S/L ratio having greater arsenic removal. On the contrary, bare SBA-15 only adsorbed ~30% of DEHP on average. When DEHP was present as a co-solute with arsenic, DEHP adsorption to bare SBA-15 was increased. For SBA-15 regeneration, adsorbed arsenic was recovered with EDTA elution, whereas adsorbed DEHP was destructed with Fenton oxidation.

  11. Lactobacillus plantarum LB95 impairs the virulence potential of Gram-positive and Gram-negative food-borne pathogens in HT-29 and Vero cell cultures.

    PubMed

    Dutra, Virna; Silva, Ana Carla; Cabrita, Paula; Peres, Cidália; Malcata, Xavier; Brito, Luisa

    2016-01-01

    Listeria monocytogenes, Salmonella enterica and verocytotoxigenic Escherichia coli (VTEC) are amongst the most important agents responsible for food outbreaks occurring worldwide. In this work, two Lactobacillus spp. strains (LABs), Lactobacillus plantarum (LB95) and Lactobacillus paraplantarum (LB13), previously isolated from spontaneously fermenting olive brines, and two reference probiotic strains, Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, were investigated for their ability to attenuate the virulence of the aforementioned pathogens using animal cell culture assays. In competitive exclusion assays, the relative percentages of adhesion and invasion of S. enterica subsp. enterica serovar Enteritidis were significantly reduced when the human HT-29 cell line was previously exposed to LB95. The relative percentage of invasion by Listeria monocytogenes was significantly reduced when HT-29 cells were previously exposed to LB95. In the cytotoxicity assays, the cell-free supernatant of the co-culture (CFSC)of VTEC with LB95 accounted for the lowest value obtained amongst the co-cultures of VTEC with LABs, and was significantly lower than the value obtained with the co-culture of VTEC with the two probiotic reference strains. The cytotoxicity of CFSC of VTEC with both LB95 and LB13 exhibited values not significantly different from the cell-free supernatant of the nonpathogenic E. coli B strain. Our results suggested that LB95 may be able to attenuate the virulence of Gram-positive and Gram-negative food-borne pathogens; together with other reported features of these strains, our data reveal their possible use in probiotic foods due to their interesting potential in preventing enteric infections in humans.

  12. Lactobacillus plantarum LB95 impairs the virulence potential of Gram-positive and Gram-negative food-borne pathogens in HT-29 and Vero cell cultures.

    PubMed

    Dutra, Virna; Silva, Ana Carla; Cabrita, Paula; Peres, Cidália; Malcata, Xavier; Brito, Luisa

    2016-01-01

    Listeria monocytogenes, Salmonella enterica and verocytotoxigenic Escherichia coli (VTEC) are amongst the most important agents responsible for food outbreaks occurring worldwide. In this work, two Lactobacillus spp. strains (LABs), Lactobacillus plantarum (LB95) and Lactobacillus paraplantarum (LB13), previously isolated from spontaneously fermenting olive brines, and two reference probiotic strains, Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, were investigated for their ability to attenuate the virulence of the aforementioned pathogens using animal cell culture assays. In competitive exclusion assays, the relative percentages of adhesion and invasion of S. enterica subsp. enterica serovar Enteritidis were significantly reduced when the human HT-29 cell line was previously exposed to LB95. The relative percentage of invasion by Listeria monocytogenes was significantly reduced when HT-29 cells were previously exposed to LB95. In the cytotoxicity assays, the cell-free supernatant of the co-culture (CFSC)of VTEC with LB95 accounted for the lowest value obtained amongst the co-cultures of VTEC with LABs, and was significantly lower than the value obtained with the co-culture of VTEC with the two probiotic reference strains. The cytotoxicity of CFSC of VTEC with both LB95 and LB13 exhibited values not significantly different from the cell-free supernatant of the nonpathogenic E. coli B strain. Our results suggested that LB95 may be able to attenuate the virulence of Gram-positive and Gram-negative food-borne pathogens; together with other reported features of these strains, our data reveal their possible use in probiotic foods due to their interesting potential in preventing enteric infections in humans. PMID:26506821

  13. Characterization of Leuconostoc oenos Isolated from Oregon Wines †

    PubMed Central

    Izuagbe, Y. S.; Dohman, T. P.; Sandine, W. E.; Heatherbell, D. A.

    1985-01-01

    This study was designed to characterize isolates of Leuconostoc species from Oregon wines. Gram-positive cocci were isolated, and their biochemical properties and abilities to decompose malic acid were determined. All of the isolates were heterofermentative, catalase negative, and facultatively anaerobic and occurred in pairs and chains. They produced acid from glucose, fructose, mannose, ribose, cellobiose, trehalose, and salicin but not from sucrose or lactose. They did not produce ammonia from arginine or dextran from sucrose. They grew at pH values of less than 4 and in 10% ethanol. Most but not all strains produced lactic acid and carbon dioxide from malic acid, as determined by paper chromatography and respirometry, respectively. These malolactic bacteria were considered to be strains of Leuconostoc oenos. We compared these isolates with reference strains for relative growth at pH values of 4.0, 3.5, 3.0, and 2.8 at 22°C. The isolates were similar in their growth responses at the two highest pH levels. At pH 3.0 and 2.8, however, the strains failed to grow but revealed variable abilities to dissimilate malic acid. PMID:16346886

  14. The PECACE domain: a new family of enzymes with potential peptidoglycan cleavage activity in Gram-positive bacteria

    PubMed Central

    Pagliero, Estelle; Dideberg, Otto; Vernet, Thierry; Di Guilmi, Anne Marie

    2005-01-01

    Background The metabolism of bacterial peptidoglycan is a dynamic process, synthases and cleavage enzymes are functionally coordinated. Lytic Transglycosylase enzymes (LT) are part of multienzyme complexes which regulate bacterial division and elongation. LTs are also involved in peptidoglycan turnover and in macromolecular transport systems. Despite their central importance, no LTs have been identified in the human pathogen Streptococcus pneumoniae. We report the identification of the first putative LT enzyme in S. pneumoniae and discuss its role in pneumococcal peptidoglycan metabolism. Results Homology searches of the pneumococcal genome allowed the identification of a new domain putatively involved in peptidoglycan cleavage (PECACE, PEptidoglycan CArbohydrate Cleavage Enzyme). This sequence has been found exclusively in Gram-positive bacteria and gene clusters containing pecace are conserved among Streptococcal species. The PECACE domain is, in some instances, found in association with other domains known to catalyze peptidoglycan hydrolysis. Conclusions A new domain, PECACE, putatively involved in peptidoglycan hydrolysis has been identified in S. pneumoniae. The probable enzymatic activity deduced from the detailed analysis of the amino acid sequence suggests that the PECACE domain may proceed through a LT-type or goose lyzosyme-type cleavage mechanism. The PECACE function may differ largely from the other hydrolases already identified in the pneumococcus: LytA, LytB, LytC, CBPD and PcsB. The multimodular architecture of proteins containing the PECACE domain is another example of the many activities harbored by peptidoglycan hydrolases, which is probably required for the regulation of peptidoglycan metabolism. The release of new bacterial genomes sequences will probably add new members to the five groups identified so far in this work, and new groups could also emerge. Conversely, the functional characterization of the unknown domains mentioned in this work

  15. Enhanced antibacterial and anti-biofilm activities of silver nanoparticles against Gram-negative and Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Kwon, Deug-Nam; Kim, Jin-Hoi

    2014-07-01

    Silver nanoparticles (AgNPs) have been used as antibacterial, antifungal, antiviral, anti-inflammtory, and antiangiogenic due to its unique properties such as physical, chemical, and biological properties. The present study was aimed to investigate antibacterial and anti-biofilm activities of silver nanoparticles alone and in combination with conventional antibiotics against various human pathogenic bacteria. Here, we show that a simple, reliable, cost effective and green method for the synthesis of AgNPs by treating silver ions with leaf extract of Allophylus cobbe. The A. cobbe-mediated synthesis of AgNPs (AgNPs) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Furthermore, the antibacterial and anti-biofilm activity of antibiotics or AgNPs, or combinations of AgNPs with an antibiotic was evaluated using a series of assays: such as in vitro killing assay, disc diffusion assay, biofilm inhibition, and reactive oxygen species generation in Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumonia. The results suggest that, in combination with antibiotics, there were significant antimicrobial and anti-biofilm effects at lowest concentration of AgNPs using a novel plant extract of A. cobbe, otherwise sublethal concentrations of the antibiotics. The significant enhancing effects were observed for ampicillin and vancomycin against Gram-negative and Gram-positive bacteria, respectively. These data suggest that combining antibiotics and biogenic AgNPs can be used therapeutically for the treatment of infectious diseases caused by bacteria. This study presented evidence of antibacterial and anti-biofilm effects of A. cobbe-mediated synthesis of AgNPs and their enhanced capacity against various human pathogenic bacteria. These results

  16. B. subtilis GS67 protects C. elegans from Gram-positive pathogens via fengycin-mediated microbial antagonism.

    PubMed

    Iatsenko, Igor; Yim, Joshua J; Schroeder, Frank C; Sommer, Ralf J

    2014-11-17

    Studies on Caenorhabditis elegans have provided detailed insight into host-pathogen interactions. Usually, the E. coli strain OP50 is used as food source for laboratory studies, but recent work has shown that a variety of bacteria have dramatic effects on C. elegans physiology, including immune responses. However, the mechanisms by which different bacteria impact worm resistance to pathogens are poorly understood. Although pathogen-specific immune priming is often discussed as a mechanism underlying such observations, interspecies microbial antagonism might represent an alternative mode of action. Here, we use several natural Bacillus strains to study their effects on nematode survival upon pathogen challenge. We show that B. subtilis GS67 persists in the C. elegans intestine and increases worm resistance to Gram-positive pathogens, suggesting that direct inhibition of pathogens might be the primary protective mechanism. Indeed, chemical and genetic analyses identified the lipopeptide fengycin as the major inhibitory molecule produced by B. subtilis GS67. Specifically, a fengycin-defective mutant of B. subtilis GS67 lost inhibitory activity against pathogens and was unable to protect C. elegans from infections. Furthermore, we found that purified fengycin cures infected worms in a dose-dependent manner, indicating that it acts as an antibiotic. Our results reveal a molecular mechanism for commensal-mediated C. elegans protection and highlight the importance of interspecies microbial antagonism for the outcome of animal-pathogen interactions. Furthermore, our work strengthens C. elegans as an in vivo model to reveal protective mechanisms of commensal bacteria, including those relevant to mammalian hosts.

  17. Unexpected Roles for Toll-Like Receptor 4 and TRIF in Intraocular Infection with Gram-Positive Bacteria

    PubMed Central

    Parkunan, Salai Madhumathi; Randall, C. Blake; Coburn, Phillip S.; Astley, Roger A.; Staats, Rachel L.

    2015-01-01

    Inflammation caused by infection with Gram-positive bacteria is typically initiated by interactions with Toll-like receptor 2 (TLR2). Endophthalmitis, an infection and inflammation of the posterior segment of the eye, can lead to vision loss when initiated by a virulent microbial pathogen. Endophthalmitis caused by Bacillus cereus develops as acute inflammation with infiltrating neutrophils, and vision loss is potentially catastrophic. Residual inflammation observed during B. cereus endophthalmitis in TLR2−/− mice led us to investigate additional innate pathways that may trigger intraocular inflammation. We first hypothesized that intraocular inflammation during B. cereus endophthalmitis would be controlled by MyD88- and TRIF-mediated signaling, since MyD88 and TRIF are the major adaptor molecules for all bacterial TLRs. In MyD88−/− and TRIF−/− mice, we observed significantly less intraocular inflammation than in eyes from infected C57BL/6J mice, suggesting an important role for these TLR adaptors in B. cereus endophthalmitis. These results led to a second hypothesis, that TLR4, the only TLR that signals through both MyD88 and TRIF signaling pathways, contributed to inflammation during B. cereus endophthalmitis. Surprisingly, B. cereus-infected TLR4−/− eyes also had significantly less intraocular inflammation than infected C57BL/6J eyes, indicating an important role for TLR4 in B. cereus endophthalmitis. Taken together, our results suggest that TLR4, TRIF, and MyD88 are important components of the intraocular inflammatory response observed in experimental B. cereus endophthalmitis, identifying a novel innate immune interaction for B. cereus and for this disease. PMID:26195555

  18. Target affinities of faropenem to and its impact on the morphology of gram-positive and gram-negative bacteria.

    PubMed

    Dalhoff, A; Nasu, T; Okamoto, K

    2003-07-01

    Faropenem is a new oral beta-lactam antibiotic unique from carbapenems and other available beta-lactams. Determinants of the in vitro activity of beta-lactam antibiotics include affinity to penicillin-binding proteins (PBPs) and beta-lactamase stability. In this study, the binding affinity of faropenem to various PBPs and its impact on the morphology of Staphylococcus aureus and Escherichia coli were evaluated. In general, faropenem demonstrated high binding affinity to high-molecular-weight PBPs but low affinity to low-molecular-weight PBPs. In S. aureus and Streptococcus pneumoniae, faropenem exhibited high binding affinity to PBP1, followed by PBP3 and PBP2. In E. coli, faropenem showed the highest affinity for PBP2, followed by PBP1A, PBP1B, PBP3 and PBP4. In Proteus vulgaris, binding was highest to PBP4, followed by PBP1A, PBP2 and PBP3. In Serratia marcescens, faropenem bound preferentially to PBP2 and PBP4. Exposure of S. aureus to faropenem at minimum inhibitory concentrations (MICs) of 1/8 or 1/4 resulted in irregular septum formation. At 1x MIC or higher, a larger number of lysed cells were observed. Exposure of E. coli to 1/8x MIC or 1/4x MIC also induced changes in cellular shape; the normal rod-shaped form changed to a spherical form in a time-dependent manner. After exposure of E. coli to 1x MIC for 2 h, bulging-shaped E. coli cells were observed and after 4 h of exposure cell lysis was demonstrated. In the presence of 4x MIC, spheroplast-like forms and cell lysis were observed. The morphological changes triggered by faropenem are in agreement with the PBP binding affinities reported. Thus, the high binding affinities of faropenem to PBPs from gram-negative and gram-positive bacteria are mirrored by its pronounced and concentration-dependent bactericidal effect. PMID:12886052

  19. Cellular fatty acid composition as an adjunct to the identification of asporogenous, aerobic gram-positive rods.

    PubMed

    Bernard, K A; Bellefeuille, M; Ewan, E P

    1991-01-01

    Cellular fatty acid (CFA) compositions of 561 asporogenous, aerobic gram-positive rods were analyzed by gas-liquid chromatography as an adjunct to their identification when grown on blood agar at 35 degrees C. The organisms could be divided into two groups. In the first group (branched-chain type), which included coryneform CDC groups A-3, A-4, and A-5; some strains of B-1 and B-3; "Corynebacterium aquaticum"; Brevibacterium liquefaciens; Rothia dentocariosa; and Listeria spp., the rods had sizable quantities of antiesopentadecanoic (Ca15:0) and anteisoheptadecanoic (Ca17:0) acids. Other species with these types of CFA included B. acetylicum, which contained large amounts of isotridecanoic (Ci13:0) and anteisotridecanoic (Ca13:0) acids. CFAs useful for distinguishing among Jonesia denitrificans, Oerskovia spp., some strains of CDC groups B-1 and B-3, Kurthia spp., and Propionibacterium avidum were hexadecanoic (C 16:0) acid, isopentadecanoic (Ci15:0) acid, and Ca15:0). The second group (straight-chained type), which included Actinomyces pyogenes; Arcanobacterium haemolyticum; C. bovis; C. cystitidis; C. diphtheriae; C. flavescens, "C. gentalium"; C. jeikeium; C. kutscheri; C. matruchotii; C .minutissimum; C. mycetoides; C. pilosum; C. pseudodiphtheriticum; "C. pseudogenitalium"; C. pseudotuberculosis; C. renale; CDC groups 1, 2, ANF-1, D-2, E, F-1, F-2, G-1, G-2, and I-2; C. striatum; "C. tuberculostearicum"; C. ulcerans; C. vitarumen; C. xerosis; and Erysipelothrix rhusiopathiae, was typified by significant quantities of hexadecanoic (C16:0) and oleic acids (C18:cis9), with differences in the amounts of linoleic acid (C18:2), stearic acid (C18:0), an unnamed peak (equivalent chain length, 14.966), and small quantities of other known saturated and unsaturated fatty acids. CFA composition of these organisms was sufficiently discriminatory to assist in classification but could not be used as the sole means of identification.

