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Sample records for aerobic gram negative

  1. Antimicrobial Susceptibility of Enteric Gram Negative Facultative Anaerobe Bacilli in Aerobic versus Anaerobic Conditions

    PubMed Central

    Amachawadi, Raghavendra G.; Renter, David G.; Volkova, Victoriya V.

    2016-01-01

    Antimicrobial treatments result in the host’s enteric bacteria being exposed to the antimicrobials. Pharmacodynamic models can describe how this exposure affects the enteric bacteria and their antimicrobial resistance. The models utilize measurements of bacterial antimicrobial susceptibility traditionally obtained in vitro in aerobic conditions. However, in vivo enteric bacteria are exposed to antimicrobials in anaerobic conditions of the lower intestine. Some of enteric bacteria of food animals are potential foodborne pathogens, e.g., Gram-negative bacilli Escherichia coli and Salmonella enterica. These are facultative anaerobes; their physiology and growth rates change in anaerobic conditions. We hypothesized that their antimicrobial susceptibility also changes, and evaluated differences in the susceptibility in aerobic vs. anaerobic conditions of generic E. coli and Salmonella enterica of diverse serovars isolated from cattle feces. Susceptibility of an isolate was evaluated as its minimum inhibitory concentration (MIC) measured by E-Test® following 24 hours of adaptation to the conditions on Mueller-Hinton agar, and on a more complex tryptic soy agar with 5% sheep blood (BAP) media. We considered all major antimicrobial drug classes used in the U.S. to treat cattle: β-lactams (specifically, ampicillin and ceftriaxone E-Test®), aminoglycosides (gentamicin and kanamycin), fluoroquinolones (enrofloxacin), classical macrolides (erythromycin), azalides (azithromycin), sulfanomides (sulfamethoxazole/trimethoprim), and tetracyclines (tetracycline). Statistical analyses were conducted for the isolates (n≥30) interpreted as susceptible to the antimicrobials based on the clinical breakpoint interpretation for human infection. Bacterial susceptibility to every antimicrobial tested was statistically significantly different in anaerobic vs. aerobic conditions on both media, except for no difference in susceptibility to ceftriaxone on BAP agar. A satellite experiment

  2. Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium

    SciTech Connect

    Lobos, J.H.; Leib, T.K. ); Tahmun Su )

    1992-06-01

    A novel bacterium designated strain MV1 was isolated from a sludge enrichmet takes from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4[prime]-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO[sub 2], 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.

  3. Species distribution and antimicrobial susceptibility of gram-negative aerobic bacteria in hospitalized cancer patients

    PubMed Central

    Ashour, Hossam M; El-Sharif, Amany

    2009-01-01

    Background Nosocomial infections pose significant threats to hospitalized patients, especially the immunocompromised ones, such as cancer patients. Methods This study examined the microbial spectrum of gram-negative bacteria in various infection sites in patients with leukemia and solid tumors. The antimicrobial resistance patterns of the isolated bacteria were studied. Results The most frequently isolated gram-negative bacteria were Klebsiella pneumonia (31.2%) followed by Escherichia coli (22.2%). We report the isolation and identification of a number of less-frequent gram negative bacteria (Chromobacterium violacum, Burkholderia cepacia, Kluyvera ascorbata, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, and Salmonella arizona). Most of the gram-negative isolates from Respiratory Tract Infections (RTI), Gastro-intestinal Tract Infections (GITI), Urinary Tract Infections (UTI), and Bloodstream Infections (BSI) were obtained from leukemic patients. All gram-negative isolates from Skin Infections (SI) were obtained from solid-tumor patients. In both leukemic and solid-tumor patients, gram-negative bacteria causing UTI were mainly Escherichia coli and Klebsiella pneumoniae, while gram-negative bacteria causing RTI were mainly Klebsiella pneumoniae. Escherichia coli was the main gram-negative pathogen causing BSI in solid-tumor patients and GITI in leukemic patients. Isolates of Escherichia coli, Klebsiella, Enterobacter, Pseudomonas, and Acinetobacter species were resistant to most antibiotics tested. There was significant imipenem -resistance in Acinetobacter (40.9%), Pseudomonas (40%), and Enterobacter (22.2%) species, and noticeable imipinem-resistance in Klebsiella (13.9%) and Escherichia coli (8%). Conclusion This is the first study to report the evolution of imipenem-resistant gram-negative strains in Egypt. Mortality rates were higher in cancer patients with nosocomial Pseudomonas infections than any other bacterial infections. Policies restricting

  4. Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4.

    PubMed

    Carbajal-Rodríguez, Irma; Stöveken, Nadine; Satola, Barbara; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2011-01-01

    The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.

  5. Time-to-positivity-based discrimination between Enterobacteriaceae, Pseudomonas aeruginosa and strictly anaerobic Gram-negative bacilli in aerobic and anaerobic blood culture vials.

    PubMed

    Defrance, Gilles; Birgand, Gabriel; Ruppé, Etienne; Billard, Morgane; Ruimy, Raymond; Bonnal, Christine; Andremont, Antoine; Armand-Lefèvre, Laurence

    2013-05-01

    Time-to-positivity (TTP) of first positive blood cultures growing Gram-negative bacilli (GNB) was investigated. When anaerobic vials were positive first, TTP ≤ 18 h differentiated Enterobacteriaceae from strict anaerobic Gram-negative bacilli (PPV 98.8%). When the aerobic ones were first, TTP ≤ 13 h differentiated Enterobacteriaceae from Pseudomonas aeruginosa and other GNB (PPV 80.8%).

  6. Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

    PubMed

    Faron, Matthew L; Buchan, Blake W; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L; Granato, Paul A; Wilson, Deborah A; Procop, Gary W; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A

    2015-01-01

    The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

  7. Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates

    PubMed Central

    Faron, Matthew L.; Buchan, Blake W.; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L.; Granato, Paul A.; Wilson, Deborah A.; Procop, Gary W.; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A.

    2015-01-01

    The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria. PMID:26529504

  8. Manual and automated instrumentation for identification of Enterobacteriaceae and other aerobic gram-negative bacilli.

    PubMed

    O'hara, Caroline M

    2005-01-01

    Identification of gram-negative bacilli, both enteric and nonenteric, by conventional methods is not realistic for clinical microbiology laboratories performing routine cultures in today's world. The use of commercial kits, either manual or automated, to identify these organisms is a common practice. The advent of rapid or "spot" testing has eliminated the need for some commonly isolated organisms to be identified with the systems approach. Commercially available systems provide more in-depth identification to the species level as well as detect new and unusual strains. The answers obtained from these systems may not always be correct and must be interpreted with caution. The patient demographics, laboratory workload and work flow, and technologist's skill levels should dictate the system of choice. Cost considerations introduce another variable into the equation affecting choice. Each system has its own strengths and weaknesses, and each laboratory must decide on the level of sophistication that fulfills its particular needs.

  9. Manual and Automated Instrumentation for Identification of Enterobacteriaceae and Other Aerobic Gram-Negative Bacilli

    PubMed Central

    O'Hara, Caroline M.

    2005-01-01

    Identification of gram-negative bacilli, both enteric and nonenteric, by conventional methods is not realistic for clinical microbiology laboratories performing routine cultures in today's world. The use of commercial kits, either manual or automated, to identify these organisms is a common practice. The advent of rapid or “spot” testing has eliminated the need for some commonly isolated organisms to be identified with the systems approach. Commercially available systems provide more in-depth identification to the species level as well as detect new and unusual strains. The answers obtained from these systems may not always be correct and must be interpreted with caution. The patient demographics, laboratory workload and work flow, and technologist's skill levels should dictate the system of choice. Cost considerations introduce another variable into the equation affecting choice. Each system has its own strengths and weaknesses, and each laboratory must decide on the level of sophistication that fulfills its particular needs. PMID:15653824

  10. In silico analysis of 16S rRNA gene sequencing based methods for identification of medically important aerobic Gram-negative bacteria.

    PubMed

    Teng, Jade L L; Yeung, Ming-Yiu; Yue, Geoffrey; Au-Yeung, Rex K H; Yeung, Eugene Y H; Fung, Ami M Y; Tse, Herman; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2011-09-01

    This study provides guidelines on the usefulness of full and 527 bp 16S rRNA gene sequencing and Microseq databases for identifying medically important aerobic Gram-negative bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 26.1 % and 32.6 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15.2 % and 26.1 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level. Among the major groups of aerobic Gram-negative bacteria, the methods and databases are least useful for identification of Aeromonas, Bordetella and Bartonella species. None of the Aeromonas species can be confidently or doubtfully identified, whereas only 0 % and 0-33.3 % of Bordetella species and 0-10 % and 0-10 % of Bartonella species can be confidently and doubtfully identified, respectively. On the other hand, these methods and databases are most useful for identification of members of the families Pasteurellaceae and Legionellaceae and Campylobacter species: 29.6-59.3 % and 7.4-18.5 % of members of Pasteurellaceae, 36-52 % and 12-24 % of members of Legionellaceae, and 26.7-60 % and 0-13.3 % of Campylobacter species can be confidently and doubtfully identified, respectively. Thirty-nine medically important aerobic Gram-negative bacteria that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. Twenty-three medically important aerobic Gram-negative bacteria that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. Compared with results of our previous studies on anaerobic and Gram-positive bacteria, full and 527 bp 16S rRNA gene sequencing are able to confidently identify significantly more anaerobic Gram-positive and Gram-negative bacteria than aerobic Gram

  11. 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.

  12. Epidemiology and antimicrobial susceptibility of Gram-negative aerobic bacteria causing intra-abdominal infections during 2010-2011.

    PubMed

    Hawser, Stephen; Hoban, Daryl J; Badal, Robert E; Bouchillon, Samuel K; Biedenbach, Douglas; Hackel, Meredith; Morrissey, Ian

    2015-02-01

    The study for monitoring antimicrobial resistance trends (SMART) surveillance program monitors the epidemiology and trends in antibiotic resistance of intra-abdominal pathogens to currently used therapies. The current report describes such trends during 2010-2011. A total of 25,746 Gram-negative clinical isolates from intra-abdominal infections were collected and classified as hospital-associated (HA) if the hospital length of stay (LOS) at the time of specimen collection was ≥48 hours, community-associated (CA) if LOS at the time of specimen collection was <48 hours, or unknown (no designation given by participating centre). A total of 92 different species were collected of which the most common was Escherichia coli: 39% of all isolates in North America to 55% in Africa. Klebsiella pneumoniae was the second most common pathogen: 11% of all isolates from Europe to 19% of all isolates from Asia. Isolates were from multiple intra-abdominal sources of which 32% were peritoneal fluid, 20% were intra-abdominal abscesses, and 16.5% were gall bladder infections. Isolates were further classified as HA (55% of all isolates), CA (39% of all isolates), or unknown (6% of all isolates). The most active antibiotics tested were imipenem, ertapenem, amikacin, and piperacillin-tazobactam. Resistance rates to all other antibiotics tested were high. Considering the current data set and high-level resistance of intra-abdominal pathogens to various antibiotics, further monitoring of the epidemiology of intra-abdominal infections and their susceptibility to antibiotics through SMART is warranted.

  13. [Culture and differentiation of obligatory aerobic gram-negative rods from human material; a scheme for application in routine diagnosis (author's transl)].

    PubMed

    von Graevenitz, A; Grehn, M

    1976-12-01

    The diagnosis of obligately aerobic Gram-negative rods in the clinical laboratory may encounter difficulties since media used for Enterobacteriacae are only partially usable for the diagnosis of this group of bacteria (Psuedomonas, Xanthomonas, Alcaligenes, Achromobacter, Brucella, Bordetella, Flavobacterium, Moraxella, Acinetobacter, and some still unnamed taxa). We have developed a diagnostic scheme, based on recent publications in the field and representing an extension of earlier tables from this and other laboratories, which attempts to classify a maximal number of obligately aerobic Gram-negative rods with a minimal number of tests. The scheme, employed on 4051 strains, used blood agar and MacConkey Agar as isolation media. Growth characteristics on these media and microscopic morphology may be of help, but only the type of growth on Triple Sugar Iron (or Kligler's) Agar is characteristic for the group as a whole (no growth in the butt, alkalinization or no pH change on the slant). A primary identification series employs tests for oxidase (Kovacs), oxidation of glucose and xylose (in OF medium), deoxyribonuclease and indole (in DNase Test Agar with Methyl Green), nitrate reduction (in Indole Nitrite Medium), motility (hanging drop), and fluorescein production (on Flo Agar). Results of Kirby-Bauer antimicrobial sensitivity testing serve as additional (colistin) or confirmatory criteria. Incubation is at 30 degrees C for 24-48 hrs. If a diagnosis is not possible than, a secondary series, including tests for lysine decarboxylase (tablets), 4 hr urease, esculin hydrolysis, growth at 42 C and on SS Agar, gelatin liquefaction, and flagellar staining may have to be used, and read after 4-24 hrs at 30 degrees C. Five tables, drawn up according to oxidase, glucose, and xylose reactions, serve to identify the species or taxa. Biotypes cannot be differentiated. The scheme will need updating as more knowledge of these bacteria will become available.

  14. Direct inoculation method using BacT/ALERT 3D and BD Phoenix System allows rapid and accurate identification and susceptibility testing for both Gram-positive cocci and Gram-negative rods in aerobic blood cultures.