  20. Acylation of SC4 dodecapeptide increases bactericidal potency against Gram-positive bacteria, including drug-resistant strains.

    PubMed Central

    Lockwood, Nathan A; Haseman, Judith R; Tirrell, Matthew V; Mayo, Kevin H

    2004-01-01

    We have conjugated dodecyl and octadecyl fatty acids to the N-terminus of SC4, a potently bactericidal, helix-forming peptide 12-mer (KLFKRHLKWKII), and examined the bactericidal activities of the resultant SC4 'peptide-amphiphile' molecules. SC4 peptide-amphiphiles showed up to a 30-fold increase in bactericidal activity against Gram-positive strains (Staphylococcus aureus, Streptococcus pyogenes and Bacillus anthracis), including S. aureus strains resistant to conventional antibiotics, but little or no increase in bactericidal activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fatty acid conjugation improved endotoxin (lipopolysaccharide) neutralization by 3- to 6-fold. Although acylation somewhat increased lysis of human erythrocytes, it did not increase lysis of endothelial cells, and the haemolytic effects occurred at concentrations 10- to 100-fold higher than those required for bacterial cell lysis. For insight into the mechanism of action of SC4 peptide-amphiphiles, CD, NMR and fluorescence spectroscopy studies were performed in micelle and liposome models of eukaryotic and bacterial cell membranes. CD indicated that SC4 peptide-amphiphiles had the strongest helical tendencies in liposomes mimicking bacterial membranes, and strong membrane integration of the SC4 peptide-amphiphiles was observed using tryptophan fluorescence spectroscopy under these conditions; results that correlated with the increased bactericidal activities of SC4 peptide-amphiphiles. NMR structural analysis in micelles demonstrated that the two-thirds of the peptide closest to the fatty acid tail exhibited a helical conformation, with the positively-charged side of the amphipathic helix interacting more with the model membrane surface. These results indicate that conjugation of a fatty acid chain to the SC4 peptide enhances membrane interactions, stabilizes helical structure in the membrane-bound state and increases bactericidal potency. PMID:14609430

  1. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    PubMed Central

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

    Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID

  2. Enhanced antibacterial and anti-biofilm activities of silver nanoparticles against Gram-negative and Gram-positive bacteria

    PubMed Central

    2014-01-01

    Silver nanoparticles (AgNPs) have been used as antibacterial, antifungal, antiviral, anti-inflammtory, and antiangiogenic due to its unique properties such as physical, chemical, and biological properties. The present study was aimed to investigate antibacterial and anti-biofilm activities of silver nanoparticles alone and in combination with conventional antibiotics against various human pathogenic bacteria. Here, we show that a simple, reliable, cost effective and green method for the synthesis of AgNPs by treating silver ions with leaf extract of Allophylus cobbe. The A. cobbe-mediated synthesis of AgNPs (AgNPs) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Furthermore, the antibacterial and anti-biofilm activity of antibiotics or AgNPs, or combinations of AgNPs with an antibiotic was evaluated using a series of assays: such as in vitro killing assay, disc diffusion assay, biofilm inhibition, and reactive oxygen species generation in Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumonia. The results suggest that, in combination with antibiotics, there were significant antimicrobial and anti-biofilm effects at lowest concentration of AgNPs using a novel plant extract of A. cobbe, otherwise sublethal concentrations of the antibiotics. The significant enhancing effects were observed for ampicillin and vancomycin against Gram-negative and Gram-positive bacteria, respectively. These data suggest that combining antibiotics and biogenic AgNPs can be used therapeutically for the treatment of infectious diseases caused by bacteria. This study presented evidence of antibacterial and anti-biofilm effects of A. cobbe-mediated synthesis of AgNPs and their enhanced capacity against various human pathogenic bacteria. These results

  3. B. subtilis GS67 protects C. elegans from Gram-positive pathogens via fengycin-mediated microbial antagonism.

    PubMed

    Iatsenko, Igor; Yim, Joshua J; Schroeder, Frank C; Sommer, Ralf J

    2014-11-17

    Studies on Caenorhabditis elegans have provided detailed insight into host-pathogen interactions. Usually, the E. coli strain OP50 is used as food source for laboratory studies, but recent work has shown that a variety of bacteria have dramatic effects on C. elegans physiology, including immune responses. However, the mechanisms by which different bacteria impact worm resistance to pathogens are poorly understood. Although pathogen-specific immune priming is often discussed as a mechanism underlying such observations, interspecies microbial antagonism might represent an alternative mode of action. Here, we use several natural Bacillus strains to study their effects on nematode survival upon pathogen challenge. We show that B. subtilis GS67 persists in the C. elegans intestine and increases worm resistance to Gram-positive pathogens, suggesting that direct inhibition of pathogens might be the primary protective mechanism. Indeed, chemical and genetic analyses identified the lipopeptide fengycin as the major inhibitory molecule produced by B. subtilis GS67. Specifically, a fengycin-defective mutant of B. subtilis GS67 lost inhibitory activity against pathogens and was unable to protect C. elegans from infections. Furthermore, we found that purified fengycin cures infected worms in a dose-dependent manner, indicating that it acts as an antibiotic. Our results reveal a molecular mechanism for commensal-mediated C. elegans protection and highlight the importance of interspecies microbial antagonism for the outcome of animal-pathogen interactions. Furthermore, our work strengthens C. elegans as an in vivo model to reveal protective mechanisms of commensal bacteria, including those relevant to mammalian hosts. PMID:25448001

  4. Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria.

    PubMed

    Fischer, Carol L; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2012-03-01

    There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity--the sphingoid bases D-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid--against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. D-sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.

  5. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

    PubMed Central

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M.; Nichols, Wright W.

    2015-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  6. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria.

    PubMed

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

    Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l(-1) O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l(-1). However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID

  7. Antibacterial Activity of Sphingoid Bases and Fatty Acids against Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    Fischer, Carol L.; Drake, David R.; Dawson, Deborah V.; Blanchette, Derek R.; Brogden, Kim A.

    2012-01-01

    There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity—the sphingoid bases d-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid—against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. d-Sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection. PMID:22155833

  8. Unravelling a vicious circle: animal feed marketed in Costa Rica contains irregular concentrations of tetracyclines and abundant oxytetracycline-resistant Gram-positive bacteria.

    PubMed

    Granados-Chinchilla, Fabio; Alfaro, Margarita; Chavarría, Guadalupe; Rodríguez, César

    2014-01-01

    Diverse tetracyclines are used to prevent and control bacterial infections in livestock and farmed fish. These drugs are administered through the diet, but farmers seldom check whether feed contains antibiotic-resistant bacteria that may colonise their crops or transfer their resistance traits to species of veterinary relevance. To examine whether antibiotic dosage defines the abundance of antibiotic-resistant bacteria in animal feed, we determined the concentration of parental compounds and epimers of oxytetracycline (OTC), doxycycline, tetracycline and chlortetracycline, as well as the abundance and resistance level of OTC-resistant bacteria in samples of fish (n = 21), poultry (n = 21), swine (n = 21), and shrimp feed (n = 21) marketed in Costa Rica. Fish feed contained the highest amounts of tetracyclines (119-8365 mg kg(-1)) and the largest proportion of bacteria resistant to 10 μg ml(-1) (1.8-92.4%) or 100 μg ml(-1) of OTC (12.5-63.8%). Poultry (78-438 mg kg(-1)) and swine (41-1076 mg kg(-1)) feed had intermediate concentrations of tetracyclines and OTC-resistant bacteria (0.2-66% and 0.3-49%, respectively), whereas shrimp feed showed the lowest amounts of tetracyclines (21.5-50.3 mg kg(-1)), no OTC and no culturable OTC-resistant bacteria. In line with these results, the MIC50 of OTC for 150 isolates from fish and poultry feed was > 256 µg ml(-1), while that of 150 bacteria isolated from swine feed was 192 µg ml(-1). Phenotypic tests, fatty acid profiles and proteotypic analyses by matrix-assisted laser desorption/ionisation-time of flight mass-spectroscopy revealed that most OTC-resistant isolates were Gram-positive bacteria of low G+C% content from the genera Staphylococcus and Bacillus. Clear correlations between OTC dosage and feed colonisation with OTC-resistant bacteria were seen in medicated feed for fish (r = 0.179-0.651). Nonetheless, some unmedicated feed for fish, swine and poultry contained large populations of OTC-resistant bacteria

  9. A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

    PubMed Central

    Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

    2013-01-01

    We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

  10. Prior statin use and 90-day mortality in Gram-negative and Gram-positive bloodstream infection: a prospective observational study.

    PubMed

    Mehl, A; Harthug, S; Lydersen, S; Paulsen, J; Åsvold, B O; Solligård, E; Damås, J K; Edna, T-H

    2015-03-01

    In several studies on patients with bloodstream infection (BSI), prior use of statins has been associated with improved survival. Gram-positive and Gram-negative bacteria alert the innate immune system in different ways. We, therefore, studied whether the relation between prior statin use and 90-day total mortality differed between Gram-positive and Gram-negative BSI. We conducted a prospective observational cohort study of 1,408 adults with BSI admitted to Levanger Hospital between January 1, 2002, and December 31, 2011. Data on the use of statins and other medications at admission, comorbidities, functional status, treatment, and outcome were obtained from the patients' hospital records. The relation of statin use with 90-day mortality differed between Gram-negative and Gram-positive BSI (p-value for interaction 0.01). Among patients with Gram-negative BSI, statin users had significantly lower 90-day total mortality [odds ratio (OR) 0.42, 95 % confidence interval (CI) 0.23-0.75, p = 0.003]. The association remained essentially unchanged after adjusting for the effect of sex, age, functional status before the infection, and underlying diseases that were considered confounders (adjusted OR 0.38, 95 % CI 0.20-0.72, p = 0.003). A similar analysis of patients with Gram-positive BSI showed no association of statin use with mortality (adjusted OR 1.22, 95 % CI 0.69-2.17, p = 0.49). The present study suggests that prior statin use is associated with a lower 90-day total mortality in Gram-negative BSI, but not in Gram-positive BSI.

  11. Antimicrobial susceptibility of clinical isolates from earthquake victims in Wenchuan.

    PubMed

    Kang, M; Xie, Y; Mintao, C; Chen, Z; Chen, H; Fan, H; Chen, W; Guo, X

    2009-01-01

    On 12 May 2008, an earthquake measuring 8.0 on the Richter scale struck Wenchuan County, Sichuan, China. Between 12 May and 11 June, 1823 victims were hospitalized in West China Hospital. These patients were severely injured, and most of their wounds were contaminated. Here, the results of bacteriological identification and antibiotic susceptibility testing of 725 non-duplicate isolates from earthquake victims are presented. Gram-negative bacilli were most frequently isolated (71.3%). Only 18.9% of isolates were Gram-positive bacteria; Candida spp. accounted for 9.7%, and Gram-negative cocci for 0.1%. After anaerobic culture, four Clostridium sordellii strains and one Clostridium bifermentans strain were isolated from deep wounds. Specimen culture from earthquake victims revealed a spectrum of pathogens and antibiotic susceptibilities that was different from that usually encountered in West China Hospital, especially concerning methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producers, and multidrug-resistant (MDR) non-fermenting Gram-negative bacilli. The pathophysiology of the injuries in earthquake victims was different from that in the patients who were not earthquake victims. A combination of environmental bacteria with a high proportion of Gram-negative bacteria was often observed in the earthquake victims. Approximately 26% of all earthquake victims were shown to be carriers of MDR microorganisms. Therefore, appropriate microbiological assessment upon admission, and identification of patients to be put in quarantine, is of paramount importance.

  12. Antimicrobial susceptibility of clinical isolates from earthquake victims in Wenchuan.

    PubMed

    Kang, M; Xie, Y; Mintao, C; Chen, Z; Chen, H; Fan, H; Chen, W; Guo, X

    2009-01-01

    On 12 May 2008, an earthquake measuring 8.0 on the Richter scale struck Wenchuan County, Sichuan, China. Between 12 May and 11 June, 1823 victims were hospitalized in West China Hospital. These patients were severely injured, and most of their wounds were contaminated. Here, the results of bacteriological identification and antibiotic susceptibility testing of 725 non-duplicate isolates from earthquake victims are presented. Gram-negative bacilli were most frequently isolated (71.3%). Only 18.9% of isolates were Gram-positive bacteria; Candida spp. accounted for 9.7%, and Gram-negative cocci for 0.1%. After anaerobic culture, four Clostridium sordellii strains and one Clostridium bifermentans strain were isolated from deep wounds. Specimen culture from earthquake victims revealed a spectrum of pathogens and antibiotic susceptibilities that was different from that usually encountered in West China Hospital, especially concerning methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producers, and multidrug-resistant (MDR) non-fermenting Gram-negative bacilli. The pathophysiology of the injuries in earthquake victims was different from that in the patients who were not earthquake victims. A combination of environmental bacteria with a high proportion of Gram-negative bacteria was often observed in the earthquake victims. Approximately 26% of all earthquake victims were shown to be carriers of MDR microorganisms. Therefore, appropriate microbiological assessment upon admission, and identification of patients to be put in quarantine, is of paramount importance. PMID:19220339

  13. Quantification of Gram-positive bacteria: adaptation and evaluation of a preparation strategy using high amounts of clinical tissue

    PubMed Central

    2014-01-01

    Background A preparation method for quantification of bacteria in tissues is obligatory to reduce tissue mass, concentrate the target, purify, remove inhibitory substances and to achieve constant target recovery rates. No preparation method has been available until now for a high mass of tissue applicable for routine use and analytical veterinary diagnostics. Results This study describes an easy-to-use tissue preparation protocol to quantify Gram-positive bacteria from a large volume of tissue matrix. A previously published sample preparation method (Matrix-Lysis) from food science was successfully adapted for clinical use on tissues from pigs, including cerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissue. This tissue preparation method now permits quantification of pathogens from 5 g of organic matrix, which is a 20–200 fold increase by weight compared to other methods. It is based on solubilization of the sample matrix with either a chaotrope plus detergent or divalent salts as solubilization agents. The method was designed as a modular system, offering the possibility to change lysis buffers, according to tissue solubilization characteristics and the intended detection method (molecular or culture). Using Listeria monocytogenes as model organism, viable cell quantification or DNA extraction and quantitative real-time PCR were performed after Matrix-Lysis to determine recovery rates and detection limit (LOD). The adapted Matrix-Lysis protocol resulted in high recovery rates (mean value: 76% ± 39%) for all tested organs, except kidney, and recovery was constant over 5 log scales for all tested buffer systems. The LOD for Matrix-Lysis with subsequent plate count method (PCM) was as low as 1 CFU/5 g, while for qPCR based detection the LOD was 102 bacterial cell equivalents (BCE)/5 g for two buffer systems. Conclusions This tissue preparation is inexpensive and can be easily used for routine and analytical veterinary

  14. Conjunctival sac bacterial flora isolated prior to cataract surgery

    PubMed Central

    Suto, Chikako; Morinaga, Masahiro; Yagi, Tomoko; Tsuji, Chieko; Toshida, Hiroshi

    2012-01-01

    Objective To determine the trends of conjunctival sac bacterial flora isolated from patients prior to cataract surgery. Subjects and methods The study comprised 579 patients (579 eyes) who underwent cataract surgery. Specimens were collected by lightly rubbing the inferior palpebral conjunctival sac with a sterile cotton swab 2 weeks before surgery, and then cultured for isolation of bacteria and antimicrobial sensitivity testing. The bacterial isolates and percentage of drug-resistant isolates were compared among age groups and according to whether or not patients had diabetes mellitus, hyperlipidemia, dialysis therapy, oral steroid use, dry eye syndrome, or allergic conjunctivitis. Results The bacterial isolation rate was 39.2%. There were 191 strains of Gram-positive cocci, accounting for the majority of all isolates (67.0%), among which methicillin-sensitive coagulase-negative staphylococci was the most frequent (127 strains, 44.5%), followed by methicillin-resistant coagulase-negative staphylococci (37 strains, 12.7%). All 76 Gram-positive bacillary isolates (26.7%) were from the genus Corynebacterium. Among the 16 Gram-negative bacillary isolates (5.9%), the most frequent was Escherichia coli (1.0%). The bacterial isolation rate was higher in patients >60 years old, and was lower in patients with dry eye syndrome, patients under topical treatment for other ocular disorders, and patients with hyperlipidemia. There was no significant difference in bacterial isolation rate with respect to the presence/absence of diabetes mellitus, steroid therapy, dialysis, or a history of allergic conjunctivitis. Methicillin-resistant coagulase-negative staphylococci showed a significantly higher detection rate in diabetic patients than nondiabetic patients (20.3% versus 7.0%, P < 0.05). The percentage of all isolates resistant to levofloxacin, cefmenoxime, and tobramycin was 14.0%, 15.2%, and 17.9%, respectively, with no significant differences among these drugs. Conclusion

  15. Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

    PubMed Central

    De Kimpe, S. J.; Thiemermann, C.; Vane, J. R.

    1995-01-01

    ) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs. PMID:8719795

  16. [A new method for the disruption of cell walls of gram-positive bacteria and mycobacteria on the point of nucleic acid extraction: sand method].

    PubMed

    Şahin, Fikret; Kıyan, Mehmet; Karasartova, Djursun; Çalgın, M Kerem; Akhter, Shameem; Türegün Atasoy, Buse

    2016-01-01

    Nowadays molecular methods are widely used in the rapid diagnosis of infectious agents. Polymerase chain reaction (PCR) is the most preferred method for this purpose. Obtaining sufficient and pure DNA or RNA is important for the PCR. Different DNA extraction protocols such as phenol-chloroform, proteinase K, glass beads and boiling have been used successfully for DNA isolation from gram-negative bacteria. However since gram-positive bacteria have a thicker layer of peptidoglycan and mycobacteria have complex glycolipids in their cell walls, for the isolation of DNA or RNA from these microorganisms, the complex cell wall structure must be eliminated. For this purpose, the bacterial cell wall must be completely or partially removed forming sferoblast using lysostaphin in the Staphylococcus genus as gram-positive bacteria and using a chemical like cetyltrimethyl ammonium bromide for the Mycobacterium genus. In this study, we planned to use sand particles for the mechanical elimination of the cell wall without any need for chemicals and we called this procedure as "sand method". For the purpose of DNA extraction, the fine-grained sand was washed with ddH(2)O without losing small particles and then sterilized by autoclaving. For the purpose of RNA extraction; the sand was washed with ddH(2)O, incubated for 30 minutes with 10% HCl, and then autoclaved. A methicillin-resistant Staphylococcus aureus (MRSA) strain previously isolated and identified from a clinical specimen was mixed in 100 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. The MRSA-sand mix was treated with proteinase K and phenol-chloroform, and ethanol precipitation protocol was then followed for obtaining DNA. For comparison of the sand method with the other methods, the same amount of bacteria used in the sand method was incubated for one hour with lysostaphin, and then the proteinase K DNA extraction method were completed in the same

  17. [A new method for the disruption of cell walls of gram-positive bacteria and mycobacteria on the point of nucleic acid extraction: sand method].

    PubMed

    Şahin, Fikret; Kıyan, Mehmet; Karasartova, Djursun; Çalgın, M Kerem; Akhter, Shameem; Türegün Atasoy, Buse

    2016-01-01

    Nowadays molecular methods are widely used in the rapid diagnosis of infectious agents. Polymerase chain reaction (PCR) is the most preferred method for this purpose. Obtaining sufficient and pure DNA or RNA is important for the PCR. Different DNA extraction protocols such as phenol-chloroform, proteinase K, glass beads and boiling have been used successfully for DNA isolation from gram-negative bacteria. However since gram-positive bacteria have a thicker layer of peptidoglycan and mycobacteria have complex glycolipids in their cell walls, for the isolation of DNA or RNA from these microorganisms, the complex cell wall structure must be eliminated. For this purpose, the bacterial cell wall must be completely or partially removed forming sferoblast using lysostaphin in the Staphylococcus genus as gram-positive bacteria and using a chemical like cetyltrimethyl ammonium bromide for the Mycobacterium genus. In this study, we planned to use sand particles for the mechanical elimination of the cell wall without any need for chemicals and we called this procedure as "sand method". For the purpose of DNA extraction, the fine-grained sand was washed with ddH(2)O without losing small particles and then sterilized by autoclaving. For the purpose of RNA extraction; the sand was washed with ddH(2)O, incubated for 30 minutes with 10% HCl, and then autoclaved. A methicillin-resistant Staphylococcus aureus (MRSA) strain previously isolated and identified from a clinical specimen was mixed in 100 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. The MRSA-sand mix was treated with proteinase K and phenol-chloroform, and ethanol precipitation protocol was then followed for obtaining DNA. For comparison of the sand method with the other methods, the same amount of bacteria used in the sand method was incubated for one hour with lysostaphin, and then the proteinase K DNA extraction method were completed in the same

  18. Effectiveness of sequential intravenous-to-oral antibiotic switch therapy in hospitalized patients with gram-positive infection: the SEQUENCE cohort study.

    PubMed

    Rodriguez-Pardo, D; Pigrau, C; Campany, D; Diaz-Brito, V; Morata, L; de Diego, I C; Sorlí, L; Iftimie, S; Pérez-Vidal, R; García-Pardo, G; Larrainzar-Coghen, T; Almirante, B

    2016-08-01

    Switching from intravenous to oral antibiotic therapy may improve inpatient management and reduce hospital stays and the complications of intravenous treatment. We aimed to assess the effectiveness of intravenous-to-oral antibiotic switch therapy and an early discharge algorithm in hospitalized patients with gram-positive infection. We performed a prospective cohort study with a retrospective comparison cohort, recruited from eight tertiary, acute-care Spanish referral hospitals. All patients included had culture-confirmed methicillin-resistant gram-positive infection, or methicillin-susceptible gram-positive infection and beta-lactam allergy and had received intravenous treatment with glycopeptides, lipopeptides, or linezolid. The study comprised two cohorts: the prospective cohort to assess the effectiveness of a sequential intravenous-to-oral antibiotic switch algorithm and early discharge, and a retrospective cohort in which the algorithm had not been applied, used as the comparator. A total of 247 evaluable patients were included; 115 in the prospective and 132 in the retrospective cohort. Forty-five retrospective patients (34 %) were not changed to oral antibiotics, and 87 (66 %) were changed to oral antibiotics without following the proposed algorithm. The duration of hospitalization was significantly shorter in the prospective cohort compared to the retrospective group that did not switch to oral drugs (16.7 ± 18.7 vs 23 ± 13.4 days, P  < 0.001). No differences were observed regarding the incidence of catheter-related bacteraemia (4.4 % vs 2.6 %, P = 0.621). Our results suggest that an intravenous-to-oral antibiotic switch strategy is effective for reducing the length of hospital stay in selected hospitalized patients with gram-positive infection.

  19. A Functional dlt Operon, Encoding Proteins Required for Incorporation of d-Alanine in Teichoic Acids in Gram-Positive Bacteria, Confers Resistance to Cationic Antimicrobial Peptides in Streptococcus pneumoniae

    PubMed Central

    Kovács, Márta; Halfmann, Alexander; Fedtke, Iris; Heintz, Manuel; Peschel, Andreas; Vollmer, Waldemar; Hakenbeck, Regine; Brückner, Reinhold

    2006-01-01

    Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli ′lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides. PMID:16885447

  20. Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

    PubMed Central

    Hankins, Jessica V.; Madsen, James A.; Giles, David K.; Brodbelt, Jennifer S.; Trent, M. Stephen

    2012-01-01

    Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for d-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms. PMID:22589301

  1. Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

    PubMed

    Roller, C; Ludwig, W; Schleifer, K H

    1992-06-01

    An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.

  2. Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G + C content.

    PubMed

    Meier, H; Amann, R; Ludwig, W; Schleifer, K H

    1999-05-01

    Almost one thousand 16S rRNA sequences of Gram-positive bacteria with a low DNA G + C content from public databases were analyzed using the ARB software package. A signature region was identified between positions 354 and 371 (E. coli numbering) for the Bacillus sub-branch of the Gram-positive bacteria with a low DNA G + C content, the former orders Bacillales and Lactobacillales. Three oligonucleotide probes, namely LGC354A, LGC354B, and LGC354C, were designed to target this diagnostic site. Their fluorescent derivatives were suitable for whole cell detection by fluorescence in situ hybridization (FISH). Hybridization conditions were adjusted for differentiation of target and related non-target reference species. When applying FISH to whole bacterial cells in a sample of activated sludge from a communal wastewater treatment plant, members of the Bacillus sub-branch were detected at levels from 0.01% of cells in samples fixed with paraformaldehyde to over 8 percent in the same samples fixed with ethanol and treated with lysozyme. The problems of quantitative in situ analysis of Gram-positive bacteria with a low DNA G + C content in biofilm flocs are discussed and recommendations made. Members of the Bacillus sub-branch were detected in different abundances in activated sludge samples from different wastewater plants.

  3. Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria.

    PubMed

    Torcato, Inês M; Huang, Yen-Hua; Franquelim, Henri G; Gaspar, Diana; Craik, David J; Castanho, Miguel A R B; Troeira Henriques, Sónia

    2013-03-01

    BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required. PMID:23246973

  4. Predict Gram-Positive and Gram-Negative Subcellular Localization via Incorporating Evolutionary Information and Physicochemical Features Into Chou's General PseAAC.

    PubMed

    Sharma, Ronesh; Dehzangi, Abdollah; Lyons, James; Paliwal, Kuldip; Tsunoda, Tatsuhiko; Sharma, Alok

    2015-12-01

    In this study, we used structural and evolutionary based features to represent the sequences of gram-positive and gram-negative subcellular localizations. To do this, we proposed a normalization method to construct a normalize Position Specific Scoring Matrix (PSSM) using the information from original PSSM. To investigate the effectiveness of the proposed method we compute feature vectors from normalize PSSM and by applying support vector machine (SVM) and naïve Bayes classifier, respectively, we compared achieved results with the previously reported results. We also computed features from original PSSM and normalized PSSM and compared their results. The archived results show enhancement in gram-positive and gram-negative subcellular localizations. Evaluating localization for each feature, our results indicate that employing SVM and concatenating features (amino acid composition feature, Dubchak feature (physicochemical-based features), normalized PSSM based auto-covariance feature and normalized PSSM based bigram feature) have higher accuracy while employing naïve Bayes classifier with normalized PSSM based auto-covariance feature proves to have high sensitivity for both benchmarks. Our reported results in terms of overall locative accuracy is 84.8% and overall absolute accuracy is 85.16% for gram-positive dataset; and, for gram-negative dataset, overall locative accuracy is 85.4% and overall absolute accuracy is 86.3%.

  5. Real-world performance of a microarray-based rapid diagnostic for Gram-positive bloodstream infections and potential utility for antimicrobial stewardship.

    PubMed

    Aitken, Samuel L; Hemmige, Vagish S; Koo, Hoonmo L; Vuong, Nancy N; Lasco, Todd M; Garey, Kevin W

    2015-01-01

    The Verigene Gram-positive blood culture assay (BC-GP) is a microarray-based rapid diagnostic test, which includes targets for 12 bacterial species and 3 resistance determinants. We prospectively compared the diagnostic accuracy of the BC-GP to routine microbiologic methods and evaluated the potential of the BC-GP for antimicrobial stewardship programs. A total of 143 consecutive patients with Gram-positive bacteremia were included in the analysis. BC-GP correctly identified 127/128 (99.2%) of organisms from monomicrobial blood cultures and 9/14 (64.3%) from polymicrobial, including all methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Stewardship interventions were possible in 51.0% of patients, most commonly stopping or preventing unnecessary vancomycin or starting a targeted therapy. In Monte Carlo simulations, unnecessary antibiotics could be stopped at least 24 hours earlier in 65.6% of cases, and targeted therapy could be started at least 24 hours earlier in 81.2%. BC-GP is a potentially useful test for antibiotic stewardship in patients with Gram-positive bacteremia.

  6. Synthesis of well-dispersed silver nanorods of different aspect ratios and their antimicrobial properties against Gram positive and negative bacterial strains.