    PubMed

    Yonetani, Shota; Okazaki, Mitsuhiro; Araki, Koji; Makino, Hiroshi; Fukugawa, Yoko; Okuyama, Takahiro; Ohnishi, Hiroaki; Watanabe, Takashi

    2012-06-01

    This study describes a direct inoculation method using the automated BacT/ALERT 3D and the BD Phoenix System in combination for identification and susceptibility testing of isolates from positive blood cultures. Organism identification and susceptibility results were compared with the conventional method for 211 positive aerobic blood cultures. Of 110 Gram-positive cocci (GPCs), 98 (89.1%) isolates were correctly identified to the species level. Of 101 Gram-negative rods (GNRs), 98 (97.0%) isolates were correctly identified to the species level. The overall categorical agreement in antimicrobial susceptibility testing among the 110 GPCs was 92.7%, with 0.04% very major and 0.7% major error rates. The overall categorical agreement among 78 isolates of enterobacteria and 23 isolates of nonfermenters in GNRs was 99.5% and 91.1%, respectively, with no major errors identified. We conclude that, compared with previously reported direct inoculation methods, our method is superior in identification and susceptibility testing of GPCs.

  15. Gram-Negative Flagella Glycosylation

    PubMed Central

    Merino, Susana; Tomás, Juan M.

    2014-01-01

    Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation. PMID:24557579

  16. Gram-negative flagella glycosylation.

    PubMed

    Merino, Susana; Tomás, Juan M

    2014-02-19

    Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.

  17. 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.

  18. [Distribution of ubiquinones (coenzyme Q) in Gram negative bacillae].

    PubMed

    Denis, F A; D'Oultremont, P A; Debacq, J J; Cherel, J M; Brisou, J

    1975-01-01

    The coenzyme Q system was examined on 55 strains of Gram negative aerobic or facultatively anaerobic rods. No bacteria contain Co-Q7 nor Co-Q10. Ubiquinone Q8 predominates in Flavobacterium and in Enterobacteriaceae; Q9 was the only homolog found in the Pseudomonas, and predominates in the Acinetobacter.

  19. Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and iso-octylthioglycolic acid by aerobic gram-negative bacteria.

    PubMed

    Toups, Mario; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2010-04-01

    Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater's toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.

  20. Antimicrobial Susceptibilities of Aerobic and Facultative Gram-Negative Bacilli from Intra-abdominal Infections in Patients from Seven Regions in China in 2012 and 2013.

    PubMed

    Zhang, Hui; Yang, Qiwen; Liao, Kang; Ni, Yuxing; Yu, Yunsong; Hu, Bijie; Sun, Ziyong; Huang, Wenxiang; Wang, Yong; Wu, Anhua; Feng, Xianju; Luo, Yanping; Hu, Zhidong; Chu, Yunzhuo; Chen, Shulan; Cao, Bin; Su, Jianrong; Gui, Bingdong; Duan, Qiong; Zhang, Shufang; Shao, Haifeng; Kong, Haishen; Badal, Robert E; Xu, Yingchun

    2015-10-19

    To evaluate the antimicrobial susceptibility of Gram-negative bacilli that caused hospital-acquired and community-acquired intra-abdominal infections (IAIs) in China between 2012 and 2013, we determined the susceptibilities to 12 antimicrobials and the extended-spectrum β-lactamase (ESBL) statuses of 3,540 IAI isolates from seven geographic areas in China in a central laboratory using CLSI broth microdilution and interpretive standards. Most infections were caused by Escherichia coli (46.3%) and Klebsiella pneumoniae (19.7%). Rates of ESBL-producing E. coli (P = 0.031), K. pneumoniae (P = 0.017), and Proteus mirabilis (P = 0.004) were higher in hospital-acquired IAIs than in community-acquired IAIs. Susceptibilities of enterobacteriaceae to ertapenem, amikacin, piperacillin-tazobactam, and imipenem were 71.3% to 100%, 81.3% to 100%, 64.7% to 100%, and 83.1% to 100%, respectively, but imipenem was ineffective against P. mirabilis (<20%). Although most ESBL-positive hospital-acquired isolates were resistant to third- and fourth-generation cephalosporins, the majority were susceptible to cefoxitin (47.9% to 83.9%). Susceptibilities of ESBL-positive isolates to ampicillin-sulbactam (<10%) were low, whereas susceptibilities to ciprofloxacin (0% to 54.6%) and levofloxacin (0% to 63.6%) varied substantially. The prevalences of cephalosporin-susceptible E. coli and K. pneumoniae were higher in the northeastern and southern regions than in the central and eastern regions, reflecting the ESBL-positive rates in these areas, and were lowest in the Jiangsu-Zhejiang (Jiang-Zhe) area where the rates of carbapenem resistance were also highest. Ertapenem, amikacin, piperacillin-tazobactam, and imipenem are the most efficacious antibiotics for treating IAIs in China, especially those caused by E. coli or K. pneumoniae. Resistance to cephalosporins and carbapenems is more common in the Jiang-Zhe area than in other regions in China.

  1. Antimicrobial Susceptibilities of Aerobic and Facultative Gram-Negative Bacilli from Intra-abdominal Infections in Patients from Seven Regions in China in 2012 and 2013

    PubMed Central

    Zhang, Hui; Yang, Qiwen; Liao, Kang; Ni, Yuxing; Yu, Yunsong; Hu, Bijie; Sun, Ziyong; Huang, Wenxiang; Wang, Yong; Wu, Anhua; Feng, Xianju; Luo, Yanping; Hu, Zhidong; Chu, Yunzhuo; Chen, Shulan; Cao, Bin; Su, Jianrong; Gui, Bingdong; Duan, Qiong; Zhang, Shufang; Shao, Haifeng; Kong, Haishen; Badal, Robert E.

    2015-01-01

    To evaluate the antimicrobial susceptibility of Gram-negative bacilli that caused hospital-acquired and community-acquired intra-abdominal infections (IAIs) in China between 2012 and 2013, we determined the susceptibilities to 12 antimicrobials and the extended-spectrum β-lactamase (ESBL) statuses of 3,540 IAI isolates from seven geographic areas in China in a central laboratory using CLSI broth microdilution and interpretive standards. Most infections were caused by Escherichia coli (46.3%) and Klebsiella pneumoniae (19.7%). Rates of ESBL-producing E. coli (P = 0.031), K. pneumoniae (P = 0.017), and Proteus mirabilis (P = 0.004) were higher in hospital-acquired IAIs than in community-acquired IAIs. Susceptibilities of enterobacteriaceae to ertapenem, amikacin, piperacillin-tazobactam, and imipenem were 71.3% to 100%, 81.3% to 100%, 64.7% to 100%, and 83.1% to 100%, respectively, but imipenem was ineffective against P. mirabilis (<20%). Although most ESBL-positive hospital-acquired isolates were resistant to third- and fourth-generation cephalosporins, the majority were susceptible to cefoxitin (47.9% to 83.9%). Susceptibilities of ESBL-positive isolates to ampicillin-sulbactam (<10%) were low, whereas susceptibilities to ciprofloxacin (0% to 54.6%) and levofloxacin (0% to 63.6%) varied substantially. The prevalences of cephalosporin-susceptible E. coli and K. pneumoniae were higher in the northeastern and southern regions than in the central and eastern regions, reflecting the ESBL-positive rates in these areas, and were lowest in the Jiangsu-Zhejiang (Jiang-Zhe) area where the rates of carbapenem resistance were also highest. Ertapenem, amikacin, piperacillin-tazobactam, and imipenem are the most efficacious antibiotics for treating IAIs in China, especially those caused by E. coli or K. pneumoniae. Resistance to cephalosporins and carbapenems is more common in the Jiang-Zhe area than in other regions in China. PMID:26482308

  2. Surveillance of antimicrobial susceptibility of aerobic and facultative Gram-negative bacilli isolated from patients with intra-abdominal infections in China: the 2002-2009 Study for Monitoring Antimicrobial Resistance Trends (SMART).

    PubMed

    Yang, Qiwen; Wang, Hui; Chen, Minjun; Ni, Yuxing; Yu, Yunsong; Hu, Bijie; Sun, Ziyong; Huang, Wenxiang; Hu, Yunjian; Ye, Huifen; Badal, Robert E; Xu, Yingchun

    2010-12-01

    The objective of this study was to investigate the distribution and susceptibility of aerobic and facultative Gram-negative bacilli (GNB) isolated from patients with intra-abdominal infections (IAIs) in China. From 2002 to 2009, minimum inhibitory concentrations of 14 antibiotics for 3420 aerobic and facultative GNB from up to eight hospitals in six cities were determined by the broth microdilution method. Enterobacteriaceae comprised 82.9% (2834/3420) of the total isolates, with Escherichia coli (49.2%) being the most commonly isolated species followed by Klebsiella pneumoniae (17.0%), Enterobacter cloacae (5.8%) and Citrobacter freundii (2.3%). Amongst the antimicrobial agents tested, the three carbapenems (ertapenem, imipenem and meropenem) were the most active agents against Enterobacteriaceae, with susceptibility rates of 96.1-99.6% (2002-2009), 98.2-100% (2002-2009) and 99.6-100% (2002-2004), respectively, followed by amikacin (86.8-95.1%) and piperacillin/tazobactam (84.5-94.3%). Susceptibility rates of all tested third- and fourth-generation cephalosporins against Enterobacteriaceae declined by nearly 30%, with susceptibility rates of 40.2%, 39.1%, 56.3% and 51.8% in 2009 for ceftriaxone, cefotaxime, ceftazidime and cefepime, respectively. The occurrence of extended-spectrum β-lactamases increased rapidly, especially for E. coli (from 20.8% in 2002 to 64.9% in 2009). Susceptibility of E. coli to ciprofloxacin decreased from 57.6% in 2002 to 24.2% in 2009. The least active agent against Enterobacteriaceae was ampicillin/sulbactam (SAM) (25.3-44.3%). In conclusion, Enterobacteriaceae were the major pathogens causing IAIs, and carbapenems retained the highest susceptibility rates over the 8-year study period. Third- and fourth-generation cephalosporins, fluoroquinolones and SAM may not be ideal choices for empirical therapy of IAIs in China.

  3. Gemmatimonas aurantiaca gen. nov., sp. nov., a gram-negative, aerobic, polyphosphate-accumulating micro-organism, the first cultured representative of the new bacterial phylum Gemmatimonadetes phyl. nov.

    PubMed

    Zhang, Hui; Sekiguchi, Yuji; Hanada, Satoshi; Hugenholtz, Philip; Kim, Hongik; Kamagata, Yoichi; Nakamura, Kazunori

    2003-07-01

    A phylogenetically novel aerobic bacterium was isolated from an anaerobic-aerobic sequential batch reactor operated under enhanced biological phosphorus removal conditions for wastewater treatment. The isolation strategy used targeted slowly growing polyphosphate-accumulating bacteria by combining low-speed centrifugations and prolonged incubation on a low-nutrient medium. The isolate, designated strain T-27T, was a gram-negative, rod-shaped aerobe. Cells often appeared to divide by budding replication. Strain T-27T grew at 25-35 degrees C with an optimum growth temperature of 30 degrees C, whilst no growth was observed below 20 degrees C or above 37 degrees C within 20 days incubation. The pH range for growth was 6.5-9.5, with an optimum at pH 7.0. Strain T-27T was able to utilize a limited range of substrates, such as yeast extract, polypepton, succinate, acetate, gelatin and benzoate. Neisser staining was positive and 4,6-diamidino-2-phenylindole-stained cells displayed a yellow fluorescence, indicative of polyphosphate inclusions. Menaquinone 9 was the major respiratory quinone. The cellular fatty acids of the strain were mainly composed of iso-C15:0, C16:1 and C14:0. The G + C content of the genomic DNA was 66 mol%. Comparative analyses of 16S rRNA gene sequences indicated that strain T-27T belongs to candidate division BD (also called KS-B), a phylum-level lineage in the bacterial domain, to date comprised exclusively of environmental 16S rDNA clone sequences. Here, a new genus and species are proposed, Gemmatimonas aurantiaca (type strain T-27T=JCM 11422T=DSM 14586T) gen. nov., sp. nov., the first cultivated representative of the Gemmatimonadetes phyl. nov. Environmental sequence data indicate that this phylum is widespread in nature and has a phylogenetic breadth (19% 16S rDNA sequence divergence) that is greater than well-known phyla such as the Actinobacteria (18% divergence).

  4. Revisiting the gram-negative lipoprotein paradigm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The processing of lipoproteins (lpps) in Gram-negative bacteria is generally considered to be an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein n-acyltransferase. The mature lipoproteins are then sorted...

  5. Revisiting the Gram-Negative Lipoprotein Paradigm

    PubMed Central

    LoVullo, Eric D.; Wright, Lori F.; Isabella, Vincent; Huntley, Jason F.

    2015-01-01

    ABSTRACT The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS Δlnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the Δlnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. IMPORTANCE This paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria. PMID:25755189

  6. Oligoflexus tunisiensis gen. nov., sp. nov., a Gram-negative, aerobic, filamentous bacterium of a novel proteobacterial lineage, and description of Oligoflexaceae fam. nov., Oligoflexales ord. nov. and Oligoflexia classis nov.

    PubMed Central

    Nakai, Ryosuke; Nishijima, Miyuki; Tazato, Nozomi; Handa, Yutaka; Karray, Fatma; Sayadi, Sami; Isoda, Hiroko

    2014-01-01

    A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria. The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia. PMID:25013226

  7. Antimicrobial susceptibility and extended-spectrum beta-lactamase rates in aerobic gram-negative bacteria causing intra-abdominal infections in Vietnam: report from the Study for Monitoring Antimicrobial Resistance Trends (SMART 2009-2011).