    PubMed

    Ojha, Animesh K; Forster, Stefan; Kumar, Sumeet; Vats, Siddharth; Negi, Sangeeta; Fischer, Ingo

    2013-12-20

    In the present contribution, we describe the synthesis of highly dispersed silver nanorods (NRs) of different aspect ratios using a chemical route. The shape and size of the synthesized NRs were characterized by Transmission Electron Microscopy (TEM) and UV-visible spectroscopy. Longitudinal and transverse absorptions bands confirm the rod type structure. The experimentally recorded UV-visible spectra of NRs solutions were fitted by using an expression of the extinction coefficient for rod like nano structures under the dipole approximation. Simulated and experimentally observed UV-visible spectra were compared to determine the aspect ratios (R) of NRs. The average values of R for NR1, NR2 and NR3 solutions are estimated to be 3.0 ± 0.1, 1.8 ± 0.1 and 1.2 ± 0.1, respectively. These values are in good agreement with those obtained by TEM micrographs. The silver NRs of known aspect ratios are used to study antimicrobial activities against B. subtilis (gram positive) and E. coli (gram negative) microbes. We observed that the NRs of intermediate aspect ratio (R = 1.8) have greater antimicrobial effect against both, B. subtilis (gram positive) and E. coli (gram negative). The NRs of aspect ratio, R = 3.0 has better antimicrobial activities against gram positive than on the gram negative.

  7. Multidrug-Resistance and Toxic Metal Tolerance of Medically Important Bacteria Isolated from an Aquaculture System

    PubMed Central

    Resende, Juliana Alves; Silva, Vânia L.; Fontes, Cláudia Oliveira; Souza-Filho, Job Alves; de Oliveira, Tamara Lopes Rocha; Coelho, Cíntia Marques; César, Dionéia Evangelista; Diniz, Cláudio Galuppo

    2012-01-01

    The use of antimicrobials and toxic metals should be considered carefully in aquaculture and surrounding environments. We aimed to evaluate medically relevant bacteria in an aquaculture system and their susceptibility to antimicrobials and toxic metals. Selective cultures for enterobacteria (ENT), non-fermenting Gram-negative rods (NFR) and Gram-positive cocci (GPC) were obtained from water samples collected in two different year seasons. The isolated bacteria were biochemically identified and antimicrobial and toxic metal susceptibility patterns were determined. Overall, 407 representative strains were recovered. In general, bacteria isolated from fish ponds showed higher multiple antibiotic resistance indices when compared to those isolated from a water-fed canal. Resistance to penicillin and azithromycin was observed more frequently in the GPC group, whereas resistance to ampicillin and ampicillin/sulbactam or gentamicin was observed more frequently in the ENT and NFR groups, respectively. All the isolated bacteria were tolerant to nickel, zinc, chromium and copper at high levels (≥1,024 μg mL−1), whereas tolerance to cadmium and mercury varied among the isolated bacteria (2–1,024 μg mL−1). Multidrug-resistant bacteria were more frequent and diverse in fish ponds than in the water-fed canal. A positive correlation was observed between antimicrobial resistance and metal tolerance. The data point out the need for water treatment associated with the aquaculture system. PMID:22972388

  8. Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria.

    PubMed

    Viera, Gustavo Hitzschky Fernandes; Mourão, Jozeanne Alves; Angelo, Angela Maria; Costa, Renata Albuquerque; Vieira, Regine Helena Silva dos Fernandes

    2010-01-01

    Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) and pods of soursop (Annona muricata) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 microL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis. Antibacterial activity (inhibition halo > 13 mm) against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus and V. cholerae, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated.

  9. Genetic characteristics of Streptococcus dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.).

    PubMed

    Tsai, M-A; Wang, P-C; Yoshida, T; Chen, S-C

    2015-12-01

    Disease outbreaks occurred during 2007-2013 in Taiwan with 2.5-10% mortality among the cage cultured cobia, Rachycentron canadum (L.), characterized by the presence of polyserositis, pericarditis and peritonitis. The micro-organisms isolated from internal organs were Gram-positive cocci. The isolates were confirmed as Streptococcus dysgalactiae by a polymerase chain reaction assay that yielded the expected specific 259 bp amplicon. Additionally, partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish was also compared and produced 100% sequence identity with S. dysgalactiae (GenBank accession number AB252398). The genetic characterization was then determined by pulsed-field gel electrophoresis (PFGE) analysis. Based on PFGE, the Apa I or Sma I digestion patterns of chromosomal DNA of these isolates were grouped into three main clusters. Taiwanese strains were divided into two clusters, and the tet(M) gene was detected in cluster 1 (pulsotypes: A1-A2 and S1-S3), but not in cluster 2 strains (pulsotypes: A3-A4 and S4-S5). Three Japanese strains from amberjack, Seriola dumerili (Risso), were grouped into cluster 3 (pulsotypes: A5-A7 and S6-S8) and displayed no mortality to cobia in the challenge experiment. Conversely, Taiwanese strains from cobia and snubnose pompano, Trachinotus blochii (L.), displayed a mortality rate of 50-87.5% in cobia. PMID:25087459

  10. Genetic characteristics of Streptococcus dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.).

    PubMed

    Tsai, M-A; Wang, P-C; Yoshida, T; Chen, S-C

    2015-12-01

    Disease outbreaks occurred during 2007-2013 in Taiwan with 2.5-10% mortality among the cage cultured cobia, Rachycentron canadum (L.), characterized by the presence of polyserositis, pericarditis and peritonitis. The micro-organisms isolated from internal organs were Gram-positive cocci. The isolates were confirmed as Streptococcus dysgalactiae by a polymerase chain reaction assay that yielded the expected specific 259 bp amplicon. Additionally, partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish was also compared and produced 100% sequence identity with S. dysgalactiae (GenBank accession number AB252398). The genetic characterization was then determined by pulsed-field gel electrophoresis (PFGE) analysis. Based on PFGE, the Apa I or Sma I digestion patterns of chromosomal DNA of these isolates were grouped into three main clusters. Taiwanese strains were divided into two clusters, and the tet(M) gene was detected in cluster 1 (pulsotypes: A1-A2 and S1-S3), but not in cluster 2 strains (pulsotypes: A3-A4 and S4-S5). Three Japanese strains from amberjack, Seriola dumerili (Risso), were grouped into cluster 3 (pulsotypes: A5-A7 and S6-S8) and displayed no mortality to cobia in the challenge experiment. Conversely, Taiwanese strains from cobia and snubnose pompano, Trachinotus blochii (L.), displayed a mortality rate of 50-87.5% in cobia.

  11. Oxidative stress-mediated selective antimicrobial ability of nano-VO2 against Gram-positive bacteria for environmental and biomedical applications

    NASA Astrophysics Data System (ADS)

    Li, Jinhua; Zhou, Huaijuan; Wang, Jiaxing; Wang, Donghui; Shen, Ruxiang; Zhang, Xianlong; Jin, Ping; Liu, Xuanyong

    2016-06-01

    Vanadium dioxide (VO2) is a unique thermochromic material as a result of its semiconductor-metal transition, holding great promise for energy-saving intelligent windows. Herein, pure nano-VO2 from discrete nanoparticles to continuous films were successfully deposited on quartz glass by controlling the sputtering parameters. It was demonstrated that, for Gram-positive S. aureus and S. epidermidis, the nano-VO2 could effectively disrupt bacteria morphology and membrane integrity, and eventually cause death. By contrast, the nano-VO2 did not exhibit significant toxicity towards Gram-negative E. coli and P. aeruginosa. To our knowledge, this is the first report on a selective antimicrobial effect of nano-VO2 materials on Gram-positive bacteria. Based on the experimental results, a plausible mechanism was proposed for the antimicrobial selectivity, which might originate from the different sensitivity of Gram-positive and Gram-negative bacteria to intracellular reactive oxygen species (ROS) level. Elevated intracellular ROS levels exceed the threshold that bacteria can self-regulate to maintain cellular redox homeostasis and thus cause oxidative stress, which can be alleviated by the intervention of glutathione (GSH) antioxidant. In addition, nano-VO2 did not produce significant cytotoxicity (hemolysis) against human erythrocytes within 12 h. Meanwhile, potential cytotoxicity against HIBEpiC revealed a time- and dose-dependent behavior that might be controlled and balanced by careful design. The findings in the present work may contribute to understanding the antimicrobial behavior of nano-VO2, and to expanding the new applications of VO2-based nanomaterials in environmental and biomedical fields.

  12. Rapid detection and differentiation of Gram-negative and Gram-positive pathogenic bacteria in urine using TaqMan probe.

    PubMed

    Shigemura, K; Shirakawa, T; Okada, H; Tanaka, K; Kamidono, S; Arakawa, S; Gotoh, A

    2005-03-01

    Urinary tract infection has been shown to be quite complicated and often difficult to diagnose and treat. For appropriate diagnosis, it is very important to find the correct Gram stain classification as soon as possible, especially in severe cases where there is a possibility of severe sepsis developing. In order to solve this problem, we developed a new method to detect a Gram stain of bacteria obtained from 1 ml of urine from urinary tract infection patients using a consensus real-time PCR protocol with a TaqMan probe that allows detection of spiked bacterial 16S DNA from urine. We extracted DNA of 55 urine samples obtained from patients with complicated urinary tract infection and at the same time performed urine culture testing. After DNA extraction, they were subjected to real-time PCR using a TaqMan discrimination system. Sixteen kinds of bacteria were cultured from the urine culture testing. Of these bacteria, eight were classified as Gram-positive bacteria and the other eight were classified as Gram-negative bacteria. Of the 55 samples, the TaqMan technique result showed 27 samples that were classified as Gram-negative bacteria; 11 samples that were Gram-positive, 10 that included both Gram-negative and -positive bacteria, and 7 that showed no amplification. The classifications of all samples corresponded exactly to those determined by urine culture testing. The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of Gram-positive or -negative bacteria in urine of urinary tract infection patients. This assay can differentiate those species tested, but whether the presence of other (untested) bacteria could lead to misinterpretation is unknown. For further investigation, it is important to test other (untested) bacteria in the near future.

  13. Rapid detection and differentiation of Gram-negative and Gram-positive pathogenic bacteria in urine using TaqMan probe.

    PubMed

    Shigemura, K; Shirakawa, T; Okada, H; Tanaka, K; Kamidono, S; Arakawa, S; Gotoh, A

    2005-03-01

    Urinary tract infection has been shown to be quite complicated and often difficult to diagnose and treat. For appropriate diagnosis, it is very important to find the correct Gram stain classification as soon as possible, especially in severe cases where there is a possibility of severe sepsis developing. In order to solve this problem, we developed a new method to detect a Gram stain of bacteria obtained from 1 ml of urine from urinary tract infection patients using a consensus real-time PCR protocol with a TaqMan probe that allows detection of spiked bacterial 16S DNA from urine. We extracted DNA of 55 urine samples obtained from patients with complicated urinary tract infection and at the same time performed urine culture testing. After DNA extraction, they were subjected to real-time PCR using a TaqMan discrimination system. Sixteen kinds of bacteria were cultured from the urine culture testing. Of these bacteria, eight were classified as Gram-positive bacteria and the other eight were classified as Gram-negative bacteria. Of the 55 samples, the TaqMan technique result showed 27 samples that were classified as Gram-negative bacteria; 11 samples that were Gram-positive, 10 that included both Gram-negative and -positive bacteria, and 7 that showed no amplification. The classifications of all samples corresponded exactly to those determined by urine culture testing. The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of Gram-positive or -negative bacteria in urine of urinary tract infection patients. This assay can differentiate those species tested, but whether the presence of other (untested) bacteria could lead to misinterpretation is unknown. For further investigation, it is important to test other (untested) bacteria in the near future. PMID:15750767

  14. Gram-positive siderophore-shuttle with iron-exchange from Fe-siderophore to apo-siderophore by Bacillus cereus YxeB.

    PubMed

    Fukushima, Tatsuya; Allred, Benjamin E; Sia, Allyson K; Nichiporuk, Rita; Andersen, Ulla N; Raymond, Kenneth N

    2013-08-20

    Small molecule iron-chelators, siderophores, are very important in facilitating the acquisition of Fe(III), an essential element for pathogenic bacteria. Many Gram-negative outer-membrane transporters and Gram-positive lipoprotein siderophore-binding proteins have been characterized, and the binding ability of outer-membrane transporters and siderophore-binding proteins for Fe-siderophores has been determined. However, there is little information regarding the binding ability of these proteins for apo-siderophores, the iron-free chelators. Here we report that Bacillus cereus YxeB facilitates iron-exchange from Fe-siderophore to apo-siderophore bound to the protein, the first Gram-positive siderophore-shuttle system. YxeB binds ferrioxamine B (FO, Fe-siderophore)/desferrioxamine B (DFO, apo-siderophore) in vitro. Disc-diffusion assays and growth assays using the yxeB mutant reveal that YxeB is responsible for importing the FO. Cr-DFO (a FO analog) is bound by YxeB in vitro and B. cereus imports or binds Cr-DFO in vivo. In vivo uptake assays using Cr-DFO and FO and growth assays using DFO and Cr-DFO show that B. cereus selectively imports and uses FO when DFO is present. Moreover, in vitro competition assays using Cr-DFO and FO clearly demonstrate that YxeB binds only FO, not Cr-DFO, when DFO is bound to the protein. Iron-exchange from FO to DFO bound to YxeB must occur when DFO is initially bound by YxeB. Because the metal exchange rate is generally first order in replacement ligand concentration, protein binding of the apo-siderophore acts to dramatically enhance the iron exchange rate, a key component of the Gram-positive siderophore-shuttle mechanism.

  15. Gram-positive siderophore-shuttle with iron-exchange from Fe-siderophore to apo-siderophore by Bacillus cereus YxeB

    PubMed Central

    Fukushima, Tatsuya; Allred, Benjamin E.; Sia, Allyson K.; Nichiporuk, Rita; Andersen, Ulla N.; Raymond, Kenneth N.

    2013-01-01

    Small molecule iron-chelators, siderophores, are very important in facilitating the acquisition of Fe(III), an essential element for pathogenic bacteria. Many Gram-negative outer-membrane transporters and Gram-positive lipoprotein siderophore-binding proteins have been characterized, and the binding ability of outer-membrane transporters and siderophore-binding proteins for Fe-siderophores has been determined. However, there is little information regarding the binding ability of these proteins for apo-siderophores, the iron-free chelators. Here we report that Bacillus cereus YxeB facilitates iron-exchange from Fe-siderophore to apo-siderophore bound to the protein, the first Gram-positive siderophore-shuttle system. YxeB binds ferrioxamine B (FO, Fe-siderophore)/desferrioxamine B (DFO, apo-siderophore) in vitro. Disc-diffusion assays and growth assays using the yxeB mutant reveal that YxeB is responsible for importing the FO. Cr-DFO (a FO analog) is bound by YxeB in vitro and B. cereus imports or binds Cr-DFO in vivo. In vivo uptake assays using Cr-DFO and FO and growth assays using DFO and Cr-DFO show that B. cereus selectively imports and uses FO when DFO is present. Moreover, in vitro competition assays using Cr-DFO and FO clearly demonstrate that YxeB binds only FO, not Cr-DFO, when DFO is bound to the protein. Iron-exchange from FO to DFO bound to YxeB must occur when DFO is initially bound by YxeB. Because the metal exchange rate is generally first order in replacement ligand concentration, protein binding of the apo-siderophore acts to dramatically enhance the iron exchange rate, a key component of the Gram-positive siderophore-shuttle mechanism. PMID:23924612

  16. Oxidative stress-mediated selective antimicrobial ability of nano-VO2 against Gram-positive bacteria for environmental and biomedical applications.

    PubMed

    Li, Jinhua; Zhou, Huaijuan; Wang, Jiaxing; Wang, Donghui; Shen, Ruxiang; Zhang, Xianlong; Jin, Ping; Liu, Xuanyong

    2016-06-01

    Vanadium dioxide (VO2) is a unique thermochromic material as a result of its semiconductor-metal transition, holding great promise for energy-saving intelligent windows. Herein, pure nano-VO2 from discrete nanoparticles to continuous films were successfully deposited on quartz glass by controlling the sputtering parameters. It was demonstrated that, for Gram-positive S. aureus and S. epidermidis, the nano-VO2 could effectively disrupt bacteria morphology and membrane integrity, and eventually cause death. By contrast, the nano-VO2 did not exhibit significant toxicity towards Gram-negative E. coli and P. aeruginosa. To our knowledge, this is the first report on a selective antimicrobial effect of nano-VO2 materials on Gram-positive bacteria. Based on the experimental results, a plausible mechanism was proposed for the antimicrobial selectivity, which might originate from the different sensitivity of Gram-positive and Gram-negative bacteria to intracellular reactive oxygen species (ROS) level. Elevated intracellular ROS levels exceed the threshold that bacteria can self-regulate to maintain cellular redox homeostasis and thus cause oxidative stress, which can be alleviated by the intervention of glutathione (GSH) antioxidant. In addition, nano-VO2 did not produce significant cytotoxicity (hemolysis) against human erythrocytes within 12 h. Meanwhile, potential cytotoxicity against HIBEpiC revealed a time- and dose-dependent behavior that might be controlled and balanced by careful design. The findings in the present work may contribute to understanding the antimicrobial behavior of nano-VO2, and to expanding the new applications of VO2-based nanomaterials in environmental and biomedical fields. PMID:27240639

  17. Oxidative stress-mediated selective antimicrobial ability of nano-VO2 against Gram-positive bacteria for environmental and biomedical applications.

    PubMed

    Li, Jinhua; Zhou, Huaijuan; Wang, Jiaxing; Wang, Donghui; Shen, Ruxiang; Zhang, Xianlong; Jin, Ping; Liu, Xuanyong

    2016-06-01

    Vanadium dioxide (VO2) is a unique thermochromic material as a result of its semiconductor-metal transition, holding great promise for energy-saving intelligent windows. Herein, pure nano-VO2 from discrete nanoparticles to continuous films were successfully deposited on quartz glass by controlling the sputtering parameters. It was demonstrated that, for Gram-positive S. aureus and S. epidermidis, the nano-VO2 could effectively disrupt bacteria morphology and membrane integrity, and eventually cause death. By contrast, the nano-VO2 did not exhibit significant toxicity towards Gram-negative E. coli and P. aeruginosa. To our knowledge, this is the first report on a selective antimicrobial effect of nano-VO2 materials on Gram-positive bacteria. Based on the experimental results, a plausible mechanism was proposed for the antimicrobial selectivity, which might originate from the different sensitivity of Gram-positive and Gram-negative bacteria to intracellular reactive oxygen species (ROS) level. Elevated intracellular ROS levels exceed the threshold that bacteria can self-regulate to maintain cellular redox homeostasis and thus cause oxidative stress, which can be alleviated by the intervention of glutathione (GSH) antioxidant. In addition, nano-VO2 did not produce significant cytotoxicity (hemolysis) against human erythrocytes within 12 h. Meanwhile, potential cytotoxicity against HIBEpiC revealed a time- and dose-dependent behavior that might be controlled and balanced by careful design. The findings in the present work may contribute to understanding the antimicrobial behavior of nano-VO2, and to expanding the new applications of VO2-based nanomaterials in environmental and biomedical fields.

  18. Evaluation of the in vitro growth of urinary tract infection-causing gram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU) medium.

    PubMed

    Ipe, Deepak S; Ulett, Glen C

    2016-08-01

    Bacteriuria is a hallmark of urinary tract infection (UTI) and asymptomatic bacteriuria (ABU), which are among the most frequent infections in humans. A variety of gram-negative and gram-positive bacteria are associated with these infections but Escherichia coli contributes up to 80% of cases. Multiple bacterial species including E. coli can grow in human urine as a means to maintain colonization during infections. In vitro bacteriuria studies aimed at modeling microbial growth in urine have utilized various compositions of synthetic human urine (SHU) and a Composite SHU formulation was recently proposed. In this study, we sought to validate the recently proposed Composite SHU as a medium that supports the growth of several bacterial species that are known to grow in normal human urine and/or artificial urine. Comparative growth assays of gram-negative and gram-positive bacteria E. coli, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus agalactiae, Staphylococcus saprophyticus and Enterococcus faecalis were undertaken using viable bacterial count and optical density measurements over a 48h culture period. Three different SHU formulations were tested in various culture vessels, shaking conditions and volumes and showed that Composite SHU can support the robust growth of gram-negative bacteria but requires supplementation with 0.2% yeast extract to support the growth of gram-positive bacteria. Experiments are also presented that show an unexpected but major influence of P. mirabilis towards the ability to measure bacterial growth in generally accepted multiwell assays using absorbance readings, predicted to have a basis in the release of volatile organic compound(s) from P. mirabilis during growth in Composite SHU medium. This study represents an essential methodological validation of a more chemically defined type of synthetic urine that can be applied to study mechanisms of bacteriuria and we conclude will offer a useful in vitro model to investigate the

  19. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    SciTech Connect

    Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng; Nelson, Ornella D.; Li, Zhi; Lin, Hening; Callister, Stephen J.; Richardson, Ruth E.

    2015-01-01

    Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.

  20. Review of meta-analyses of vancomycin compared with new treatments for Gram-positive skin and soft-tissue infections: Are we any clearer?

    PubMed

    Tsoulas, Christos; Nathwani, Dilip

    2015-07-01

    Vancomycin has been considered the standard of care for treatment of Gram-positive skin and soft-tissue infections (SSTIs). Its value has been questioned over the last decade owing to well acknowledged limitations in efficacy and tolerability and the emergence of newer meticillin-resistant Staphylococcus aureus (MRSA)-active antibacterial agents. However, no single agent has shown better results versus vancomycin in SSTI trials. The aim of this review was to identify and summarise data from meta-analyses (MAs) for the treatment of Gram-positive and MRSA SSTIs. A systematic search identified 21 published MAs examining the use of newer antibiotics and vancomycin in SSTIs. In terms of clinical and microbiological efficacy, linezolid (in Gram-positive and MRSA SSTIs) and telavancin (in MRSA SSTIs) were shown to be more effective than vancomycin. The safety of newer antimicrobials in general was comparable with vancomycin, except for telavancin, which was associated with more severe adverse events (AEs), and tigecycline owing to an all-cause mortality imbalance observed in all infections but not confirmed in SSTIs. Specific AEs were related to the use of newer agents, such as nephrotoxicity for telavancin, creatine phosphokinase elevations for daptomycin, and thrombocytopenia with linezolid. Some evidence suggests that daptomycin could be associated with reduced treatment duration, and linezolid with reduced length of intravenous treatment and hospital length of stay compared with vancomycin. Considering the limitations of this type of research and the comparative efficacy results demonstrated in head-to-head randomised controlled trials, data are still not sufficient to support the widespread use of new agents over vancomycin.

  1. Evaluation of the in vitro growth of urinary tract infection-causing gram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU) medium.

    PubMed

    Ipe, Deepak S; Ulett, Glen C

    2016-08-01

    Bacteriuria is a hallmark of urinary tract infection (UTI) and asymptomatic bacteriuria (ABU), which are among the most frequent infections in humans. A variety of gram-negative and gram-positive bacteria are associated with these infections but Escherichia coli contributes up to 80% of cases. Multiple bacterial species including E. coli can grow in human urine as a means to maintain colonization during infections. In vitro bacteriuria studies aimed at modeling microbial growth in urine have utilized various compositions of synthetic human urine (SHU) and a Composite SHU formulation was recently proposed. In this study, we sought to validate the recently proposed Composite SHU as a medium that supports the growth of several bacterial species that are known to grow in normal human urine and/or artificial urine. Comparative growth assays of gram-negative and gram-positive bacteria E. coli, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus agalactiae, Staphylococcus saprophyticus and Enterococcus faecalis were undertaken using viable bacterial count and optical density measurements over a 48h culture period. Three different SHU formulations were tested in various culture vessels, shaking conditions and volumes and showed that Composite SHU can support the robust growth of gram-negative bacteria but requires supplementation with 0.2% yeast extract to support the growth of gram-positive bacteria. Experiments are also presented that show an unexpected but major influence of P. mirabilis towards the ability to measure bacterial growth in generally accepted multiwell assays using absorbance readings, predicted to have a basis in the release of volatile organic compound(s) from P. mirabilis during growth in Composite SHU medium. This study represents an essential methodological validation of a more chemically defined type of synthetic urine that can be applied to study mechanisms of bacteriuria and we conclude will offer a useful in vitro model to investigate the

  2. Activity of the Lichen Usnea steineri and its Major Metabolites against Gram-positive, Multidrug-resistant Bacteria.

    PubMed

    Tozatti, Marcos G; Ferreira, Daniele S; Flauzino, Lúzio G Bocalon; Moraes, Thaís da Silva; Martins, Carlos H G; Groppo, Milton; Andrade e Silva, Márcio L; Januário, Ana H; Pauletti, Patricia M; Cunhaa, Wilson R

    2016-04-01

    The antimicrobial activity and possible synergistic effects of extracts and compounds isolated from Usnea steineri were evaluated against four resistant bacterial species. A phytochemical study of the acetone extract of U. steineri resulted in the isolation and characterization of difractaic acid and (+)-usnic acid as the main compounds. The acetone extract showed strong activity (less than 10 µg/mL) against resistant strains of Staphylococcus epidermidis and Enterococcus faecalis, and (+)-usnic acid exhibited strong activity against S. epidermidis (MIC 3.12 µg/mL), S. aureus and S. haemnolyticus (MIC 12.5 µg/mL). Combinations of penicillin and tetracycline with (+)-usnic acid did not show any synergistic antimicrobial effects. Difractaic acid was inactive. Our results showed that the acetone extract of U. steineri possesses significant in vitro antimicrobial activity, which is likely related to the presence of (+)-usnic acid. PMID:27396202

  3. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans.

    PubMed Central

    Jäger, W; Schäfer, A; Pühler, A; Labes, G; Wohlleben, W

    1992-01-01

    The expression of the structural gene (sacB) encoding Bacillus subtilis levansucrase in two gram-positive soil bacteria, Corynebacterium glutamicum ATCC 13032 and Streptomyces lividans 1326, was investigated. sacB expression in the presence of sucrose is lethal to C. glutamicum but not to S. lividans. While S. lividans secretes levansucrase into the medium, we could show that the enzyme is retained by C. glutamicum cells. Our results imply that the sacB gene can be used as a positive selection system in coryneform bacteria. PMID:1644774

  4. Pleural effusion adenosine deaminase: a candidate biomarker to discriminate between Gram-negative and Gram-positive bacterial infections of the pleural space

    PubMed Central

    Li, Ruolin; Wang, Junli; Wang, Xinfeng; Wang, Maoshui

    2016-01-01

    OBJECTIVES: Delay in the treatment of pleural infection may contribute to its high mortality. In this retrospective study, we aimed to evaluate the diagnostic accuracy of pleural adenosine deaminase in discrimination between Gram-negative and Gram-positive bacterial infections of the pleural space prior to selecting antibiotics. METHODS: A total of 76 patients were enrolled and grouped into subgroups according to Gram staining: 1) patients with Gram-negative bacterial infections, aged 53.2±18.6 years old, of whom 44.7% had empyemas and 2) patients with Gram-positive bacterial infections, aged 53.5±21.5 years old, of whom 63.1% had empyemas. The pleural effusion was sampled by thoracocentesis and then sent for adenosine deaminase testing, biochemical testing and microbiological culture. The Mann-Whitney U test was used to examine the differences in adenosine deaminase levels between the groups. Correlations between adenosine deaminase and specified variables were also quantified using Spearman’s correlation coefficient. Moreover, receiver operator characteristic analysis was performed to evaluate the diagnostic accuracy of pleural effusion adenosine deaminase. RESULTS: Mean pleural adenosine deaminase levels differed significantly between Gram-negative and Gram-positive bacterial infections of the pleural space (191.8±32.1 U/L vs 81.0±16.9 U/L, p<0.01). The area under the receiver operator characteristic curve was 0.689 (95% confidence interval: 0.570, 0.792, p<0.01) at the cutoff value of 86 U/L. Additionally, pleural adenosine deaminase had a sensitivity of 63.2% (46.0-78.2%); a specificity of 73.7% (56.9-86.6%); positive and negative likelihood ratios of 2.18 and 0.50, respectively; and positive and negative predictive values of 70.6% and 66.7%, respectively. CONCLUSIONS: Pleural effusion adenosine deaminase is a helpful alternative biomarker for early and quick discrimination of Gram-negative from Gram-positive bacterial infections of the pleural space

  5. Physico-Chemical-Managed Killing of Penicillin-Resistant Static and Growing Gram-Positive and Gram-Negative Vegetative Bacteria

    NASA Technical Reports Server (NTRS)

    Richmond, Robert Chaffee (Inventor); Schramm, Jr., Harry F. (Inventor); Defalco, Francis G. (Inventor); Farris, III, Alex F. (Inventor)

    2012-01-01

    Systems and methods for the use of compounds from the Hofmeister series coupled with specific pH and temperature to provide rapid physico-chemical-managed killing of penicillin-resistant static and growing Gram-positive and Gram-negative vegetative bacteria. The systems and methods represent the more general physico-chemical enhancement of susceptibility for a wide range of pathological macromolecular targets to clinical management by establishing the reactivity of those targets to topically applied drugs or anti-toxins.

  6. In vitro activity of A-56268 (TE-031), a new macrolide, compared with that of erythromycin and clindamycin against selected gram-positive and gram-negative organisms.