    PubMed

    Biedenbach, Douglas J; Bouchillon, Samuel K; Hoban, Daryl J; Hackel, Meredith; Phuong, Doan Mai; Nga, Tran Thi Thanh; Phuong, Nguyen Tran My; Phuong, Tran Thi Lan; Badal, Robert E

    2014-08-01

    Treatment options for multidrug-resistant pathogens remain problematic in many regions and individual countries, warranting ongoing surveillance and analysis. Limited antimicrobial susceptibility information is available for pathogens from Vietnam. This study determined the bacterial susceptibility of aerobic gram-negative pathogens of intra-abdominal infections among patients in Vietnam during 2009-2011. A total of 905 isolates were collected from 4 medical centers in this investigation as part of the Study for Monitoring Antimicrobial Resistance Trends. Antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) rates among the appropriate species were determined by a central laboratory using Clinical and Laboratory Standards Institute methods. Among the species collected, Escherichia coli (48.1% ESBL-positive) and Klebsiella pneumoniae (39.5% ESBL-positive) represented the majority (46.4%) of the isolates submitted for this study. Ertapenem MIC90 values were lowest for these 2 species at 0.12 and 0.25μg/mL and remained unchanged for ESBL-positive isolates. Imipenem MIC90 values were also the same for all isolates and ESBL-positive strains at 0.25 and 0.5μg/mL, respectively. Ertapenem MIC90 values for additional species with sufficient numbers for analysis, including Enterobacter cloacae, Proteus mirabilis, Acinetobacter baumannii, and Pseudomonas aeruginosa, were 1, 0.06, >4, and >4μg/mL, respectively. Analysis of beta-lactamases in a subset of 132 phenotypically ESBL-positive Enterobacteriaceae demonstrated that CTX-M variants, particularly CTX-M-27 and CTX-M-15, were the predominant enzymes. High resistance rates in Vietnam hospitals dictate continuous monitoring as antimicrobial inactivating enzymes continue to spread throughout Asia and globally.

  8. Gram-Negative Bacterial Wound Infections

    DTIC Science & Technology

    2014-05-01

    Infections PRINCIPAL INVESTIGATOR: Luis A. Actis CONTRACTING ORGANIZATION: Miami University, Oxford, OH 45056 REPORT DATE: May 2014...SUBTITLE Gram-negative bacterial wound infections 5a. CONTRACT NUMBER W81XWH-12-2-0035 5b. GRANT NUMBER W81XWH-12-2-0035 5c. PROGRAM...laboratory conditions as well as to infect and kill G. mellonella larvae and BALB/c mice in experimental infection assays. These results validate

  9. Clinical evaluation of moxalactam: evidence of decreased efficacy in gram-positive aerobic infections.

    PubMed Central

    Salzer, W; Pegram, P S; McCall, C E

    1983-01-01

    Moxalactam was used as initial, empirical therapy in 69 patients with a variety of serious bacterial infections, 32% of which were accompanied by bacteremia. Overall, the success rate was 83% and drug-related adverse effects were minimal. The drug was less efficacious in infections caused by aerobic gram-positive pathogens than it was in those caused by gram-negative pathogens. The following gram-positive organisms were associated with special problems during moxalactam therapy: Streptococcus pneumoniae (development of meningitis and a relapse of pneumonia with a more resistant strain), Staphylococcus epidermidis (in vivo emergence of moxalactam resistance, and the enterococci (failure of therapy and a fatal superinfection. Moxalactam performed well in infections caused by most gram-negative organisms, including aminoglycoside-resistant strains, but the previously reported emergence of gram-negative bacillary resistance to moxalactam during therapy was reconfirmed in our series with Serratia marcescens. The use of moxalactam in the treatment of gram-negative meningitis was further supported by a patient with meningitis-ventriculitis caused by Bacteroides fragilis who was cured with moxalactam after failure on chloramphenicol. PMID:6222696

  10. Molecular Organization of Gram-Negative Peptidoglycan

    SciTech Connect

    Gan, L.; Chen, S.; Jensen, G.J.

    2009-05-18

    The stress-bearing component of the bacterial cell wall--a multi-gigadalton bag-like molecule called the sacculus--is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: 'Layered' and 'Scaffold', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozen-hydrated sacculi through electron cryotomography. In the Gram-negative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.

  11. [Septic arthritis in two young children caused by unusual gram-negative pathogens].

    PubMed

    Bruijn, J; Verhage, J; Bosboom, R W; Brus, F

    2000-07-29

    Two children, a girl aged 2 years and a boy aged 10 months, were moderately ill with signs of inflammation of the left and the right knee, respectively. Both had had pharyngitis, and the boy also had paronychia of the right foot. The Gram preparation of synovial fluid showed Gram-positive cocci in the girl, while Kingella kingae was cultured. In the boy, a Moraxella was cultured from the synovial fluid using an aerobic blood culture system. Both recovered without sequelae after adequate antibiotic treatment. The micro-organisms cultured were Gram-negative bacteria, which are rarely seen in septic arthritis and are difficult to demonstrate. In young children, septic arthritis often presents with mild symptoms and inconclusive laboratory findings. Even if the Gram preparation of the synovial fluid shows no micro-organisms, unusual pathogens may be isolated by means of an aerobic blood culture system.

  12. Rapid diagnosis of gram negative pneumonia by assay of endotoxin in bronchoalveolar lavage fluid.

    PubMed Central

    Pugin, J; Auckenthaler, R; Delaspre, O; van Gessel, E; Suter, P M

    1992-01-01

    BACKGROUND: Diagnosis of ventilator associated pneumonia can be made by quantitative cultures of bronchoalveolar lavage fluid or of protected specimen brushings, though cultures require 24-48 hours to provide results. In 80% of cases aerobic Gram negative bacteria are the cause. METHODS: A rapid diagnostic method of assessing the endotoxin content of lavage fluid by Limulus assay is described. Forty samples of lavage fluid were obtained from patients with multiple trauma requiring mechanical ventilation for a prolonged period. Pneumonia was diagnosed on the basis of clinical, radiological, and bacteriological findings, including quantitative cultures of lavage fluid. RESULTS: A relation was observed between the concentration of endotoxin in lavage fluid and the quantity of Gram negative bacteria. The median endotoxin content of lavage fluid in Gram negative bacterial pneumonia was 15 endotoxin units (EU)/ml; the range observed in individual patients was 6 to > 150 EU/ml. In patients with pneumonia due to Gram positive cocci and in non-infected patients the median endotoxin level was 0.17 (range < or = 0.06 to 2) EU/ml. An endotoxin level greater than or equal to 6 EU/ml distinguished patients with Gram negative bacterial pneumonia from colonised patients and from those with pneumonia due to Gram positive cocci. CONCLUSION: The measurement of endotoxin in lavage fluid is a rapid (less than two hours) and accurate diagnostic method. It should allow specific and early treatment of Gram negative bacterial pneumonia. PMID:1412100

  13. Multidrug resistance in hydrocarbon-tolerant Gram-positive and Gram-negative bacteria.

    PubMed

    Stancu, Mihaela Marilena; Grifoll, Magdalena

    2011-01-01

    New Gram-positive and Gram-negative bacteria were isolated from Poeni oily sludge, using enrichment procedures. The six Gram-positive strains belong to Bacillus, Lysinibacillus and Rhodococcus genera. The eight Gram-negative strains belong to Shewanella, Aeromonas, Pseudomonas and Klebsiella genera. Isolated bacterial strains were tolerant to saturated (i.e., n-hexane, n-heptane, n-decane, n-pentadecane, n-hexadecane, cyclohexane), monoaromatic (i.e., benzene, toluene, styrene, xylene isomers, ethylbenzene, propylbenzene) and polyaromatic (i.e., naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons, and also resistant to different antimicrobial agents (i.e., ampicillin, kanamycin, rhodamine 6G, crystal violet, malachite green, sodium dodecyl sulfate). The presence of hydrophilic antibiotics like ampicillin or kanamycin in liquid LB-Mg medium has no effects on Gram-positive and Gram-negative bacteria resistance to toxic compounds. The results indicated that Gram-negative bacteria are less sensitive to toxic compounds than Gram-positive bacteria, except one bacteria belonging to Lysinibacillus genus. There were observed cellular and molecular modifications induced by ampicillin or kanamycin to isolated bacterial strains. Gram-negative bacteria possessed between two and four catabolic genes (alkB, alkM, alkB/alkB1, todC1, xylM, PAH dioxygenase, catechol 2,3-dioxygenase), compared with Gram-positive bacteria (except one bacteria belonging to Bacillus genus) which possessed one catabolic gene (alkB/alkB1). Transporter genes (HAE1, acrAB) were detected only in Gram-negative bacteria.

  14. Fused-Ring Oxazolopyrrolopyridopyrimidine Systems with Gram-Negative Activity

    PubMed Central

    Chen, Yiyuan; Moloney, Jonathan G.; Christensen, Kirsten E.; Moloney, Mark G.

    2017-01-01

    Fused polyheterocyclic derivatives are available by annulation of a tetramate scaffold, and been shown to have antibacterial activity against a Gram-negative, but not a Gram-positive, bacterial strain. While the activity is not potent, these systems are structurally novel showing, in particular, a high level of polarity, and offer potential for the optimization of antibacterial activity. PMID:28098784

  15. Gram-negative bacteria can also form pellicles.

    PubMed

    Armitano, Joshua; Méjean, Vincent; Jourlin-Castelli, Cécile

    2014-12-01

    There is a growing interest in the bacterial pellicle, a biofilm floating at the air-liquid interface. Pellicles have been well studied in the Gram-positive bacterium Bacillus subtilis, but far less in Gram-negative bacteria, where pellicle studies have mostly focused on matrix components rather than on the regulatory cascades involved. Several Gram-negative bacteria, including pathogenic bacteria, have been shown to be able to form a pellicle under static conditions. Here, we summarize the growing body of knowledge about pellicle formation in Gram-negative bacteria, especially about the components of the pellicle matrix. We also propose that the pellicle is a specific biofilm, and that its formation involves particular processes. Since this lifestyle concerns a growing number of bacteria, its properties undoubtedly deserve further investigation.

  16. Silver enhances antibiotic activity against gram-negative bacteria.

    PubMed

    Morones-Ramirez, Jose Ruben; Winkler, Jonathan A; Spina, Catherine S; Collins, James J

    2013-06-19

    A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive-specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

  17. Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma

    NASA Astrophysics Data System (ADS)

    Mai-Prochnow, Anne; Clauson, Maryse; Hong, Jungmi; Murphy, Anthony B.

    2016-12-01

    Cold atmospheric-pressure plasma (CAP) is a relatively new method being investigated for antimicrobial activity. However, the exact mode of action is still being explored. Here we report that CAP efficacy is directly correlated to bacterial cell wall thickness in several species. Biofilms of Gram positive Bacillus subtilis, possessing a 55.4 nm cell wall, showed the highest resistance to CAP, with less than one log10 reduction after 10 min treatment. In contrast, biofilms of Gram negative Pseudomonas aeruginosa, possessing only a 2.4 nm cell wall, were almost completely eradicated using the same treatment conditions. Planktonic cultures of Gram negative Pseudomonas libanensis also had a higher log10 reduction than Gram positive Staphylococcus epidermidis. Mixed species biofilms of P. aeruginosa and S. epidermidis showed a similar trend of Gram positive bacteria being more resistant to CAP treatment. However, when grown in co-culture, Gram negative P. aeruginosa was more resistant to CAP overall than as a mono-species biofilm. Emission spectra indicated OH and O, capable of structural cell wall bond breakage, were present in the plasma. This study indicates that cell wall thickness correlates with CAP inactivation times of bacteria, but cell membranes and biofilm matrix are also likely to play a role.

  18. Multiple Responses of Gram-Positive and Gram-Negative Bacteria to Mixture of Hydrocarbons

    PubMed Central

    Marilena Lăzăroaie, Mihaela

    2010-01-01

    Most of our knowledge about pollutants and the way they are biodegraded in the environment has previously been shaped by laboratory studies using hydrocarbon-degrading bacterial strains isolated from polluted sites. In present study Gram-positive (Mycobacterium sp. IBBPo1, Oerskovia sp. IBBPo2, Corynebacterium sp. IBBPo3) and Gram-negative (Chryseomonas sp. IBBPo7, Pseudomonas sp. IBBPo10, Burkholderia sp. IBBPo12) bacteria, isolated from oily sludge, were found to be able to tolerate pure and mixture of saturated hydrocarbons, as well as pure and mixture of monoaromatic and polyaromatic hydrocarbons. Isolated Gram-negative bacteria were more tolerant to mixture of saturated (n-hexane, n-hexadecane, cyclohexane), monoaromatic (benzene, toluene, ethylbenzene) and polyaromatic (naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons than Gram-positive bacteria. There were observed cellular and molecular modifications induced by mixture of saturated, monoaromatic and polyaromatic hydrocarbons to Gram-positive and Gram-negative bacteria. These modifications differ from one strain to another and even for the same bacterial strain, according to the nature of hydrophobic substrate. PMID:24031541

  19. Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma

    PubMed Central

    Mai-Prochnow, Anne; Clauson, Maryse; Hong, Jungmi; Murphy, Anthony B.

    2016-01-01

    Cold atmospheric-pressure plasma (CAP) is a relatively new method being investigated for antimicrobial activity. However, the exact mode of action is still being explored. Here we report that CAP efficacy is directly correlated to bacterial cell wall thickness in several species. Biofilms of Gram positive Bacillus subtilis, possessing a 55.4 nm cell wall, showed the highest resistance to CAP, with less than one log10 reduction after 10 min treatment. In contrast, biofilms of Gram negative Pseudomonas aeruginosa, possessing only a 2.4 nm cell wall, were almost completely eradicated using the same treatment conditions. Planktonic cultures of Gram negative Pseudomonas libanensis also had a higher log10 reduction than Gram positive Staphylococcus epidermidis. Mixed species biofilms of P. aeruginosa and S. epidermidis showed a similar trend of Gram positive bacteria being more resistant to CAP treatment. However, when grown in co-culture, Gram negative P. aeruginosa was more resistant to CAP overall than as a mono-species biofilm. Emission spectra indicated OH and O, capable of structural cell wall bond breakage, were present in the plasma. This study indicates that cell wall thickness correlates with CAP inactivation times of bacteria, but cell membranes and biofilm matrix are also likely to play a role. PMID:27934958

  20. Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma.