    PubMed Central

    Benson, C A; Segreti, J; Beaudette, F E; Hines, D W; Goodman, L J; Kaplan, R L; Trenholme, G M

    1987-01-01

    The in vitro activity of A-56268 was determined and compared with that of erythromycin and clindamycin against a limited spectrum of 401 gram-positive and gram-negative organisms. A-56268 was quite active against erythromycin-susceptible Staphylococcus aureus, Neisseria gonorrhoeae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, and group B streptococci and was moderately active against Campylobacter fetus subsp. fetus. A-56268 was consistently bactericidal only for S. pneumoniae. The activity of A-56268 was comparable to that of erythromycin against most organisms tested. PMID:2952063

  7. Clonal Diversity in Multi Drug Resistant (MDR) Enterococci Isolated from Fecal Normal Flora.

    PubMed

    Hasannejad Bibalan, Meysam; Eshaghi, Morteza; Sadeghi, Javad; Asadian, Mahla; Narimani, Tahmineh; Talebi, Malihe

    2015-01-01

    Enterococci are Gram positive and catalase- negative cocci that are found in the gastrointestinal tract of mammals and birds, and are readily isolated from soil, surface and waters. The aim of this study was to discriminate between Enterococcus isolates based on repetitive element sequence based -PCR (Rep-PCR) with the BOXA2R primer and their antibiotics profile. Enterococci isolates were obtained from 180 fecal samples. The isolates were identified by biochemical reaction and specific identification was confirmed by PCR with species specific primers. All isolates were subjected to Rep typing and antimicrobial susceptibility tests. Rep-PCR analysis of 180 isolates revealed 93 REP types with forty-five single types (ST1 to ST45) and forty-eight common types (CT1 to 48). Antibiotic susceptibility tests exhibited that 53 (29.4%), 43 (23.8%), 11 (6.1%) and 9 (5%) were resistant to erythromycin, tetracycline, gentamicin and ciprofloxacin respectively but among the isolates, sixteen were multi drug resistant (MDR). These MDR isolates showed 11 Rep types with seven single types and four common types. In addition, 81.2% of MDR isolates were from male subjects and the average age of these persons was more than fifty years. This study showed that 56.2% of MDR isolates were homogeneous with 95 % similarity, and high rate of resistance to tetracycline and erythromycin (81.2%) were observed in these isolates. The concern about these normal flora isolates are the pathogenic potential of these bacteria through the horizontal transfer of antibiotic resistance and virulence genes. PMID:27014649

  8. The Effect of Bicarbonate on the Microbial Dissolution of Autunite Mineral in the Presence of Gram-Positive Bacteria

    SciTech Connect

    Sepulveda-Medina, Paola; Katsenovich, Yelena; Wellman, Dawn M.; Lagos, Leonel

    2015-06-01

    Bacteria are key players in the processes that govern fate and transport of contaminants. The uranium release from Na and Ca-autunite by Arthrobacter oxydans strain G968 was evaluated in the presence of bicarbonate ions. This bacterium was previously isolated from Hanford Site soil and in earlier prescreening tests demonstrated low tolerance to U(VI) toxicity compared to other A.oxydans isolates. Experiments were conducted using glass serum bottles as mixed bioreactors and sterile 6-well cell culture plates with inserts separating bacteria cells from mineral solids. Reactors containing phosphorus-limiting media were amended with bicarbonate ranging between 0-10 mM and metaautunite solids to provide a U(VI) concentration of 4.4 mmol/L. Results showed that in the presence of bicarbonate, A.oxydans G968 was able to enhance the release of U(VI) from Na and Ca autunite at the same capacity as other A.oxydans isolates with relatively high tolerance to U(VI). The effect of bacterial strains on autunite dissolution decreases as the concentration of bicarbonate increases. The results illustrate that direct interaction between the bacteria and the mineral is not necessary to result in U (VI) biorelease from autunite. The formation of secondary calcium-phosphate mineral phases on the surface of the mineral during the dissolution can ultimately reduce the natural autunite mineral contact area, which bacterial cells can access. This thereby reduces the concentration of uranium released into the solution. This study provides a better understanding of the interactions between meta-autunite and microbes in conditions mimicking arid and semiarid subsurface environments of western U.S.

  9. The effect of bicarbonate on the microbial dissolution of autunite mineral in the presence of gram-positive bacteria.

    PubMed

    Sepulveda-Medina, Paola M; Katsenovich, Yelena P; Wellman, Dawn M; Lagos, Leonel E

    2015-06-01

    Bacteria are key players in the processes that govern fate and transport of contaminants. The uranium release from Na and Ca-autunite by Arthrobacter oxydans strain G968 was evaluated in the presence of bicarbonate ions. This bacterium was previously isolated from Hanford Site soil and in earlier prescreening tests demonstrated low tolerance to U(VI) toxicity compared to other A. oxydans isolates. Experiments were conducted using glass serum bottles as mixed bioreactors and sterile 6-well cell culture plates with inserts separating bacteria cells from mineral solids. Reactors containing phosphorus-limiting media were amended with bicarbonate ranging between 0 and 10 mM and meta-autunite solids to provide a U(VI) concentration of 4.4 mmol/L. Results showed that in the presence of bicarbonate, A. oxydans G968 was able to enhance the release of U(VI) from Na and Ca autunite at the same capacity as other A. oxydans isolates with relatively high tolerance to U(VI). The effect of bacterial strains on autunite dissolution decreases as the concentration of bicarbonate increases. The results illustrate that direct interaction between the bacteria and the mineral is not necessary to result in U(VI) biorelease from autunite. The formation of secondary calcium-phosphate mineral phases on the surface of the mineral during the dissolution can ultimately reduce the natural autunite mineral contact area, which bacterial cells can access. This thereby reduces the concentration of uranium released into the solution. This study provides a better understanding of the interactions between meta-autunite and microbes in conditions mimicking arid and semiarid subsurface environments of western U.S.

  10. Design, synthesis and biological evaluation of 4-benzoyl-1-dichlorobenzoylthiosemicarbazides as potent Gram-positive antibacterial agents.

    PubMed

    Paneth, Agata; Plech, Tomasz; Kaproń, Barbara; Hagel, Dominika; Kosikowska, Urszula; Kuśmierz, Edyta; Dzitko, Katarzyna; Paneth, Piotr

    2016-01-01

    Twelve 4-benzoyl-1-dichlorobenzoylthiosemicarbazides have been tested as potential antibacterials. All the compounds had MICs between 0.49 and 15.63 µg/ml toward Micrococcus luteus, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis indicating, in most cases, equipotent or even more effective action than cefuroxime. In order to clarify if the observed antibacterial effects are universal, further research were undertaken to test inhibitory potency of two most potent compounds 3 and 11 on clinical isolates of Staphylococcus aureus. Compound 11 inhibited the growth of methicillin-sensitive S. aureus (MSSA) at MICs of 1.95-7.81 µg/ml, methicillin-resistant S. aureus (MRSA) at MICs of 0.49-1.95 µg/ml and MDR-MRSA at MIC of 0.98 and 3.90 µg/ml, respectively. Finally, inhibitory efficacy of 3 and 11 on planktonic cells and biofilms formation in clinical isolates of S. aureus and Haemophilus parainfluenzae was tested. The majority of cells in biofilm populations of MSSA and MRSA were eradicated at low level of 3, with MBICs in the range of 7.82-15.63 µg/ml.

  11. Identification of an amphioxus intelectin homolog that preferably agglutinates gram-positive over gram-negative bacteria likely due to different binding capacity to LPS and PGN.

    PubMed

    Yan, Jie; Wang, Jianfeng; Zhao, Yaqi; Zhang, Jingye; Bai, Changcun; Zhang, Changqing; Zhang, Chao; Li, Kailin; Zhang, Haiqing; Du, Xiumin; Feng, Lijun

    2012-07-01

    Intelectin is a recently described galactofuranose-binding lectin that plays a role in innate immunity in vertebrates. Little is known about intelectin in invertebrates, including amphioxus, the transitional form between vertebrates and invertebrates. We cloned an amphioxus intelectin homolog, AmphiITLN-like, coding 302 amino acids with a conserved fibrinogen-related domain (FReD) in the N-terminus and an Intelectin domain in the C-terminus. In situ hybridization in adult amphioxus showed that AmphiITLN-like transcripts were highly expressed in the digestive tract and the skin. Quantitative real-time PCR revealed that AmphiITLN-like is significantly up-regulated in response to Staphylococcus aureus challenge, but only modestly to Escherichia coli. In addition, recombinant AmphiITLN-like expressed in E. coli agglutinates Gram-negative and Gram-positive bacteria to different degrees in a calcium dependent manner. Recombinant AmphiITLN-like could bind lipopolysaccharide (LPS) and peptidoglycan (PGN), the major cell wall components of Gram-negative and Gram-positive bacteria, respectively, with a higher affinity to PGN. Our work identified and characterized for the first time an amphioxus intelectin homolog, and provided insight into the evolution and function of the intelectin family.

  12. Facile synthesis of gold nanoparticles on propylamine functionalized SBA-15 and effect of surface functionality of its enhanced bactericidal activity against gram positive bacteria

    NASA Astrophysics Data System (ADS)

    Bhuyan, Diganta; Gogoi, Animesh; Saikia, Mrinal; Saikia, Ratul; Saikia, Lakshi

    2015-07-01

    The facile synthesis of an SBA-15-pr-+NH3.Au0 nano-hybrid material by spontaneous autoreduction of aqueous chloroaurate anions on propylamine functionalized SBA-15 was successfully demonstrated. The as-synthesized SBA-15-pr-+NH3.Au0 nano-hybrid material was well characterized using low and wide angle x-ray diffraction (XRD), N2 adsorption-desorption isotherms, Fourier transform infrared (FTIR), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive x-ray spectroscopy (SEM-EDX), x-ray photoelectron spectroscopy (XPS), UV-Visible spectroscopy and atomic absorption spectroscopy (AAS). The activity of the nano-hybrid material as a potent bactericidal agent was successfully tested against Gram positive/negative bacteria viz. Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The colony killing percentage of Gram positive bacteria was found to be higher than Gram negative bacteria due to the stronger electrostatic interaction between the positively-charged amine functionality of SBA-15 and the negatively charged functionality of the bacterial cell wall.

  13. Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen. [Salmonella typhimurium; Escherichia coli; Sarcina lutea; Staphylococcus aureus; Streptococcus lactis; Streptococcus faecalis

    SciTech Connect

    Dahl, T.A.; Midden, W.R. ); Hartman, P.E. )

    1989-04-01

    Gram-negative and gram-positive bacteria were found to display different sensitivities to pure singlet oxygen generated outside of cells. Killing curves for Salmonella typhimurium and Escherichia coli strains were indicative of multihit killing, whereas curves for Sarcina lutea, Staphylococcus aureus, Streptococcus lactis, and Streptococcus faecalis exhibited single-hit kinetics. The S. typhimurium deep rough strain TA1975, which lacks nearly all of the cell wall lipopolysaccharide coat and manifests concomitant enhancement of penetration by some exogenous substances, responded to singlet oxygen with initially faster inactivation than did the S. typhimurium wild-type strain, although the maximum rates of killing appeared to be quite similar. The structure of the cell wall thus plays an important role in susceptibility to singlet oxygen. The outer membrane-lipopolysaccharide portion of the gram-negative cell wall initially protects the bacteria from extracellular singlet oxygen, although it may also serve as a source for secondary reaction products which accentuate the rates of cell killing. S. typhimurium and E. coli strains lacking the cellular antioxidant, glutathione, showed no difference from strains containing glutathione in response to the toxic effects of singlet oxygen. Strains of Sarcina lutea and Staphylococcus aureus that contained carotenoids, however, were far more resistant to singlet oxygen lethality than were both carotenoidless mutants of the same species and other gram-positive species lacking high levels of protective carotenoids.

  14. Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength.

    PubMed

    Eichenbaum, Z; Federle, M J; Marra, D; de Vos, W M; Kuipers, O P; Kleerebezem, M; Scott, J R

    1998-08-01

    We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described. PMID:9687428

  15. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids.

    PubMed

    Weaver, Keith E; Kwong, Stephen M; Firth, Neville; Francia, Maria Victoria

    2009-03-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

  16. Design of a Nanostructured Active Surface against Gram-Positive and Gram-Negative Bacteria through Plasma Activation and in Situ Silver Reduction.

    PubMed

    Gilabert-Porres, Joan; Martí, Sara; Calatayud, Laura; Ramos, Victor; Rosell, Antoni; Borrós, Salvador

    2016-01-13

    Nowadays there is an increasing focus for avoiding bacterial colonization in a medical device after implantation. Bacterial infection associated with prosthesis implantation, or even along the lifetime of the implanted prosthesis, entails a serious problem, emphasized with immunocompromised patients. This work shows a new methodology to create highly hydrophobic micro-/nanostructured silver antibacterial surfaces against Gram-positive and Gram-negative bacteria, using low-pressure plasma. PDMS (polydimethylsiloxane) samples, typically used in tracheal prosthesis, are coated with PFM (pentafluorophenyl methacrylate) through PECVD (plasma enhance chemical vapor deposition) technique. PFM thin films offer highly reactive ester groups that allow them to react preferably with amine bearing molecules, such as amine sugar, to create controlled reductive surfaces capable of reducing silver salts to a nanostructured metallic silver. This micro-/nanostructured silver coating shows interesting antibacterial properties combined with an antifouling behavior causing a reduction of Gram-positive and Gram-negative bacteria viability. In addition, these types of silver-coated samples show no apparent cytotoxicity against COS-7 cells.

  17. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids

    PubMed Central

    Weaver, Keith E.; Kwong, Stephen M.; Firth, Neville; Francia, Maria Victoria

    2009-01-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multi-resistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families. PMID:19100285

  18. The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for Gram-positive bacteria over erythrocytes

    NASA Astrophysics Data System (ADS)

    Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin

    2013-04-01

    Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34254a

  19. Antimicrobial Effect of the Triterpene 3β,6β,16β-Trihydroxylup-20(29)-ene on Planktonic Cells and Biofilms from Gram Positive and Gram Negative Bacteria

    PubMed Central

    Evaristo, Francisco Flávio Vasconcelos; Albuquerque, Maria Rose Jane R.; dos Santos, Hélcio Silva; Bandeira, Paulo Nogueira; Ávila, Fábio do Nascimento; da Silva, Bruno Rocha; Vasconcelos, Ariana Azevedo; Rabelo, Érica de Menezes; Nascimento-Neto, Luiz Gonzaga; Arruda, Francisco Vassiliepe Sousa; Vasconcelos, Mayron Alves; Carneiro, Victor Alves; Cavada, Benildo Sousa; Teixeira, Edson Holanda

    2014-01-01

    This study evaluated the antimicrobial effect of 3β,6β,16β-trihydroxylup-20(29)-ene (CLF1), a triterpene isolated from Combretum leprosum Mart., in inhibiting the planktonic growth and biofilms of Gram positive bacteria Streptococcus mutans and S. mitis. The antimicrobial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antibiofilm potential was determined by quantifying total biomass and enumerating biofilm-entrapped viable bacteria. In addition, the acute toxicity of CLF1 on Artemia sp. nauplii was also determined. The results showed that CLF1 was able in inhibiting the growth of S. mutans and S. mitis with MIC and MBC of 7.8 μg/mL and 15.6 μg/mL, respectively. CLF1 was highly effective on biofilms of both bacteria. Only 7.8 μg/mL CLF1 was enough to inhibit by 97% and 90% biomass production of S. mutans and S. mitis, respectively. On the other hand, such effects were not evident on Gram negative Pseudomonas aeruginosa and Klebsiella oxytoca. The toxicity tests showed that the LC50 of CLF1 was 98.19 μg/mL. Therefore, CLF1 isolated from C. leprosum may constitute an important natural agent for the development of new therapies for caries and other infectious diseases caused by S. mutans and S. mitis. PMID:25093179

  20. Antimicrobial effect of the triterpene 3β,6β,16β-trihydroxylup-20(29)-ene on planktonic cells and biofilms from Gram positive and Gram negative bacteria.