    PubMed

    Mai-Prochnow, Anne; Clauson, Maryse; Hong, Jungmi; Murphy, Anthony B

    2016-12-09

    Cold atmospheric-pressure plasma (CAP) is a relatively new method being investigated for antimicrobial activity. However, the exact mode of action is still being explored. Here we report that CAP efficacy is directly correlated to bacterial cell wall thickness in several species. Biofilms of Gram positive Bacillus subtilis, possessing a 55.4 nm cell wall, showed the highest resistance to CAP, with less than one log10 reduction after 10 min treatment. In contrast, biofilms of Gram negative Pseudomonas aeruginosa, possessing only a 2.4 nm cell wall, were almost completely eradicated using the same treatment conditions. Planktonic cultures of Gram negative Pseudomonas libanensis also had a higher log10 reduction than Gram positive Staphylococcus epidermidis. Mixed species biofilms of P. aeruginosa and S. epidermidis showed a similar trend of Gram positive bacteria being more resistant to CAP treatment. However, when grown in co-culture, Gram negative P. aeruginosa was more resistant to CAP overall than as a mono-species biofilm. Emission spectra indicated OH and O, capable of structural cell wall bond breakage, were present in the plasma. This study indicates that cell wall thickness correlates with CAP inactivation times of bacteria, but cell membranes and biofilm matrix are also likely to play a role.

  1. Mid-infrared spectroscopic assessment of nanotoxicity in gram-negative vs. gram-positive bacteria.

    PubMed

    Heys, Kelly A; Riding, Matthew J; Strong, Rebecca J; Shore, Richard F; Pereira, M Glória; Jones, Kevin C; Semple, Kirk T; Martin, Francis L

    2014-03-07

    Nanoparticles appear to induce toxic effects through a variety of mechanisms including generation of reactive oxygen species (ROS), physical contact with the cell membrane and indirect catalysis due to remnants from manufacture. The development and subsequent increasing usage of nanomaterials has highlighted a growing need to characterize and assess the toxicity of nanoparticles, particularly those that may have detrimental health effects such as carbon-based nanomaterials (CBNs). Due to interactions of nanoparticles with some reagents, many traditional toxicity tests are unsuitable for use with CBNs. Infrared (IR) spectroscopy is a non-destructive, high throughput technique, which is unhindered by such problems. We explored the application of IR spectroscopy to investigate the effects of CBNs on Gram-negative (Pseudomonas fluorescens) and Gram-positive (Mycobacterium vanbaalenii PYR-1) bacteria. Two types of IR spectroscopy were compared: attenuated total reflection Fourier-transform infrared (ATR-FTIR) and synchrotron radiation-based FTIR (SR-FTIR) spectroscopy. This showed that Gram-positive and Gram-negative bacteria exhibit differing alterations when exposed to CBNs. Gram-positive bacteria appear more resistant to these agents and this may be due to the protection afforded by their more sturdy cell wall. Markers of exposure also vary according to Gram status; Amide II was consistently altered in Gram-negative bacteria and carbohydrate altered in Gram-positive bacteria. ATR-FTIR and SR-FTIR spectroscopy could both be applied to extract biochemical alterations induced by each CBN that were consistent across the two bacterial species; these may represent potential biomarkers of nanoparticle-induced alterations. Vibrational spectroscopy approaches may provide a novel means of fingerprinting the effects of CBNs in target cells.

  2. Insights into Newer Antimicrobial Agents Against Gram-negative Bacteria

    PubMed Central

    Taneja, Neelam; Kaur, Harsimran

    2016-01-01

    Currently, drug resistance, especially against cephalosporins and carbapenems, among gram-negative bacteria is an important challenge, which is further enhanced by the limited availability of drugs against these bugs. There are certain antibiotics (colistin, fosfomycin, temocillin, and rifampicin) that have been revived from the past to tackle the menace of superbugs, including members of Enterobacteriaceae, Acinetobacter species, and Pseudomonas species. Very few newer antibiotics have been added to the pool of existing drugs. There are still many antibiotics that are passing through various phases of clinical trials. The initiative of Infectious Disease Society of America to develop 10 novel antibiotics against gram-negative bacilli by 2020 is a step to fill the gap of limited availability of drugs. This review aims to provide insights into the current and newer drugs in pipeline for the treatment of gram-negative bacteria and also discusses the major challenging issues for their management. PMID:27013887

  3. Mechanisms of Antimicrobial Peptide Resistance in Gram-Negative Bacteria

    PubMed Central

    Band, Victor I.; Weiss, David S.

    2014-01-01

    Cationic antimicrobial peptides (CAMPs) are important innate immune defenses that inhibit colonization by pathogens and contribute to clearance of infections. Gram-negative bacterial pathogens are a major target, yet many of them have evolved mechanisms to resist these antimicrobials. These resistance mechanisms can be critical contributors to bacterial virulence and are often crucial for survival within the host. Here, we summarize methods used by Gram-negative bacteria to resist CAMPs. Understanding these mechanisms may lead to new therapeutic strategies against pathogens with extensive CAMP resistance. PMID:25927010

  4. Mechanisms of Antimicrobial Peptide Resistance in Gram-Negative Bacteria.

    PubMed

    Band, Victor I; Weiss, David S

    2015-03-01

    Cationic antimicrobial peptides (CAMPs) are important innate immune defenses that inhibit colonization by pathogens and contribute to clearance of infections. Gram-negative bacterial pathogens are a major target, yet many of them have evolved mechanisms to resist these antimicrobials. These resistance mechanisms can be critical contributors to bacterial virulence and are often crucial for survival within the host. Here, we summarize methods used by Gram-negative bacteria to resist CAMPs. Understanding these mechanisms may lead to new therapeutic strategies against pathogens with extensive CAMP resistance.

  5. Fluorescence studies of gram-positive and gram-negative bacteria

    NASA Astrophysics Data System (ADS)

    Blust, Brittni

    2012-02-01

    Autofluorescence is a relatively unexplored technique for identification. It is nondestructive, noncontact, fast, and has the potential to be integrated in small handheld devices. On the other hand, the autofluorescent signal is sometimes very week, or it can be overwhelmed by the emission of a surrounding medium. We are exploring the possibility to develop an optical method for identification of the Gram-type of bacterial cultures based on the autofluorescence. We have enhanced the detectivity of a standard fluorimeter using combination of bandpass and long pass filters. In this particular study, we are investigating if the previously observed difference in the autofluorescent spectra of Gram-positive and Gram-negative bacteria is dependent on the age of the culture. We have selected two types of bacteria, Kocuria rhizophila and Alcagenes faecalis, and we have monitored in equal time intervals of their development the autofluorescence spectra. The stages of development were monitored separately by measuring the turbidity and creating a growth curve. The goal of this study is to find out if the previously observed difference in the autofluorescence spectra of Gram-positive and Gram-negative bacteria is dependent on the stage of the development of the bacterial culture.

  6. Genome Sequences of Nine Gram-Negative Vaginal Bacterial Isolates

    PubMed Central

    Deitzler, Grace E.; Ruiz, Maria J.; Lu, Wendy; Weimer, Cory; Park, SoEun; Robinson, Lloyd S.; Hallsworth-Pepin, Kymberlie; Wollam, Aye; Mitreva, Makedonka

    2016-01-01

    The vagina is home to a wide variety of bacteria that have great potential to impact human health. Here, we announce reference strains (now available through BEI Resources) and draft genome sequences for 9 Gram-negative vaginal isolates from the taxa Citrobacter, Klebsiella, Fusobacterium, Proteus, and Prevotella. PMID:27688330

  7. Antimicrobial Photodynamic Therapy to Kill Gram-negative Bacteria

    PubMed Central

    Sperandio, Felipe F; Huang, Ying-Ying; Hamblin, Michael R

    2013-01-01

    Antimicrobial photodynamic therapy (PDT) or photodynamic inactivation (PDI) is a new promising strategy to eradicate pathogenic microorganisms such as Gram-positive and Gram-negative bacteria, yeasts and fungi. The search for new approaches that can kill bacteria but do not induce the appearance of undesired drug-resistant strains suggests that PDT may have advantages over traditional antibiotic therapy. PDT is a non-thermal photochemical reaction that involves the simultaneous presence of visible light, oxygen and a dye or photosensitizer (PS). Several PS have been studied for their ability to bind to bacteria and efficiently generate reactive oxygen species (ROS) upon photostimulation. ROS are formed through type I or II mechanisms and may inactivate several classes of microbial cells including Gram-negative bacteria such as Pseudomonas aeruginosa, which are typically characterized by an impermeable outer cell membrane that contains endotoxins and blocks antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall. This review covers significant peer-reviewed articles together with US and World patents that were filed within the past few years and that relate to the eradication of Gram-negative bacteria via PDI or PDT. It is organized mainly according to the nature of the PS involved and includes natural or synthetic food dyes; cationic dyes such as methylene blue and toluidine blue; tetrapyrrole derivatives such as phthalocyanines, chlorins, porphyrins, chlorophyll and bacteriochlorophyll derivatives; functionalized fullerenes; nanoparticles combined with different PS; other formulations designed to target PS to bacteria; photoactive materials and surfaces; conjugates between PS and polycationic polymers or antibodies; and permeabilizing agents such as EDTA, PMNP and CaCl2. The present review also covers the different laboratory animal models normally used to treat Gram-negative bacterial infections with antimicrobial PDT. PMID

  8. Antimicrobial photodynamic therapy to kill Gram-negative bacteria.

    PubMed

    Sperandio, Felipe F; Huang, Ying-Ying; Hamblin, Michael R

    2013-08-01

    Antimicrobial photodynamic therapy (PDT) or photodynamic inactivation (PDI) is a new promising strategy to eradicate pathogenic microorganisms such as Gram-positive and Gram-negative bacteria, yeasts and fungi. The search for new approaches that can kill bacteria but do not induce the appearance of undesired drug-resistant strains suggests that PDT may have advantages over traditional antibiotic therapy. PDT is a non-thermal photochemical reaction that involves the simultaneous presence of visible light, oxygen and a dye or photosensitizer (PS). Several PS have been studied for their ability to bind to bacteria and efficiently generate reactive oxygen species (ROS) upon photo-stimulation. ROS are formed through type I or II mechanisms and may inactivate several classes of microbial cells including Gram-negative bacteria such as Pseudomonas aeruginosa, which are typically characterized by an impermeable outer cell membrane that contains endotoxins and blocks antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall. This review covers significant peer-reviewed articles together with US and World patents that were filed within the past few years and that relate to the eradication of Gram-negative bacteria via PDI or PDT. It is organized mainly according to the nature of the PS involved and includes natural or synthetic food dyes; cationic dyes such as methylene blue and toluidine blue; tetrapyrrole derivatives such as phthalocyanines, chlorins, porphyrins, chlorophyll and bacteriochlorophyll derivatives; functionalized fullerenes; nanoparticles combined with different PS; other formulations designed to target PS to bacteria; photoactive materials and surfaces; conjugates between PS and polycationic polymers or antibodies; and permeabilizing agents such as EDTA, PMNP and CaCl₂. The present review also covers the different laboratory animal models normally used to treat Gram-negative bacterial infections with antimicrobial PDT.

  9. 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.

  10. 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

  11. Resistance to aminoglycoside antibiotics of gram-negative bacilli isolated in Canadian hospitals.

    PubMed Central

    Duncan, I B; Cheung, E Y; Haldane, E V; Jackson, F L; McNaughton, R D; Morisset, R A; Noble, M A; Rennie, R P; Ronald, A R; Smith, J A

    1981-01-01

    A survey was made of the frequency of resistance to amikacin, gentamicin and tobramycin among aerobic gram-negative bacilli isolated over a 4-week period in 1979 at six large, geographically separated Canadian hospitals. In the entire series of 4407 isolates the frequency of resistance was 2.5% to amikacin, 8.1% to gentamicin, 5.9% to tobramycin and 1.7% to all three. Most (81%) of the resistant bacteria were acquired by the patients after admission to hospital. The frequency of resistance to the three aminoglycoside antibiotics in each hospital largely reflected the local rate of cross-infection by endemic strains of resistant bacteria. PMID:7237336

  12. Bacteriocins from Gram-Negative Bacteria: A Classification?

    NASA Astrophysics Data System (ADS)

    Rebuffat, Sylvie

    Bacteria produce an arsenal of toxic peptides and proteins, which are termed bacteriocins and play a role in mediating the dynamics of microbial populations and communities. Bacteriocins from Gram-negative bacteria arise mainly from Enterobacteriaceae. They assemble into two main families: high molecular mass modular proteins (30-80 kDa) termed colicins and low molecular mass peptides (between 1 and 10 kDa) termed microcins. The production of colicins is mediated by the SOS response regulon, which plays a role in the response of many bacteria to DNA damages. Microcins are highly stable hydrophobic peptides that are produced under stress conditions, particularly nutrient depletion. Colicins and microcins are found essentially in Escherichia coli, but several other Gram-negative species also produce bacteriocin-like substances. This chapter presents the basis of a classification of colicins and microcins.

  13. [Detection of resistance phenotypes in gram-negative bacteria].

    PubMed

    Navarro, Ferran; Calvo, Jorge; Cantón, Rafael; Fernández-Cuenca, Felipe; Mirelis, Beatriz

    2011-01-01

    Detecting resistance in gram-negative microorganisms has a strong clinical and epidemiological impact, but there is still a great deal of debate about the most sensitive phenotypic method and whether in vitro susceptibility results should be interpreted. The present work reviews the phenotypes and mechanisms of resistance to beta-lactams, quinolones and aminoglycosides in gram-negative bacilli and also revises the different phenotypic methods used for their detection. A clinical interpretation of in vitro susceptibility results is also discussed. Extended-spectrum and inhibitor resistant beta-lactamases, AmpC type beta-lactamases and carbapenemases are thoroughly reviewed. As regards quinolones, the resistance mediated both by plasmids and by mutations in the DNA gyrase and the topoisomerase IV genes is also reviewed. This report includes resistance patterns to aminoglycosides caused by modifying enzymes. Phenotypic detection of beta-lactam resistance in Neisseria spp. and Haemophilus influenzae is also reviewed in a separate section.

  14. Gram-negative bacterial molecules associate with Alzheimer disease pathology

    PubMed Central

    Stamova, Boryana; Jin, Lee-Way; DeCarli, Charles; Phinney, Brett; Sharp, Frank R.

    2016-01-01

    Objective: We determined whether Gram-negative bacterial molecules are associated with Alzheimer disease (AD) neuropathology given that previous studies demonstrate Gram-negative Escherichia coli bacteria can form extracellular amyloid and Gram-negative bacteria have been reported as the predominant bacteria found in normal human brains. Methods: Brain samples from gray and white matter were studied from patients with AD (n = 24) and age-matched controls (n = 18). Lipopolysaccharide (LPS) and E coli K99 pili protein were evaluated by Western blots and immunocytochemistry. Human brain samples were assessed for E coli DNA followed by DNA sequencing. Results: LPS and E coli K99 were detected immunocytochemically in brain parenchyma and vessels in all AD and control brains. K99 levels measured using Western blots were greater in AD compared to control brains (p < 0.01) and K99 was localized to neuron-like cells in AD but not control brains. LPS levels were also greater in AD compared to control brain. LPS colocalized with Aβ1-40/42 in amyloid plaques and with Aβ1-40/42 around vessels in AD brains. DNA sequencing confirmed E coli DNA in human control and AD brains. Conclusions: E coli K99 and LPS levels were greater in AD compared to control brains. LPS colocalized with Aβ1-40/42 in amyloid plaques and around vessels in AD brain. The data show that Gram-negative bacterial molecules are associated with AD neuropathology. They are consistent with our LPS-ischemia-hypoxia rat model that produces myelin aggregates that colocalize with Aβ and resemble amyloid-like plaques. PMID:27784770

  15. Gram-negative sepsis: a dilemma of modern medicine.

    PubMed Central

    Bone, R C

    1993-01-01

    Gram-negative sepsis is an increasingly common problem, with up to 300,000 cases occurring each year in the United States alone. Despite the ongoing development of new antibiotics, mortality from gram-negative sepsis remains unacceptably high. To stimulate earlier therapeutic intervention by physicians, a new set of broad definitions has been proposed to define the systemic inflammatory response characteristic of sepsis. In this review, the signs and symptoms of this progressive, injurious process are reviewed and its management is discussed, as are the mechanisms by which bacterial endotoxin triggers the biochemical events that lead to such serious complications as shock, adult respiratory distress syndrome, and disseminated intravascular coagulation. These events often occur even when appropriate antimicrobial therapy has been instituted. An increased understanding of the structure of endotoxin and its role in the development of sepsis, together with advances in hybridoma technology, has led to the development of monoclonal antibodies that bind to endotoxin and significantly attenuate its adverse effects. These agents promise to substantially reduce the morbidity and mortality associated with gram-negative sepsis. PMID:8457980

  16. The talking language in some major Gram-negative bacteria.

    PubMed

    Banerjee, Goutam; Ray, Arun Kumar

    2016-08-01

    Cell-cell interaction or quorum sensing (QS) is a vital biochemical/physiological process in bacteria that is required for various physiological functions, including nutrient uptake, competence development, biofilm formation, sporulation, as well as for toxin secretion. In natural environment, bacteria live in close association with other bacteria and interaction among them is crucial for survival. The QS-regulated gene expression in bacteria is a cell density-dependent process and the initiation process depends on the threshold level of the signaling molecule, N-acyl-homoserine lactone (AHL). The present review summarizes the QS signal and its respective circuit in Gram-negative bacteria. Most of the human pathogens belong to Gram-negative group, and only a few of them cause disease through QS system. Thus, inhibition of pathogenic bacteria is important. Use of antibiotics creates a selective pressure (antibiotics act as natural selection factor to promote one group of bacteria over another group) for emerging multidrug-resistant bacteria and will not be suitable for long-term use. The alternative process of inhibition of QS in bacteria using different natural and synthetic molecules is called quorum quenching. However, in the long run, QS inhibitors or blockers may also develop resistance, but obviously it will solve some sort of problems. In this review, we also have stated the mode of action of quorum-quenching molecule. The understanding of QS network in pathogenic Gram-negative bacteria will help us to solve many health-related problems in future.

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

    PubMed

    Yang, Haiyan; Tong, Meiping; Kim, Hyunjung

    2012-11-06

    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.

  18. Gram-negative intestinal indigenous microbiota from two Siluriform fishes in a tropical reservoir

    PubMed Central

    Duarte, Silvana; Silva, Flávia Cristina de Paula e; Zauli, Danielle Alves Gomes; Nicoli, Jacques Robert; Araújo, Francisco Gerson

    2014-01-01

    The Gram-negative intestinal microbiota of Hypostomus auroguttatus and Pimelodus maculatus, a detritivorous and an omnivorous fish species, respectively, were compared between fishes from the reservoir and the stretch of the river below the dam of the Funil hydroelectric plant, Rio de Janeiro, Brazil. Four selective culture media were used under aerobic and two under anaerobic conditions. The omnivorous species had microbiota with higher population levels compared to the detritivorous species. The number of morphotypes and population levels of total bacteria, vibrio and Bacteroides tended to be higher in summer and autumn in the reservoir, and not different in the river. The number of morphotypes of enterobacteria and total bacteria were higher in the lotic environment compared with the lentic one. The bacteria Aeromonas hydrophila and Plesiomonas shigelloides and the obligate anaerobic Fusobacterium mortiferum were the most frequently identified microorganisms in the intestine of both H. auroguttatus and P. maculatus. Both season and habitat influenced the Gram-negative intestinal microbiota of H. auroguttatus and P. maculatus. Environmental factors influenced the Gram-negative intestinal microbiota of both species with possible impact on the interrelationship between the fishes and their digestive ecosystem, although the gut microbiota composition of fishes may result from host-specific selective pressures within the gut. PMID:25763032

  19. 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.

  20. [Diagnostic and therapeutic management of Gram-negative infections].

    PubMed

    Bassetti, Matteo; Repetto, Ernestina

    2008-04-01

    Among Gram negative bacteria, Pseudomonas aeruginosa, the extended spectrum beta-lactamases (ESBL)-producing strains, Acinetobacter spp, in particular the multiresistant Acinetobacter baumannii, and Stenotrophomonas maltophilia are the most implicated micrororganisms in the ever more increasing problem of bacterial resistance. Possible solutions have to be searched, on one hand, in the use of new drugs but, on the other hand, in the re-evaluation of those already available drugs, possibly considering a new role for old drugs such as colistine and fosfomycin. Concerning ESBL-producing strains, the most recent data provided by EARSS report, in Italy, an incidence rate of 10-25 percent. The insurgence of an infection sustained by an ESBL+ve strain is strictly related to some well known risk factors, like the hospital stay itself, the disease severity, the length of stay in ICU, intubation and mechanical ventilation, catheterization, urinary or artery, and the past exposure to antibiotics. The raise in ESBL producing strains is closely related to the increasing use of cephalosporins. In the setting of a Gram negative infection, the combination therapy guarantees a higher coverage by reducing insurgence of possible resistance mechanisms, possibly resulting synergistic, and allowing a de-escalation therapy, although to this latter other problems, such as tolerability, costs and compliance, can be related. Another basic aspect to take into account of, in order to achieve the maximal efficacy of the antibiotic treatment, is the right dosage. In the idea to look for the best approach for the antibiotic treatment of a severe infection in a hospital setting, when a Gram negative aetiology is implicated, it can be possibly presumed that the right way consists in avoiding inappropriate antibiotic therapies, making therapeutic choices based on guidelines resulted from local epidemiological data, initiating the therapy promptly, avoiding excessive use of antibiotics, possibly

  1. Characterization and identification of gram-negative, nonfermentative bacteria.

    PubMed Central

    Oberhofer, T R; Rowen, J W; Cunningham, G F

    1977-01-01

    The morphological and physiological characteristics of 593 strains of nonfermentative, gram-negative bacteria are described. A battery of 46 tests was used to identify and differentiate strains representing 8 genera and 31 species of named and group-designated bacteria. Seven selected amides and organic salts were closely examined to determine their usefulness, individually or as a battery, in characterizing and identifying the organisms. Of these, allantoin and acetamide showed the most promise in differentiating the more commonly occurring organisms from biochemically similar species. Susceptiblilty patterns to 12 antimicrobics also proved useful in differentiation, especially among atypical strains. PMID:845246

  2. Elasticity of the Rod-Shaped Gram-Negative Eubacteria

    NASA Astrophysics Data System (ADS)

    Boulbitch, A.; Quinn, B.; Pink, D.

    2000-12-01

    We report a theoretical calculation of the elasticity of the peptidoglycan network, the only stress-bearing part of rod-shaped Gram-negative eubacteria. The peptidoglycan network consists of elastic peptides and inextensible glycan strands, and it has been proposed that the latter form zigzag filaments along the circumference of the cylindrical bacterial shell. The zigzag geometry of the glycan strands gives rise to nonlinear elastic behavior. The four elastic moduli of the peptidoglycan network depend on its stressed state. For a bacterium under physiological conditions the elasticity is proportional to the bacterial turgor pressure. Our results are in good agreement with recent measurements.

  3. 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).

  4. Inhaled Antibiotics for Gram-Negative Respiratory Infections.

    PubMed

    Wenzler, Eric; Fraidenburg, Dustin R; Scardina, Tonya; Danziger, Larry H

    2016-07-01

    Gram-negative organisms comprise a large portion of the pathogens responsible for lower respiratory tract infections, especially those that are nosocomially acquired, and the rate of antibiotic resistance among these organisms continues to rise. Systemically administered antibiotics used to treat these infections often have poor penetration into the lung parenchyma and narrow therapeutic windows between efficacy and toxicity. The use of inhaled antibiotics allows for maximization of target site concentrations and optimization of pharmacokinetic/pharmacodynamic indices while minimizing systemic exposure and toxicity. This review is a comprehensive discussion of formulation and drug delivery aspects, in vitro and microbiological considerations, pharmacokinetics, and clinical outcomes with inhaled antibiotics as they apply to disease states other than cystic fibrosis. In reviewing the literature surrounding the use of inhaled antibiotics, we also highlight the complexities related to this route of administration and the shortcomings in the available evidence. The lack of novel anti-Gram-negative antibiotics in the developmental pipeline will encourage the innovative use of our existing agents, and the inhaled route is one that deserves to be further studied and adopted in the clinical arena.

  5. Outer membrane protein biogenesis in Gram-negative bacteria

    PubMed Central

    Rollauer, Sarah E.; Sooreshjani, Moloud A.; Noinaj, Nicholas; Buchanan, Susan K.

    2015-01-01

    Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM. PMID:26370935

  6. The complete general secretory pathway in gram-negative bacteria.

    PubMed Central

    Pugsley, A P

    1993-01-01

    The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins. PMID:8096622

  7. Polyethyleneimine is an effective permeabilizer of gram-negative bacteria.

    PubMed

    Helander, I M; Alakomi, H L; Latva-Kala, K; Koski, P

    1997-10-01

    The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 micrograms ml-1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.

  8. Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

    PubMed

    Tunjungputri, Rahajeng N; van de Heijden, Wouter; Urbanus, Rolf T; de Groot, Philip G; van der Ven, Andre; de Mast, Quirijn

    2016-12-29

    Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.

  9. Mechanistic antimicrobial approach of extracellularly synthesized silver nanoparticles against gram positive and gram negative bacteria.

    PubMed

    Tamboli, Dhawal P; Lee, Dae Sung

    2013-09-15

    The development of eco-friendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest in nanotechnology. In this study, an extracellular enzyme system of a newly isolated microorganism, Exiguobacterium sp. KNU1, was used for the reduction of AgNO₃ solutions to silver nanoparticles (AgNPs). The extracellularly biosynthesized AgNPs were characterized by UV-vis spectroscopy, Fourier transform infra-red spectroscopy and transmission electron microscopy. The AgNPs were approximately 30 nm (range 5-50 nm) in size, well-dispersed and spherical. The AgNPs were evaluated for their antimicrobial effects on different gram negative and gram positive bacteria using the minimum inhibitory concentration method. Reasonable antimicrobial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was observed. The morphological changes occurred in all the microorganisms tested. In particular, E. coli exhibited DNA fragmentation after being treated with the AgNPs. Finally, the mechanism for their bactericidal activity was proposed according to the results of scanning electron microscopy and single cell gel electrophoresis.