    PubMed

    Evaristo, Francisco Flávio Vasconcelos; Albuquerque, Maria Rose Jane R; dos Santos, Hélcio Silva; Bandeira, Paulo Nogueira; Avila, Fábio do Nascimento; da Silva, Bruno Rocha; Vasconcelos, Ariana Azevedo; Rabelo, Erica de Menezes; Nascimento-Neto, Luiz Gonzaga; Arruda, Francisco Vassiliepe Sousa; Vasconcelos, Mayron Alves; Carneiro, Victor Alves; Cavada, Benildo Sousa; Teixeira, Edson Holanda

    2014-01-01

    This study evaluated the antimicrobial effect of 3β,6β,16β-trihydroxylup-20(29)-ene (CLF1), a triterpene isolated from Combretum leprosum Mart., in inhibiting the planktonic growth and biofilms of Gram positive bacteria Streptococcus mutans and S. mitis. The antimicrobial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antibiofilm potential was determined by quantifying total biomass and enumerating biofilm-entrapped viable bacteria. In addition, the acute toxicity of CLF1 on Artemia sp. nauplii was also determined. The results showed that CLF1 was able in inhibiting the growth of S. mutans and S. mitis with MIC and MBC of 7.8 μg/mL and 15.6 μg/mL, respectively. CLF1 was highly effective on biofilms of both bacteria. Only 7.8 μg/mL CLF1 was enough to inhibit by 97% and 90% biomass production of S. mutans and S. mitis, respectively. On the other hand, such effects were not evident on Gram negative Pseudomonas aeruginosa and Klebsiella oxytoca. The toxicity tests showed that the LC50 of CLF1 was 98.19 μg/mL. Therefore, CLF1 isolated from C. leprosum may constitute an important natural agent for the development of new therapies for caries and other infectious diseases caused by S. mutans and S. mitis. PMID:25093179

  1. Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites.

    PubMed

    Thong-On, Arunee; Suzuki, Katsuyuki; Noda, Satoko; Inoue, Jun-ichi; Kajiwara, Susumu; Ohkuma, Moriya

    2012-01-01

    Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized. PMID:22791052

  2. Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites.

    PubMed

    Thong-On, Arunee; Suzuki, Katsuyuki; Noda, Satoko; Inoue, Jun-ichi; Kajiwara, Susumu; Ohkuma, Moriya

    2012-01-01

    Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized.

  3. Genome analysis of Desulfotomaculum gibsoniae strain GrollT a highly versatile Gram-positive sulfate-reducing bacterium

    PubMed Central

    Kuever, Jan; Visser, Michael; Loeffler, Claudia; Boll, Matthias; Worm, Petra; Sousa, Diana Z.; Plugge, Caroline M.; Schaap, Peter J.; Muyzer, Gerard; Pereira, Ines A.C.; Parshina, Sofiya N.; Goodwin, Lynne A.; Kyrpides, Nikos C.; Detter, Janine; Woyke, Tanja; Chain, Patrick; Davenport, Karen W.; Rohde, Manfred; Spring, Stefan; Klenk, Hans-Peter; Stams, Alfons J.M.

    2014-01-01

    Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain GrollT together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes. PMID:25197466

  4. Antibiotic Resistance in Enterococcus faecalis Isolated from Hospitalized Patients

    PubMed Central

    Balaei Gajan, Esrafil; Shirmohammadi, Adileh; Aghazadeh, Mohammad; Alizadeh, Mohammad; Sighari Deljavan, Alireza; Ahmadpour, Farzin

    2013-01-01

    Background and aims Enterococci are Gram-positive cocci that often occur in pairs (diplococci) or short chains. Be-side developing high level of antibiotic resistance, these bacteria can cause wide range of disease in human, thus to help provide an effective treatment for infections caused by this genus, this study was conceived to provide information on Enterococcus faecalis Antibiotic resistance to widely used antibiotics in hospitalized patients. Materials and methods Disk diffusion agar and Broth dilution methods were used to perform Antibiogram test on isolated Enterococcus faecalis. Culture medium used for Disk diffusion agar test was Muller Hinton agar, and for Broth dilution methods, Muller Hinton broth culture medium was utilized. In disk diffusion agar method, different commercial antibiotics disks produced by Pharmaceutical companies were used. Microsoft Excel software was used to perform statistical analysis. Results Based on antibiograms of 105 cases, a high resistance to Synercid, Nalidixic acid, Oxacillin and Teofilin was de-tected whereas the lowest resistance observed in Nitrofurantoin, Vancomycin, Linezolid and Teicoplanin antibiotics. Conclusion According to the results, Teicoplanin, Vancomycin, Linezolid and Nitrofurantoin are recommended against E. faecalis species. PMID:23875089

  5. On-column labeling of gram-positive bacteria with a boronic acid functionalized squarylium cyanine dye for analysis by polymer-enhanced capillary transient isotachophoresis.

    PubMed

    Saito, Shingo; Massie, Tara L; Maeda, Takeshi; Nakazumi, Hiroyuki; Colyer, Christa L

    2012-03-01

    A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency

  6. Homologous gene clusters of nicotine catabolism, including a new ω-amidase for α-ketoglutaramate, in species of three genera of Gram-positive bacteria.

    PubMed

    Cobzaru, Cristina; Ganas, Petra; Mihasan, Marius; Schleberger, Paula; Brandsch, Roderich

    2011-04-01

    Gram-positive soil bacteria Arthrobacter nicotinovorans, Nocardioides sp. JS614 and Rhodococcus opacus were shown to contain similarly organized clusters of homologous genes for nicotine catabolism. An uncharacterized gene of a predicted nitrilase within these gene clusters was cloned from A. nicotinovorans and biochemical data unexpectedly showed that the protein exhibited ω-amidase activity toward α-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported α-ketoglutaramate as substrate. A-ketoglutaramate could be generated by hydrolytic cleavage of the C-N bond of the trihydroxypyridine ring produced by nicotine catabolism in these bacteria. This ω-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert α-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.

  7. Occurrence of ferredoxin:NAD+ oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria

    PubMed Central

    Hess, Verena; Gallegos, Rene; Jones, J Andrew; Barquera, Blanca; Malamy, Michael H

    2016-01-01

    A ferredoxin:NAD+ oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD+ oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na+-dependent in C. tetanomorphum and B. fragilis but Na+-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD+ oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD+ oxidoreductase activity. PMID:26793417

  8. Crystallization and first data collection of the putative transfer protein TraN from the Gram-positive conjugative plasmid pIP501.

    PubMed

    Goessweiner-Mohr, Nikolaus; Fercher, Christian; Abajy, Mohammad Yaser; Grohmann, Elisabeth; Keller, Walter

    2012-11-01

    Conjugative plasmid transfer is the most important route for the spread of resistance and virulence genes among bacteria. Consequently, bacteria carrying conjugative plasmids are a substantial threat to human health, especially hospitalized patients. Whilst detailed information about the process has been obtained for Gram-negative type-4 secretion systems, little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. The successful purification and crystallization of the putative transfer protein TraN from the G+ conjugative model plasmid pIP501 of Enterococcus faecalis are presented. Native crystals diffracted to 1.8 Å resolution on a synchrotron beamline. The crystals belonged to space group P2(1), with unit-cell parameters a=32.88, b=54.94, c=57.71 Å, β=91.89° and two molecules per asymmetric unit.

  9. Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria II. Post-segregational killing systems.

    PubMed

    Dmowski, Michał; Jagura-Burdzy, Grazyna

    2013-01-01

    Active support is needed for low copy-number plasmids to be stably maintained in bacterial cells. The mechanisms that fulfill this role are (i) partition systems (PAR) acting to separate plasmid molecules to daughter cells and (ii) toxin-andidote (TA) (post-segregational killing-PSK) systems which arrest cell growth until the plasmid reaches the correct copy-number or kill the cells that have not inherited the plasmid. Our knowledge of toxin-antidote systems comes mainly from studies on Gram-negative bacteria. However, some addiction systems of Gram-positive bacteria have been characterized in detail or recently identified. Altogether, they bring new interesting data on toxin-antidote functioning in bacteria.

  10. The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

    PubMed

    Górecki, Kamil; Hägerhäll, Cecilia; Drakenberg, Torbjörn

    2014-01-15

    (23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one. PMID:24139955

  11. High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

    PubMed Central

    Dobrijevic, Dragana; Di Liberto, Gaetana; Tanaka, Kosei; de Wouters, Tomas; Dervyn, Rozenn; Boudebbouze, Samira; Binesse, Johan; Blottière, Hervé M.; Jamet, Alexandre; Maguin, Emmanuelle; van de Guchte, Maarten

    2013-01-01

    Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems. PMID

  12. Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns

    PubMed Central

    Spencer, J.; Schwarzacher, W.

    2016-01-01

    ABSTRACT In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli. Finally, low numbers of magnetically labeled E. coli bacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria. IMPORTANCE Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved

  13. Comparative proteomic analysis of Desulfotomaculum reducens MI-1: Insights into the metabolic versatility of a gram-positive sulfate- and metal-reducing bacterium

    DOE PAGES

    Otwell, Anne E.; Callister, Stephen J.; Zink, Erika M.; Smith, Richard D.; Richardson, Ruth E.

    2016-02-19

    In this study, the proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfatemore » (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.« less

  14. Homologs of the Rml Enzymes from Salmonella enterica Are Responsible for dTDP-β-l-Rhamnose Biosynthesis in the Gram-Positive Thermophile Aneurinibacillus thermoaerophilus DSM 10155

    PubMed Central

    Graninger, Michael; Kneidinger, Bernd; Bruno, Katharina; Scheberl, Andrea; Messner, Paul

    2002-01-01

    The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of l-rhamnose- and d-glycero-d-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-l-rhamnose (dTDP-l-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-l-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91T produces d-rhamnose- and 3-acetamido-3,6-dideoxy-d-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91T for l-rhamnose and d-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91T is not able to synthesize dTDP-l-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation. PMID:12147463

  15. A Systems Biological Approach Reveals Multiple Crosstalk Mechanism between Gram-Positive and Negative Bacterial Infections: An Insight into Core Mechanism and Unique Molecular Signatures

    PubMed Central

    Thangam, Berla; Ahmed, Shiek S. S. J.

    2014-01-01

    Background Bacterial infections remain a major threat and a leading cause of death worldwide. Most of the bacterial infections are caused by gram-positive and negative bacteria, which are recognized by Toll-like receptor (TLR) 2 and 4, respectively. Activation of these TLRs initiates multiple pathways that subsequently lead to effective immune response. Although, both the TLRs share common signaling mechanism yet they may exhibit specificity as well, resulting in the release of diverse range of inflammatory mediators which could be used as candidate biomolecules for bacterial infections. Results We adopted systems biological approach to identify signaling pathways mediated by TLRs to determine candidate molecules associated with bacterial infections. We used bioinformatics concepts, including literature mining to construct protein-protein interaction network, prioritization of TLRs specific nodes using microarray data and pathway analysis. Our constructed PPI network for TLR 2 (nodes: 4091 and edges: 66068) and TLR 4 (node: 4076 and edges: 67898) showed 3207 common nodes, indicating that both the TLRs might share similar signaling events that are attributed to cell migration, MAPK pathway and several inflammatory cascades. Our results propose the potential collaboration between the shared signaling pathways of both the receptors may enhance the immune response against invading pathogens. Further, to identify candidate molecules, the TLRs specific nodes were prioritized using microarray differential expressed genes. Of the top prioritized TLR 2 molecules, 70% were co-expressed. A similar trend was also observed within TLR 4 nodes. Further, most of these molecules were preferentially found in blood plasma for feasible diagnosis. Conclusions The analysis reveals the common and unique mechanism regulated by both the TLRs that provide a broad perspective of signaling events in bacterial infections. Further, the identified candidate biomolecules could potentially aid

  16. Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

    PubMed Central

    Weeks-Gorospe, Jenni N.; Hurtig, Heather R.; Iverson, Amy R.; Schuneman, Margaret J.; Webby, Richard J.; McCullers, Jonathan A.

    2012-01-01

    A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens. PMID:22674997

  17. A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria.

    PubMed

    Li, Mo-Fei; Zhang, Min; Wang, Chun-Lin; Sun, Li

    2012-02-01

    Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. PMID:22146700

  18. A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.

    PubMed

    Ramachandran, Gayetri; Singh, Praveen K; Luque-Ortega, Juan Roman; Yuste, Luis; Alfonso, Carlos; Rojo, Fernando; Wu, Ling J; Meijer, Wilfried J J

    2014-10-01

    Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed. PMID:25340403

  19. Comparison of Small-Intestinal Submucosa and Expanded Polytetrafluoroethylene as a Vascular Conduit in the Presence of Gram-Positive Contamination

    PubMed Central

    Shell, Daniel H.; Croce, Martin A.; Cagiannos, Catherine; Jernigan, T Wright; Edwards, Norma; Fabian, Timothy C.

    2005-01-01

    Objective: As a vascular conduit, expanded polytetrafluoroethylene (ePTFE) is susceptible to graft infection with Gram-positive organisms. Biomaterials, such as porcine small-intestinal submucosa (SIS), have been successfully used clinically as tissue substitutes outside the vascular arena. Summary Background Data: In the present study, we compared a small-diameter conduit of SIS to ePTFE in the presence of Gram-positive contamination to evaluate infection resistance, incorporation and remodeling, morphometry, graft patency, and neointimal hyperplasia (NH) development. Methods: Adult male mongrel pigs were randomized to receive either SIS or ePTFE (3-cm length, 6-mm diameter) and further randomized to 1 of 3 groups: Control (no graft inoculation), Staphylococcus aureus, or mucin-producing S epidermidis (each graft inoculation with 108 colonies/mL). Pressure measurements were obtained proximal and distal to the graft to create the iliac/aorta pressure ratio. Morphometric analysis of the neointima and histopathologic examinations was performed. Other outcomes included weekly WBC counts, graft incorporation, and quantitative culture of explanted grafts. Results: Eighteen animals were randomized. All grafts were patent throughout the 6-week study period. Infected SIS grafts had less NH and little change in their iliac/aorta indices compared with infected ePTFE grafts. Quantitative cultures at euthanasia demonstrated no growth in either SIS group compared with 1.7 × 104 colonies for ePTFE S aureus and 6 × 102 for ePTFE S epi (each P < 0.001). All SIS grafts were incorporated. Histology demonstrated remodeling into host artery with smooth muscle and capillary ingrowth in all SIS groups. Scanning electron micrography illustrated smooth and complete endothelialization of all SIS grafts. Conclusions: Compared with ePTFE, SIS induces host tissue remodeling, exhibits a decreased neointimal response to infection, and is resistant to bacterial colonization. SIS may provide a

  20. Comparative Proteomic Analysis of Desulfotomaculum reducens MI-1: Insights into the Metabolic Versatility of a Gram-Positive Sulfate- and Metal-Reducing Bacterium

    PubMed Central

    Otwell, Anne E.; Callister, Stephen J.; Zink, Erika M.; Smith, Richard D.; Richardson, Ruth E.