  10. Dynamics of Gram-negative bacteria population density in a soil in the course of the succession initiated by chitin and cellulose

    NASA Astrophysics Data System (ADS)

    Konstantin, Ivanov; Lubov, Polyanskaya

    2014-05-01

    The functions of actinomycetes in polymer destruction in soil traditionally considered as the dominant, compare to another groups of bacteria. Gram-positive bacteria also have ecological functions in destruction of soil organic matter. The role of Gram-negative bacteria has been researched in the microbial succession in terms of polymers destruction, which are widely spreads in soils: chitin and cellulose. The method with nalidixic acid as an inhibitor of DNA division of Gram-negative bacteria was modified. By modified method microbial succession of Gram-negative bacteria in the different horizons of a chernozem under aerobic and anaerobic conditions was researched. Chitin and cellulose as the source of nutrients with moistening was used in experiments. The introduction of chitin had no positive effect on the population density of Gram-negative bacteria in a chernozem, but it advanced the date of their appearance in microbial succession: the maximum of Gram-negative bacteria population density was registered on the 3rd- 7th day of the experiment with adding chitin. Compare to the control, which one was without any nutrient adding this dynamics registered much earlier. Consequently, the introduction of chitin as an additional source of nutrition promoted revealing of the Gram-negative bacteria in soil already at the early stages of the succession. In the course of the succession, when the fungal mycelium begins to die off, the actinomycetic mycelium increases in length, i.e., Gram-negative bacteria are replaced at this stage with Gram-positive ones, the leading role among which belongs to actinomycetes. The growth rate of Gram-negative bacteria is higher than that of actinomycetes, so they start chitin utilization at the early stages of the succession, whereas actinomycetes dominate at the late stages. The population density of Gram-negative bacteria was lower under the anaerobic conditions as compared with that in the aerobic ones. The population density of Gram-negative

  11. A Gestalt approach to Gram-negative entry.

    PubMed

    Silver, Lynn L

    2016-12-15

    A major obstacle confronting the discovery and development of new antibacterial agents to combat resistant Gram-negative (GN) organisms is the lack of a rational process for endowing compounds with properties that allow (or promote) entry into the bacterial cytoplasm. The major permeability difference between GN and Gram-positive (GP) bacteria is the GN outer membrane (OM) which is a permeability barrier itself and potentiates efflux pumps that expel compounds. Based on the fact that OM-permeable and efflux-deleted GNs are sensitive to many anti-GP drugs, recent efforts to approach the GN entry problem have focused on ways of avoiding efflux and transiting or compromising the OM, with the tacit assumption that this could allow entry of compounds into the GN cytoplasm. But bypassing the OM and efflux obstacles does not take into account the additional requirement of penetrating the cytoplasmic membrane (CM) whose sieving properties appear to be orthogonal to that of the OM. That is, tailoring compounds to transit the OM may well compromise their ability to enter the cytoplasm. Thus, a Gestalt approach to understanding the chemical requirements for GN entry seems a useful adjunct. This might consist of characterizing compounds which reach the cytoplasm, grouping (or binning) by routes of entry and formulating chemical 'rules' for those bins. This will require acquisition of data on large numbers of compounds, using non-activity-dependent methods of measuring accumulation in the cytoplasm.

  12. 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...

  13. Rapid method for identification of gram-negative, nonfermentative bacilli.

    PubMed Central

    Otto, L A; Pickett, M J

    1976-01-01

    A rapid system (OA), based on oxidative attack of substrates, was developed for identification of gram-negative, nonfermentative bacillia (NFB). One hundred and twelve strains of NFB from 25 species (representing the genera Pseudomonas, Alcaligenes, Acinetobacter, Bordetella, Flavobacterium, Moraxella, and Xanthomonas) were assayed by OA, buffered single substrate, and oxidative/fermentative methods. The 38 substrates consisted of salts of organic acids, nitrogen-containing compounds, alcohols, and carbohydrates. Ninety-four percent of the test strains were identified by the OA method in 24 h, and 99% were identifiable in 48 h. Reproducibility was 99%. Correlation with buffered single substrate was 98% (all substrates) and 90% with the oxidative/fermentative method (carbohydrates only). Biochemical profiles of all strains are presented, as well as tables showing the most useful tests for identification. PMID:780371

  14. Detection of pathogenic gram negative bacteria using infrared thermography

    NASA Astrophysics Data System (ADS)

    Lahiri, B. B.; Divya, M. P.; Bagavathiappan, S.; Thomas, Sabu; Philip, John

    2012-11-01

    Detection of viable bacteria is of prime importance in all fields of microbiology and biotechnology. Conventional methods of enumerating bacteria are often time consuming and labor-intensive. All living organisms generate heat due to metabolic activities and hence, measurement of heat energy is a viable tool for detection and quantification of bacteria. In this article, we employ a non-contact and real time method - infrared thermography (IRT) for measurement of temperature variations in four clinically significant gram negative pathogenic bacteria, viz. Vibrio cholerae, Vibrio mimicus, Proteus mirabilis and Pseudomonas aeruginosa. We observe that, the energy content, defined as the ratio of heat generated by bacterial metabolic activities to the heat lost from the liquid medium to the surrounding, vary linearly with the bacterial concentration in all the four pathogenic bacteria. The amount of energy content observed in different species is attributed to their metabolisms and morphologies that affect the convection velocity and hence heat transport in the medium.

  15. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

    PubMed Central

    Whitney, J.C.; Howell, P.L.

    2014-01-01

    The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane β-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-N-acetylglucosamine (PNAG) secretion systems. PMID:23117123

  16. Polymyxins: a new hope in combating Gram-negative superbugs?

    PubMed

    Velkov, Tony; Roberts, Kade D; Thompson, Philip E; Li, Jian

    2016-06-01

    Polymyxins have emerged as an important last-line of defense against Gram-negative 'superbugs'. Unfortunately, the effective use of polymyxins in the clinic has been hampered by their nephrotoxic side effects. Over the last 10 years various industry and academic groups across the globe have been trying to develop new polymyxins that are safer and more efficacious than the currently approved polymyxin B and colistin. However these drug discovery programs are yet to deliver a new and improved polymyxin drug into the clinic. In this piece we provide an overview of the current state of these polymyxin drug discovery programs from a medicinal chemistry perspective as well as some thoughts on how future drug discovery efforts may ultimately find success.

  17. Gram-Negative Bacterial Sensors for Eukaryotic Signal Molecules

    PubMed Central

    Lesouhaitier, Olivier; Veron, Wilfried; Chapalain, Annelise; Madi, Amar; Blier, Anne-Sophie; Dagorn, Audrey; Connil, Nathalie; Chevalier, Sylvie; Orange, Nicole; Feuilloley, Marc

    2009-01-01

    Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction mechanism involved in

  18. Kinase activity profiling of gram-negative pneumonia.

    PubMed

    Hoogendijk, Arie J; Diks, Sander H; Peppelenbosch, Maikel P; Van Der Poll, Tom; Wieland, Catharina W

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential to immune cells. The current study explored signal transduction events during murine Gram-negative pneumonia using a systems biology approach. Kinase activity arrays enable the analysis of 1,024 consensus sequences of protein kinase substrates. Using a kinase activity array on whole lung lysates, cellular kinase activities were determined in a mouse model of K. pneumoniae pneumonia. Notable kinase activities also were validated with phospho-specific Western blots. On the basis of the profiling data, mitogen-activated protein kinase (MAPK) signaling via p42 mitogen-activated protein kinase (p42) and p38 mitogen-activated protein kinase (p38) and transforming growth factor β (TGFβ) activity were reduced during infection, whereas v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) activity generally was enhanced. AKT signaling was represented in both metabolic and inflammatory (mitogen-activated protein kinase kinase 2 [MKK], apoptosis signal-regulating kinase/mitogen-activated protein kinase kinase kinase 5 [ASK] and v-raf murine sarcoma viral oncogene homolog B1 [b-RAF]) context. This study reaffirms the importance of classic inflammation pathways, such as MAPK and TGFβ signaling and reveals less known involvement of glycogen synthase kinase 3β (GSK-3β), AKT and SRC signaling cassettes in pneumonia.

  19. Antibacterial Efficacy of Eravacycline In Vivo against Gram-Positive and Gram-Negative Organisms

    PubMed Central

    Monogue, Marguerite L.; Hamada, Yukihiro

    2016-01-01

    Members of the tetracycline class are frequently classified as bacteriostatic. However, recent findings have demonstrated an improved antibacterial killing profile, often achieving ≥3 log10 bacterial count reduction, when such antibiotics have been given for periods longer than 24 h. We aimed to study this effect with eravacycline, a novel fluorocycline, given in an immunocompetent murine thigh infection model over 72 h against two methicillin-resistant Staphylococcus aureus (MRSA) isolates (eravacycline MICs = 0.03 and 0.25 μg/ml) and three Enterobacteriaceae isolates (eravacycline MICs = 0.125 to 0.25 μg/ml). A humanized eravacycline regimen, 2.5 mg/kg of body weight given intravenously (i.v.) every 12 h (q12h), demonstrated progressively enhanced activity over the 72-h study period. A cumulative dose response in which bacterial density was reduced by more than 3 log10 CFU at 72 h was noted over the study period in the two Gram-positive isolates, and eravacycline performed similarly to comparator antibiotics (tigecycline, linezolid, and vancomycin). A cumulative dose response with eravacycline and comparators (tigecycline and meropenem) over the study period was also observed in the Gram-negative isolates, although more variability in bacterial killing was observed for all antibacterial agents. Overall, a bacterial count reduction of ≥3 log was achieved in one of the three isolates with both eravacycline and tigecycline, while meropenem achieved a similar endpoint against two of the three isolates. Bactericidal activity is typically defined in vitro over 24 h; however, extended regimen studies in vivo may demonstrate an improved correlation with clinical outcomes by better identification of antimicrobial effects. PMID:27353265

  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. Linear alkanesulfonates as carbon and energy sources for gram-positive and gram-negative bacteria.

    PubMed

    Reichenbecher, W; Murrell, J C

    1999-01-01

    Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source. Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3-C6 and the substituted sulfonates taurine and isethionate as carbon and energy source. A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail. Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus. After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans. Both bacteria also utilized a wide range of sulfonates as sulfur source. Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds. Cell-free extracts of strain P53 exhibited high sulfite oxidase activity [2.34 U (mg protein)-1] when assayed with ferricyanide, but not with cytochrome c. Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine. Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate. Strain P40 cells also accumulated sulfite under these conditions. Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase. When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation.

  2. Binding of polymyxin B nonapeptide to gram-negative bacteria.

    PubMed Central

    Vaara, M; Viljanen, P

    1985-01-01

    The binding of the outer membrane-disorganizing peptide polymyxin B nonapeptide (PMBN) to gram-negative bacteria was studied by using tritium-labeled PMBN. Smooth Salmonella typhimurium had a binding capacity of ca. 6 nmol of PMBN per mg (dry weight) of bacteria, which corresponds to ca. 1 X 10(6) to 2 X 10(6) molecules of PMBN per single cell. The binding was of relatively high affinity (Kd, 1.3 microM). The isolated outer membrane of S. typhimurium bound ca. 100 nmol of PMBN per mg of outer membrane protein (Kd, 1.1 microM), whereas the cytoplasmic membrane bound 9 to 10 times less. Other bacteria which are susceptible to the action of PMBN (Escherichia coli strains, Pseudomonas aeruginosa, Haemophilus influenzae) also bound large amounts of PMBN. The S. typhimurium pmrA mutant, Neisseria gonorrhoeae, and Proteus mirabilis (all known as resistant to polymyxin and PMBN) bound 3.3, 4, and 12 times less than S. typhimurium, respectively. The binding of PMBN to S. typhimurium was effectively inhibited by low concentrations of polymyxin B, compound EM49 (octapeptin), polylysine, and protamine. Spermine, Ca2+, and Mg2+ also inhibited the PMBN binding although they were ca. 160, 700, and 2,400 times less active (based on molarity) than polymyxin B, respectively. No binding inhibition was found at the tested concentrations of streptomycin, tetralysine, spermidine, or cadaverine. PMID:2988430

  3. Screening for Gram-negative bacteria: Impact of preanalytical parameters

    PubMed Central

    Warnke, Philipp; Johanna Pohl, Friederike Pola; Kundt, Guenther; Podbielski, Andreas

    2016-01-01

    Screening recommendations for multidrug-resistant Gram-negative bacteria comprise microbiological analyses from rectal swabs. However, essential specifications of the preanalytic steps of such screenings, i.e. the sampling technique, sampling devices and sampling site, are lacking. For standardized and optimum screening conditions these parameters are indispensable. Here, the optimum parameters were examined irrespective of the antibiotic resistance patterns of the target bacteria in order to establish a general basis for this type of screening. Swabs with rayon, polyurethane-cellular-foam and nylon-flocked tips were tested. Different sampling locations were evaluated, i.e. perianal, intraanal and deep intraanal. Subjects were swabbed and quantities of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii were assessed. Overall prevalences of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii were 94%, 16%, 12%, and 2%, respectively. Bacterial recovery rates were independent from the sampling-timepoint during hospital stay. Polyurethane-cellular-foam or nylon-flocked swabs recovered significantly more bacteria as compared to rayon swabs. Intraanal swabbing resulted in significantly higher bacterial quantities as compared to perianal swabbing. In contrast, for the detection of A. baumannii, perianal swabbing seems more suitable than intraanal swabbing. Gender-related differences in bacterial recovery could be detected from perianal but not from intraanal swabs. PMID:27460776

  4. Pili in Gram-negative and Gram-positive bacteria - structure, assembly and their role in disease.

    PubMed

    Proft, T; Baker, E N

    2009-02-01

    Many bacterial species possess long filamentous structures known as pili or fimbriae extending from their surfaces. Despite the diversity in pilus structure and biogenesis, pili in Gram-negative bacteria are typically formed by non-covalent homopolymerization of major pilus subunit proteins (pilins), which generates the pilus shaft. Additional pilins may be added to the fiber and often function as host cell adhesins. Some pili are also involved in biofilm formation, phage transduction, DNA uptake and a special form of bacterial cell movement, known as 'twitching motility'. In contrast, the more recently discovered pili in Gram-positive bacteria are formed by covalent polymerization of pilin subunits in a process that requires a dedicated sortase enzyme. Minor pilins are added to the fiber and play a major role in host cell colonization.This review gives an overview of the structure, assembly and function of the best-characterized pili of both Gram-negative and Gram-positive bacteria.

  5. [Nonfermentative gram-negative bacteria: isolation rates and antibiotic sensitivity].

    PubMed

    Bogomolova, N S; Bol'shakov, L V; Kuznetsova, S M; Oreshkina, T D

    2010-01-01

    The isolation rates of nonfermentative gram-negative bacteria (NFGNB) are analyzed in the inpatients treated at the B. V. Petrovsky Russian Surgery Research Center in 2005-2009 and antibiotic resistance trends in nosocomial strains of NFGNB are traced in the above period. The study of the etiological structure of nosocomial infections has shown that the past 2 years (2008 and 2009) were marked by a clear tendency for the preponderance of gram-positive coccal pathogens (46.8 and 53.9%) with a considerable (1.5-2-fold) reduction in the proportion of representatives of enterobacteria (31.5 and 24.5%) and NFGB (13.4 and 11.3%), but with an increase in the proportion of fungi up to 7.1 and 8.6%, respectively. Among the NFGNBs, P. aeruginosa remains ohe of the most common pathogens for nosocomial infections although its portion in the number of all etiologically significant microorganisms was substantially reduced (from 13% in 2005 to 4.6% in 2009). It continues to remain one of the most common causative agents for infections of the urinary tract (e.g., after renal transplantation) and upper and lower respiratory tract (e.g. nosocomial pneumonia) and for those developing after surgical interventions (postoperative wound suppuration discharged along the drainages, from a T-sized tube, etc.). Among the NFGNBs, Acinetobacter spp. was the second frequently isolated pathogen, the isolation rate for which also decreased from 7.9% in 2005 to 2.6% in 2009. Polymyxin B and carbapenems (imipenem, meropenem, and doripenem) showed the highest activity against the vast majority of the test strains; however, there was an absolutely clear declining trend in the proportion of carbapenem-sensitive strains among virtually all the NFGNBs under study. According to the proportion of imipenem-, meropenem-, and doripenem-sensitive nosocomial P. aeroginosa strains (66.7, 46.6, and 44.7%, respectively), doripenem had the least activity. Acinetobacter spp. strains sensitive to these drugs showed

  6. Dustborne and Airborne Gram-Positive and Gram-Negative Bacteria in High versus Low ERMI Homes

    PubMed Central

    Adhikari, Atin; Kettleson, Eric M.; Vesper, Stephen; Kumar, Sudhir; Popham, David L.; Schaffer, Christopher; Indugula, Reshmi; Chatterjee, Kanistha; Allam, Karteek K.; Grinshpun, Sergey A.; Reponen, Tiina

    2014-01-01

    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 into low (<5) and high (>5) ERMI groups based on the average ERMI values as well as 2011 ERMI values. Dust and air samples were collected from the homes in 2011 and all samples were analyzed for Gram-positive and Gram-negative bacteria using QPCR assays, endotoxin by the LAL assay, and N-acetyl-muramic acid using HPLC. In addition, air samples were analyzed for culturable bacteria. When average ERMI values were considered, the concentration and load of Gram-positive bacteria determined with QPCR in house dust, but not air, were significantly greater in high ERMI homes than in low ERMI homes. Furthermore, the concentration of endotoxin, but not muramic acid, in the dust was significantly greater in high ERMI than in low ERMI homes. In contrast, when ERMI values of 2011 were considered, Gram-negative bacteria determined with QPCR in air, endotoxin in air, and muramic acid in dust were significantly greater in high ERMI homes. The results suggest that both short-term and long-term mold contamination in homes could be linked with the bacterial concentrations in house dust, however, only the current mold status was associated with bacterial concentrations in air. Although correlations were found between endotoxin and Gram-negative bacteria as well as between muramic acid and Gram-positive bacteria in the entire data set, diverging associations were observed between the different measures of bacteria and the home moldiness. It is likely that concentrations of cells obtained by QPCR and concentrations of cell wall components are not equivalent and represent too broad categories to understand the bacterial composition and sources of the home microbiota. PMID:24642096

  7. Dustborne and airborne Gram-positive and Gram-negative bacteria in high versus low ERMI homes.

    PubMed

    Adhikari, Atin; Kettleson, Eric M; Vesper, Stephen; Kumar, Sudhir; Popham, David L; Schaffer, Christopher; Indugula, Reshmi; Chatterjee, Kanistha; Allam, Karteek K; Grinshpun, Sergey A; Reponen, Tiina

    2014-06-01

    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 into low (<5) and high (>5) ERMI groups based on the average ERMI values as well as 2011 ERMI values. Dust and air samples were collected from the homes in 2011 and all samples were analyzed for Gram-positive and Gram-negative bacteria using QPCR assays, endotoxin by the LAL assay, and N-acetyl-muramic acid using HPLC. In addition, air samples were analyzed for culturable bacteria. When average ERMI values were considered, the concentration and load of Gram-positive bacteria determined with QPCR in house dust, but not air, were significantly greater in high ERMI homes than in low ERMI homes. Furthermore, the concentration of endotoxin, but not muramic acid, in the dust was significantly greater in high ERMI than in low ERMI homes. In contrast, when ERMI values of 2011 were considered, Gram-negative bacteria determined with QPCR in air, endotoxin in air, and muramic acid in dust were significantly greater in high ERMI homes. The results suggest that both short-term and long-term mold contamination in homes could be linked with the bacterial concentrations in house dust, however, only the current mold status was associated with bacterial concentrations in air. Although correlations were found between endotoxin and Gram-negative bacteria as well as between muramic acid and Gram-positive bacteria in the entire data set, diverging associations were observed between the different measures of bacteria and the home moldiness. It is likely that concentrations of cells obtained by QPCR and concentrations of cell wall components are not equivalent and represent too broad categories to understand the bacterial composition and sources of the home microbiota.

  8. Biochemical characterization of Gram-positive and Gram-negative plant-associated bacteria with micro-Raman spectroscopy.

    PubMed

    Paret, Mathews L; Sharma, Shiv K; Green, Lisa M; Alvarez, Anne M

    2010-04-01

    Raman spectra of Gram-positive and Gram-negative plant bacteria have been measured with micro-Raman spectrometers equipped with 785 and 514.5 nm lasers. The Gram-positive bacteria Microbacterium testaceum, Paenibacillus validus, and Clavibacter michiganensis subsp. michiganensis have strong carotenoid bands in the regions 1155-1157 cm(-1) and 1516-1522 cm(-1) that differentiate them from other tested Gram-negative bacteria. In the Raman spectrum of Gram-positive bacteria Bacillus megaterium excited with 785 nm laser, the Raman bands at 1157 and 1521 cm(-1) are weak in intensity compared to other Gram-positive bacteria, and these bands did not show significant resonance Raman enhancement in the spectrum recorded with 514.5 nm laser excitation. The Gram-positive bacteria could be separated from each other based on the bands associated with the in-phase C=C (v(1)) vibrations of the polyene chain of carotenoids. None of the Gram-negative bacteria tested had carotenoid bands. The bacteria in the genus Xanthomonas have a carotenoid-like pigment, xanthomonadin, identified in Xanthomonas axonopodis pv. dieffenbachiae, and it is a unique Raman marker for the bacteria. The representative bands for xanthomonadin were the C-C stretching (v(2)) vibrations of the polyene chain at 1135-1136 cm(-1) and the in-phase C=C (v(1)) vibrations of the polyene chain at 1529-1531 cm(-1), which were distinct from the carotenoid bands of other tested bacteria. The tyrosine peak in the region 1170-1175 cm(-1) was the only other marker present in Gram-negative bacteria that was absent in all tested Gram-positives. A strong-intensity exopolysaccharide-associated marker at 1551 cm(-1) is a distinguishable feature of Enterobacter cloacae. The Gram-negative Agrobacterium rhizogenes and Ralstonia solanacearum were differentiated from each other and other tested bacteria on the basis of presence or absence and relative intensities of peaks. The principal components analysis (PCA) of the spectra

  9. Probing interaction of gram-positive and gram-negative bacterial cells with ZnO nanorods.

    PubMed

    Jain, Aanchal; Bhargava, Richa; Poddar, Pankaj

    2013-04-01

    In the present work, the physiological effects of the ZnO nanorods on the Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Aerobacter aerogenes) bacterial cells have been studied. The analysis of bacterial growth curves for various concentrations of ZnO nanorods indicates that Gram positive and Gram negative bacterial cells show inhibition at concentrations of ~64 and ~256 μg/mL respectively. The marked difference in susceptibility towards nanorods was also validated by spread plate and disk diffusion methods. In addition, the scanning electron micrographs show a clear damage to the cells via changed morphology of the cells from rod to coccoid etc. The confocal optical microscopy images of these cells also demonstrate the reduction in live cell count in the presence of ZnO nanorods. These, results clearly indicate that the antibacterial activity of ZnO nanorods is higher towards Gram positive bacterium than Gram negative bacterium which indicates that the structure of the cell wall might play a major role in the interaction with nanostructured materials and shows high sensitivity to the particle concentration.

  10. Antibiotic Trends Amid Multidrug-Resistant Gram-Negative Infections in Intensive Care Units.

    PubMed

    Fowler, Leanne H; Lee, Susan

    2017-03-01

    Isolates from ICUs most commonly find multidrug-resistant (MDR) gram-negative bacteria. The purpose of this article is to discuss the significant impact MDR gram-negative infections are having on ICUs, the threat on health and mortality, and effective and new approaches aimed to combat MDR gram-negative infections in critically ill populations. Inappropriate antibiotic therapies for suspected or documented infections are the leading cause of the emergence of bacterial resistance. A variety of strategies are aimed at combatting this international burden via antibiotic stewardship programs. Studies are demonstrating promise against the virulence MDR gram-negative infections have posed.

  11. Multidrug-resistant Gram-negative bacteria in solid organ transplant recipients with bacteremias.

    PubMed

    Wan, Q Q; Ye, Q F; Yuan, H

    2015-03-01

    Bloodstream infections (BSIs) remain as life-threatening complications and are associated with significant morbidity and mortality among solid organ transplant (SOT) recipients. Multidrug-resistant (MDR) Gram-negative bacteria can cause serious bacteremias in these recipients. Reviews have aimed to investigate MDR Gram-negative bacteremias; however, they were lacking in SOT recipients in the past. To better understand the characteristics of bacteremias due to MDR Gram-negative bacteria, optimize preventive and therapeutic strategies, and improve the outcomes of SOT recipients, this review summarize the epidemiology, clinical and laboratory characteristics, and explores the mechanisms, prevention, and treatment of MDR Gram-negative bacteria.

  12. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance.

    PubMed

    Blair, Jessica M A; Richmond, Grace E; Piddock, Laura J V

    2014-01-01

    Gram-negative bacteria express a plethora of efflux pumps that are capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentration, allowing bacteria to survive at higher antibiotic concentrations. Overexpression of some efflux pumps can cause clinically relevant levels of antibiotic resistance in Gram-negative pathogens. This review discusses the role of efflux in resistance of clinical isolates of Gram-negative bacteria, the regulatory mechanisms that control efflux pump expression, the recent advances in our understanding of efflux pump structure and how inhibition of efflux is a promising future strategy for tackling multidrug resistance in Gram-negative pathogens.

  13. Potential strategies for the eradication of multidrug-resistant Gram-negative bacterial infections.

    PubMed

    Huwaitat, Rawan; McCloskey, Alice P; Gilmore, Brendan F; Laverty, Garry

    2016-07-01

    Antimicrobial resistance is one of the leading threats to society. The increasing burden of multidrug-resistant Gram-negative infection is particularly concerning as such bacteria are demonstrating resistance to nearly all currently licensed therapies. Various strategies have been hypothesized to treat multidrug-resistant Gram-negative infections including: targeting the Gram-negative outer membrane; neutralization of lipopolysaccharide; inhibition of bacterial efflux pumps and prevention of protein folding. Silver and silver nanoparticles, fusogenic liposomes and nanotubes are potential strategies for extending the activity of licensed, Gram-positive selective, antibiotics to Gram-negatives. This may serve as a strategy to fill the current void in pharmaceutical development in the short term. This review outlines the most promising strategies that could be implemented to solve the threat of multidrug-resistant Gram-negative infections.

  14. 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.

  15. Different Use of Cell Surface Glycosaminoglycans As Adherence Receptors to Corneal Cells by Gram Positive and Gram Negative Pathogens

    PubMed Central

    García, Beatriz; Merayo-Lloves, Jesús; Rodríguez, David; Alcalde, Ignacio; García-Suárez, Olivia; Alfonso, José F.; Baamonde, Begoña; Fernández-Vega, Andrés; Vazquez, Fernando; Quirós, Luis M.

    2016-01-01

    The epithelium of the cornea is continuously exposed to pathogens, and adhesion to epithelial cells is regarded as an essential first step in bacterial pathogenesis. In this article, the involvement of glycosaminoglycans in the adhesion of various pathogenic bacteria to corneal epithelial cells is analyzed. All microorganisms use glycosaminoglycans as receptors, but arranged in different patterns depending on the Gram-type of the bacterium. The heparan sulfate chains of syndecans are the main receptors, though other molecular species also seem to be involved, particularly in Gram-negative bacteria. Adherence is inhibited differentially by peptides, including heparin binding sequences, indicating the participation of various groups of Gram-positive, and -negative adhesins. The length of the saccharides produces a major effect, and low molecular weight chains inhibit the binding of Gram-negative microorganisms but increase the adherence of Gram-positives. Pathogen adhesion appears to occur preferentially through sulfated domains, and is very dependent on N- and 6-O-sulfation of the glucosamine residue and, to a lesser extent, 2-O sulfation of uronic acid. These data show the differential use of corneal receptors, which could facilitate the development of new anti-infective strategies. PMID:27965938

  16. Evaluation of the API 20E system for the identification of gram-negative nonfermenters from animal origin.

    PubMed Central

    Devenish, J A; Barnum, D A

    1982-01-01

    The API 20E system was evaluated on isolates from animals of aerobic nonfermentative and cytochrome oxidase positive Gram-negative rods. An accuracy of identification of 80% (214/268 isolates) was achieved for those organisms included in the 1976-1977 API profile index. Members of the genera Pseudomonas and Acinetobacter were identified with 100% accuracy. Organisms not included in the API profile gave either an unacceptable profile number or were incorrectly identified as Moraxella spp. When the inoculum size was increased there was better identification. PMID:7042055

  17. Pharmacodynamic studies of trovafloxacin and grepafloxacin in vitro against Gram-positive and Gram-negative bacteria.