    2016-01-01

    The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals. PMID:26925055

  1. A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

    PubMed

    Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

    2015-01-01

    A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and

  2. Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium.

    PubMed

    Liang, Jie-Liang; Nie, Yong; Wang, Miaoxiao; Xiong, Guangming; Wang, Yi-Ping; Maser, Edmund; Wu, Xiao-Lei

    2016-01-01

    n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48 bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (± 0.03) [k(D)  = 1.06 (± 0.16) μM, k(D) ' = 0.05 (± 0.01) μM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria.

  3. iLoc-Gpos: a multi-layer classifier for predicting the subcellular localization of singleplex and multiplex Gram-positive bacterial proteins.

    PubMed

    Wu, Zhi-Cheng; Xiao, Xuan; Chou, Kuo-Chen

    2012-01-01

    By introducing the "multi-layer scale", as well as hybridizing the information of gene ontology and the sequential evolution information, a novel predictor, called iLoc-Gpos, has been developed for predicting the subcellular localization of Gram positive bacterial proteins with both single-location and multiple-location sites. For facilitating comparison, the same stringent benchmark dataset used to estimate the accuracy of Gpos-mPLoc was adopted to demonstrate the power of iLoc-Gpos. The dataset contains 519 Gram-positive bacterial proteins classified into the following four subcellular locations: (1) cell membrane, (2) cell wall, (3) cytoplasm, and (4) extracell; none of proteins included has ≥25% pairwise sequence identity to any other in a same subset (subcellular location). The overall success rate by jackknife test on such a stringent benchmark dataset by iLoc-Gpos was over 93%, which is about 11% higher than that by GposmPLoc. As a user-friendly web-server, iLoc-Gpos is freely accessible to the public at http://icpr.jci.edu.cn/bioinfo/iLoc- Gpos or http://www.jci-bioinfo.cn/iLoc-Gpos. Meanwhile, a step-by-step guide is provided on how to use the web-server to get the desired results. Furthermore, for the user � s convenience, the iLoc-Gpos web-server also has the function to accept the batch job submission, which is not available in the existing version of Gpos-mPLoc web-server.

  4. Streptococcus oligofermentans sp. nov., a novel oral isolate from caries-free humans.

    PubMed

    Tong, Huichun; Gao, Xuejun; Dong, Xiuzhu

    2003-07-01

    Five streptococcal strains were isolated from dental plaque and saliva of caries-free humans. The cells were gram-positive, non-spore-forming, non-motile cocci that were arranged in short chains. The strains were catalase-negative, facultatively anaerobic and produced lactic acid exclusively from glucose fermentation. Biochemical analysis that used both conventional methods and the commercial API 20 Strep system showed that the five strains fermented only a few kinds of sugar. The mean DNA G + C content of the five novel strains was 39.5 +/- 0.8 mol%. Phylogenetic analysis based on 16S rDNA sequence homology indicated that the new isolates represented a novel member of the mitis group of the genus Streptococcus, related most closely to the recently described species Streptococcus sinensis. DNA-DNA relatedness between novel strain LMG 21535T and type strains of phylogenetically related species of oral streptococci was 7.1-16.4%. Therefore a novel Streptococcus species, Streptococcus oligofermentans sp. nov., is proposed. The type strain is LMG 21535T=AS 1.3089T.

  5. In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria.

    PubMed

    Huband, Michael D; Bradford, Patricia A; Otterson, Linda G; Basarab, Gregory S; Kutschke, Amy C; Giacobbe, Robert A; Patey, Sara A; Alm, Richard A; Johnstone, Michele R; Potter, Marie E; Miller, Paul F; Mueller, John P

    2015-01-01

    AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development. PMID:25385112

  6. Staphylococcus massiliensis sp. nov., isolated from a human brain abscess.

    PubMed

    Al Masalma, Mouhamad; Raoult, Didier; Roux, Véronique

    2010-05-01

    Gram-positive, catalase-positive, coagulase-negative, non-motile, non-fermentative and novobiocin-susceptible cocci were isolated from a human brain abscess sample (strain 5402776(T)). This novel strain was analysed by a polyphasic taxonomic approach. The respiratory quinones detected were MK-7 (93 %) and MK-6 (7 %) and the major fatty acids were C(15 : 0) iso (60.5 %), C(17 : 0) iso (8.96 %) C(15 : 0) anteiso (7.93 %) and C(19 : 0) iso (6.78 %). The peptidoglycan type was A3alpha l-Lys-Gly(2-3)-l-Ser-Gly. Based on cellular morphology and biochemical criteria, the new isolate was assigned to the genus Staphylococcus, although it did not correspond to any recognized species. The G+C content of the DNA was 36.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the new isolate was most closely related to Staphylococcus piscifermentans, Staphylococcus condimenti, Staphylococcus carnosus subsp. carnosus, S. carnosus subsp. utilis and Staphylococcus simulans (97.7 %, 97.6 %, 97.6 %, 97.6 % and 96.5 % sequence similarity, respectively). Comparison of tuf, hsp60, rpoB, dnaJ and sodA gene sequences was also performed. In phylogenetic analysis inferred from tuf, dnaJ and rpoB gene sequence comparisons, strain 5402776(T) clustered with Staphylococcus pettenkoferi (93.7 %, 82.5 % and 89 % sequence similarity, respectively) and on phylogenetic analysis inferred from sodA gene sequence comparisons, it clustered with Staphylococcus chromogenes (82.8 %). On the basis of phenotypic and genotypic data, this isolate represents a novel species for which the name Staphylococcus massiliensis sp. nov. is proposed (type strain 5402776(T)=CCUG 55927(T)=CSUR P23(T)). PMID:19666814

  7. Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land)

    NASA Technical Reports Server (NTRS)

    Siebert, J.; Hirsch, P.; Friedmann, E. I. (Principal Investigator)

    1988-01-01

    Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

  8. Efficient Photodynamic Therapy against Gram-Positive and Gram-Negative Bacteria Using THPTS, a Cationic Photosensitizer Excited by Infrared Wavelength

    PubMed Central

    Schastak, Stanislaw; Ziganshyna, Svitlana; Gitter, Burkhard; Wiedemann, Peter; Claudepierre, Thomas

    2010-01-01

    The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. Concentrations of 1, 10 and 100µM of tetrahydroporphyrin-tetratosylat (THPTS) and different incubation times (30, 90 and 180min) were used to measure photodynamic efficiency against two Gram-positive strains of S.aureus (MSSA and MRSA), and two Gram-negative strains of E.coli and P.aeruginosa. We found that phototoxicity of the drug is independent of the antibiotic resistance pattern when incubated in PBS for the investigated strains. Also, an incubation with 100µM THPTS followed by illumination, yielded a 6lg (≥99.999%) decrease in the viable numbers of all bacteria strains tested, indicating that the THPTS drug has a high degree of photodynamic inactivation. We then modulated incubation time, photosensitizer concentration and monitored the effect of serum on the THPTS activity. In doing so, we established the conditions to obtain the strongest bactericidal effect. Our results suggest that this new and highly pure synthetic compound should improve the efficiency of photodynamic therapy against multiresistant bacteria and has a significant potential for clinical applications in the treatment of nosocomial infections. PMID:20652031

  9. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    PubMed

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara

    2008-01-01

    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  10. Growth of Ag-nanoparticles in an aqueous solution and their antimicrobial activities against Gram positive, Gram negative bacterial strains and Candida fungus.

    PubMed

    Aazam, Elham Shafik; Zaheer, Zoya

    2016-04-01

    Silver nanoparticles (AgNPs) were synthesized using Ocimum sanctum (Tulsi) leaves aqueous extract as reducing as well as a capping agent in absence and presence of cetyltrimethylammonium bromide (CTAB). The resulting nanomaterials were characterized by UV-visible spectrophotometer, and transmission electron microscope. The UV-Vis spectroscopy revealed the formation of AgNPs at 400-450 nm. TEM photographs indicate that the truncated triangular silver nanoplates and/or spherical morphology of the AgNPs with an average diameter of 25 nm have been distorted markedly in presence of CTAB. The AgNPs were almost mono disperse in nature. Antimicrobial activities of AgNPs were determined by using two bacteria (Gram positive Staphylococcus aureus MTCC-3160), Gram negative Escherichia coli MTCC-450) and one species of Candida fungus (Candida albicans ATCC 90030) with Kirby-Bauer or disc diffusion method. The zone of inhibition seems extremely good showing a relatively large zone of inhibition in both Staphylococcus aureus, Escherichia coli, and Candida albicans strains. PMID:26796584

  11. Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501.

    PubMed

    Goessweiner-Mohr, Nikolaus; Grumet, Lukas; Pavkov-Keller, Tea; Birner-Gruenberger, Ruth; Grohmann, Elisabeth; Keller, Walter

    2013-02-01

    The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (G-) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMΔ, comprising residues 190-322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unit-cell parameters a = 39.21, b = 54.98, c = 93.47 Å, α = 89.91, β = 86.44, γ = 78.63° and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.

  12. The Comparison of the Combined Toxicity between Gram-negative and Gram-positive Bacteria: a Case Study of Antibiotics and Quorum-sensing Inhibitors.

    PubMed

    Wang, Dali; Lin, Zhifen; Ding, Xiruo; Hu, Jingyun; Liu, Ying

    2016-02-01

    Quorum-sensing inhibitors (QSIs) are being used increasingly in diverse fields, and are likely to end up in the environment, where they may encounter the antibiotics and consequently cause joint effects on biological systems. However, the potential joint effects of QSIs and antibiotics have received little attention. In this study, the joint effects of antibiotics, represented by sulfonamides (SAs) and penicillin, as well as three potential QSIs, were investigated using both Gram-negative (Escherichia coli, E. coli) and Gram-positive bacteria (Bacillus subtilis, B. subtilis). It was found that E. coli tend to be more sensitive to the individual drugs than B. subtilis, whereas the joint effects on the two bacteria showed no difference regarding the same combination of antibiotics and QSIs. In general, SAs presented additive effects with γ-Valerolactone and 2-Pyrrolidinone, but antagonistic effects with L-(+)-Prolinol; penicillin exhibited antagonistic effects with all three QSIs. Moreover, it was found that the rate of resistance in E. coli against the individual antibiotics was reduced through the addition of the QSIs, which suggests a promising use of the QSIs in the bacterial infection treatment. This study also offers a valuable reference for the risk assessment of the antibiotics and QSIs in the real environment. PMID:27491790

  13. In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides.

    PubMed

    Roller, C; Wagner, M; Amann, R; Ludwig, W; Schleifer, K H

    1994-10-01

    23S-rRNA-targeted oligonucleotide probes were designed for the phylogenetic group 'Gram-positive bacteria with high G + C content of DNA' (GPBHGC). A sequence idiosyncrasy in two adjacent base pairs in the stem of helix 69 in domain IV of the 23S rRNA is present in all hitherto analysed strains of GPBHGC. An oligonucleotide probe targeted to this region hybridized only with strains of GPBHGC and was successfully used for in situ monitoring of these cells in activated sludge. Another unique feature of the 23S rRNA molecules of GPBHGC is a large insertion in domain III. Fluorescent oligonucleotides targeted to the highly variable regions of the rRNA within the insertions of Corynebacterium glutamicum DSM 20300, Aureobacterium testaceum DSM 20166 and Brevibacterium sp. DSM 20165 hybridized specifically to their target strains, whereas probing with oligonucleotides complementary to the rRNA-coding strand of the 23S rDNA and to the spacer between 16S and 23S rRNA of C. glutamicum did not result in detectable fluorescence. This confirmed that the large 23S insertions are indeed present in 23S rRNAs of GPBHGC and provide potential target sites for highly specific nucleic acid probes.

  14. Sulfoxides, Analogues of L-Methionine and L-Cysteine As Pro-Drugs against Gram-Positive and Gram-Negative Bacteria.

    PubMed

    Anufrieva, N V; Morozova, E A; Kulikova, V V; Bazhulina, N P; Manukhov, I V; Degtev, D I; Gnuchikh, E Yu; Rodionov, A N; Zavilgelsky, G B; Demidkina, T V

    2015-01-01

    The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 - dependent methionine γ-lyase, which metabolizes it in the patient's body. The enzyme catalyzes the γ- and β-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and β-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown. PMID:26798500

  15. Mechanism-based QSAR Models for the Toxicity of Quorum Sensing Inhibitors to Gram-negative and Gram-positive Bacteria.

    PubMed

    Wang, Dali; Lin, Zhifen; Huo, Zhengyang; Wang, Ting; Yao, Zhifeng; Cong, Yongping

    2016-07-01

    Quorum sensing inhibitors (QSIs) are a promising alternative to the antibiotics and unlikely to induce drug resistance. However, toxicity studies on the QSIs remain limited; therefore in this paper we investigated the acute (15 min) and chronic (24 h) toxicity of some potential QSIs on both gram-negative (V. fischeri) and gram-positive bacteria (B. subtilis). It was found that the toxicity of the QSIs differed with the toxicity test periods. QSAR models were developed for both the acute and chronic toxicity, using the interaction energies between QSIs and the relevant proteins, and the frontier orbital energies. Based on the QSAR models, it was suggested that QSIs primarily bind with the luciferase at 15 min, but LuxR at 24 h in V. fischeri; whereas in B. subtilis, the QSIs mainly bind with LuxS. Our study provided an insight into the toxicity mechanism for QSIs during different exposure periods. PMID:27084097

  16. Antimicrobial Activity of Cationic Antimicrobial Peptides against Gram-Positives: Current Progress Made in Understanding the Mode of Action and the Response of Bacteria

    PubMed Central

    Omardien, Soraya; Brul, Stanley; Zaat, Sebastian A. J.

    2016-01-01

    Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could aid the fight against antibiotic resistant bacteria. The mode of action of AMPs as acting on the bacterial cytoplasmic membrane has often been presented as an enigma and there are doubts whether the membrane is the sole target of AMPs. Progress has been made in clarifying the possible targets of these peptides, which is reported in this review with as focus gram-positive vegetative cells and spores. Numerical estimates are discussed to evaluate the possibility that targets, other than the membrane, could play a role in susceptibility to AMPs. Concerns about possible resistance that bacteria might develop to AMPs are addressed. Proteomics, transcriptomics, and other molecular techniques are reviewed in the context of explaining the response of bacteria to the presence of AMPs and to predict what resistance strategies might be. Emergent mechanisms are cell envelope stress responses as well as enzymes able to degrade and/or specifically bind (and thus inactivate) AMPs. Further studies are needed to address the broadness of the AMP resistance and stress responses observed. PMID:27790614

  17. Discovery of Novel Cell Wall-Active Compounds Using PywaC, a Sensitive Reporter of Cell Wall Stress, in the Model Gram-Positive Bacterium Bacillus subtilis

    PubMed Central

    Czarny, T. L.; Perri, A. L.; French, S.

    2014-01-01

    The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active