    PubMed

    Odenholt, I; Cars, T; Lowdin, E

    2000-07-01

    Grepafloxacin and trovafloxacin are two novel fluoroquinolones with extended Gram-positive bacterial spectra compared with older quinolones. The aim of the present study was to investigate the different pharmacodynamic parameters of grepafloxacin in comparison with those of trovafloxacin. The following studies were performed against various Gram-positive and Gram-negative bacteria: (i) determination of the rate and extent of killing at a concentration corresponding to the 1 h non-protein-bound human serum level following an oral dose of 800 mg grepafloxacin and 300 mg trovafloxacin; (ii) determination of the rate and extent of killing of the two quinolones at different concentrations; (iii) determination of the post-antibiotic effects (PAEs); (iv) determination of the post-antibiotic sub-MIC effects (PA SMEs); (iv) determination of the rate and extent of killing in an in vitro kinetic model. It was shown that both grepafloxacin and trovafloxacin exhibited concentration-dependent killing against both Gram-positive and Gram-negative bacteria. Grepafloxacin exhibited a slower bactericidal effect against all the Gram-positive strains investigated in comparison with trovafloxacin in spite of a similar C(max)/MIC in the static experiments and a similar AUC/MIC ratio in the kinetic experiments. No major differences in the extent and rate of killing were noted against the Gram-negative strains, which were killed almost completely after 3 h except for Pseudomonas aeruginosa. A PAE of both quinolones was noted for all strains investigated. Trovafloxacin induced longer PAEs against the Gram-positive strains but shorter PAEs in comparison with those of grepafloxacin against the Gram-negative strains. A prolonging of the PAEs was noted for all bacteria when exposed to sub-MICs in the post-antibiotic phase. With a similar AUC/MIC of 310 for the penicillin-sensitive strain of Streptococcus pneumoniae and 143 for the penicillin-resistant strain, the time for 99.9% eradication for

  18. Proteomic profiling of Gram-negative bacterial outer membrane vesicles: Current perspectives.

    PubMed

    Lee, Jaewook; Kim, Oh Youn; Gho, Yong Song

    2016-10-01

    Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria. Recent progress in the studies of Gram-negative bacterial extracellular vesicles implies that OMVs may function as intercellular communicasomes in bacteria-bacteria and bacteria-host interactions. Current MS-based high-throughput proteomic analyses of Gram-negative bacterial OMVs have identified thousands of vesicular proteins and provided clues to reveal the biogenesis and pathophysiological functions of Gram-negative bacterial OMVs. The future directions of proteomics of Gram-negative bacterial OMVs may include the isolation strategy of Gram-negative bacterial OMVs to thoroughly exclude nonvesicular contaminants and proteomics of Gram-negative bacterial OMVs derived from diverse conditions as well as body fluids of bacterium-infected hosts. We hope this review will shed light on future research in this emerging field of proteomics of extracellular vesicles derived from Gram-negative bacteria and contribute to the development of OMV-based diagnostic tools and effective vaccines.

  19. Comparative in vitro activity of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin and trovafloxacin against 4151 Gram-negative and Gram-positive organisms.

    PubMed

    Blondeau, J M; Laskowski, R; Bjarnason, J; Stewart, C

    2000-02-01

    Gatifloxacin, grepafloxacin, moxifloxacin and trovafloxacin are fluoroquinolones with enhanced Gram-positive activity while retaining broad-spectrum activity against Gram-negative pathogens. Levofloxacin and ciprofloxacin are older quinolones with broad activity against Gram-negative pathogens and borderline activity against some Gram-positive organisms. We compared the in vitro activity of these compounds against 4151 Gram-negative and -positive organisms. Gatifloxacin, grepafloxacin, moxifloxacin and trovafloxacin were highly active against penicillin sensitive and resistant Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae. Ciprofloxacin and levofloxacin were active but less potent. All compounds were highly active (overall) against Gram-negative pathogens with ciprofloxacin being the most active agent against Pseudomonas aeruginosa. Our data indicate that the advanced fluoroquinolones will be important compounds for treating infections caused by Gram-positive and Gram-negative pathogens.

  20. Fluctuations in the population density of Gram-negative bacteria in a chernozem in the course of a succession initiated by moistening and chitin and cellulose introduction

    NASA Astrophysics Data System (ADS)

    Polyanskaya, L. M.; Ivanov, K. E.; Zvyagintsev, D. G.

    2012-10-01

    The role has been studied of Gram-negative bacteria in the destruction of polymers widely spread in soils: chitin and cellulose. The introduction of chitin had no positive effect on the population density of Gram-negative bacteria, but it advanced the date of their appearance: the maximum population density of Gram-negative bacteria was recorded not on the 7th-15th day as in the control but much earlier, on the 3rd-7th day of the experiment. Consequently, the introduction of chitin as an additional source of nutrition promoted revealing of the Gram-negative bacteria already at the early stages of the succession. In the course of the succession, when the fungal mycelium begins to die off, the actinomycetic mycelium increases in length, i.e., Gram-negative bacteria are replaced at this stage with Gram-positive ones, the leading role among which belongs to actinomycetes. The growth rate of Gram-negative bacteria is higher than that of actinomycetes, so they start chitin utilization at the early stages of the succession, whereas actinomycetes dominate at the late stages. The population density of Gram-negative bacteria was lower under the anaerobic conditions as compared with that in the aerobic ones. The population density of Gram-negative bacteria in the lower layer of the A horizon and in the B horizon was slightly higher only in the case of the chitin introduction. When cellulose was introduced into the soil under aerobic conditions, the population density of Gram-negative bacteria in all the layers of the A horizon was maximal from the 14th to the 22nd day of the experiment. Cellulose was utilized in the soil mostly by fungi, and this was suggested by the increase of the length of the fungal mycelium. Simultaneously, an increase in the length of the actinomycetal mycelium was observed, as these organisms also perform cellulose hydrolysis in soils. The Gram-negative bacteria began to develop at the stage of the fungal mycelium destruction, which indirectly

  1. Outcomes of single organism peritonitis in peritoneal dialysis: gram negatives versus gram positives in the Network 9 Peritonitis Study.

    PubMed

    Bunke, C M; Brier, M E; Golper, T A

    1997-08-01

    The use of the "peritonitis rate" in the management of patients undergoing peritoneal dialysis is assuming importance in comparing the prowess of facilities, care givers and new innovations. For this to be a meaningful outcome measure, the type of infection (causative pathogen) must have less clinical significance than the number of infections during a time interval. The natural history of Staphylococcus aureus, pseudomonas, and fungal peritonitis would not support that the outcome of an episode of peritonitis is independent of the causative pathogen. Could this concern be extended to other more frequently occurring pathogens? To address this, the Network 9 Peritonitis Study identified 530 episodes of single organism peritonitis caused by a gram positive organism and 136 episodes caused by a single non-pseudomonal gram negative (NPGN) pathogen. Coincidental soft tissue infections (exit site or tunnel) occurred equally in both groups. Outcomes of peritonitis were analyzed by organism classification and by presence or absence of a soft tissue infection. NPGN peritonitis was associated with significantly more frequent catheter loss, hospitalization, and technique failure and was less likely to resolve regardless of the presence or absence of a soft tissue infection. Hospitalization and death tended to occur more frequently with enterococcal peritonitis than with other gram positive peritonitis. The outcomes in the NPGN peritonitis group were significantly worse (resolution, catheter loss, hospitalization, technique failure) compared to coagulase negative staphylococcal or S. aureus peritonitis, regardless of the presence or absence of a coincidental soft tissue infection. Furthermore, for the first time, the poor outcomes of gram negative peritonitis are shown to be independent of pseudomonas or polymicrobial involvement or soft tissue infections. The gram negative organism appears to be the important factor. In addition, the outcome of peritonitis caused by S. aureus

  2. Synergy of nitric oxide and silver sulfadiazine against gram-negative, gram-positive, and antibiotic-resistant pathogens.

    PubMed

    Privett, Benjamin J; Deupree, Susan M; Backlund, Christopher J; Rao, Kavitha S; Johnson, C Bryce; Coneski, Peter N; Schoenfisch, Mark H

    2010-12-06

    The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver(I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5. Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria, while Gram-positive bacteria were more susceptible to combination therapy. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of burns and chronic wounds.

  3. Conventional tube and microplate Limulus amoebocyte lysate procedures for determination of gram-negative bacteria in milk.

    PubMed

    May, S A; Mikolajcik, E M; Richter, E R

    1989-05-01

    A comparison was made of the conventional tube and microplate Limulus amoebocyte lysate assay for detection of gram-negative bacterial lipopolysaccharide in milk. Raw whole milk samples were maintained frozen and portions were examined periodically on alternate days during 13-d storage to evaluate the reproducibility of both Limulus amoebocyte lysate procedures and to determine optimum reaction conditions for the microplate method. One-day-old, raw and locally purchased pasteurized milk samples, held at 7 degrees C, were analyzed during storage to establish the correlation of both procedures with aerobic and modified psychrotrophic plate counts. Vitamin- and mineral-fortified dairy-based products were examined using the microplate Limulus amoebocyte lysate test as a potential indicator of raw material or finished product bacterial quality and possible postprocessing contamination. Statistical analysis of the data collected comparing the conventional tube and the microplate Limulus amoebocyte lysate assay demonstrated no significant difference exists between the methods when either the modified psychrotrophic bacterial count or the aerobic plate count was used to determine gram-negative bacteria in pasteurized or raw milk (P less than .91). The microplate method, which uses half the lysate reagent, was a good indicator of the bacterial quality of milk and fortified dairy products, consistently detecting bacterial levels greater than 10(3) to 10(4)/ml.

  4. 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

  5. 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.

  6. Exploiting Quorum Sensing Interfering Strategies in Gram-Negative Bacteria for the Enhancement of Environmental Applications

    PubMed Central

    Zhang, Weiwei; Li, Chenghua

    2016-01-01

    Quorum sensing (QS) is a widespread intercellular form of communication to coordinate physiological processes and cooperative activities of bacteria at the population level, and it depends on the production, secretion, and detection of small diffusible autoinducers, such as acyl-homoserine lactones (AHLs), auto-inducing oligo-peptides (AIPs) and autoinducer 2. In this review, the function of QS autoinducers of gram-negative bacteria in different aspects of wastewater treatment systems is examined. Based on research primarily performed over the past 10 years, QS involvement in the formation of biofilm and aerobic granules and changes of the microbial community and degradation/transformation pathways is discussed. In particular, the QS pathway in the role of bacterial infections and disease prevention in aquaculture is addressed. Interference of QS autoinducer-regulated pathways is considered potential treatment for a variety of environmentally related problems. This review is expected to serve as a stepping stone for further study and development strategies based on the mediation of QS-regulated pathways to enhance applications in both wastewater treatment systems and aquaculture. PMID:26779175

  7. Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli.

    PubMed Central

    Petzel, J P; Hartman, P A

    1985-01-01

    Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3890742

  8. Ferric ammonium citrate decomposition--a taxonomic tool for gram-negative bacteria.

    PubMed

    Szentmihályi, A; Lányi, B

    1986-01-01

    The iron uptake test of Szabó and Vandra has been modified and used for the differentiation of Gram-negative bacteria. Nutrient agar containing 20 g per litre of ferric ammonium citrate was distributed into narrow tubes and solidified so as to form butts and slants. Considering the localization of the rusty-brown coloration produced after seeding and incubation, 2367 strains were classified into four groups. (1) Unchanged medium: Escherichia coli, Shigella spp., Yersinia spp., Hafnia alvei and Morganella morganii 100% each, Klebsiella spp., 50%, Enterobacter cloacae 37%, Proteus vulgaris 59%, Acinetobacter spp. 42%, Pseudomonas fluorescens 19%, some other bacteria 2-12%. (2) Rusty-brown slant, unchanged butt: Salmonella subgenera II, III and IV 98%, Citrobacter freundii 65%, E. cloacae 55%, P. vulgaris 41%, Proteus mirabilis 98%, Providencia rettgeri 100%, urease-negative Providencia 96%, Acinetobacter spp. 58%, Pseudomonas aeruginosa 100%, P. fluorescens 81%, UFP (unclassified fluorescent pseudomonads) 100%, other Pseudomonas spp. 55%. (3) Unchanged slant, brown butt: S. typhi 88%, Salmonella subgenus I 3%, Klebsiella spp. 31%, some other bacteria 2-3%. (4) Rusty-brown slant, brown butt: Salmonella subgenus I 75%, C. freundii 20%, Klebsiella spp. 12%, some other bacteria 1-5%. Colour reactions in ferric ammonium citrate agar are associated with the accumulation of ferric hydroxide: bacteria giving positive reactions on the slant took up as an average, 63 times more iron than those with negative test. The localization of colour reaction correlated partly with aerobic and anaerobic citrate utilization or decomposition in Simmons' minimal and in Kauffmann's peptone water medium.

  9. Teaching 'old' polymyxins new tricks: new-generation lipopeptides targeting gram-negative 'superbugs'.

    PubMed

    Velkov, Tony; Roberts, Kade D; Nation, Roger L; Wang, Jiping; Thompson, Philip E; Li, Jian

    2014-05-16

    The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a 'last-line' therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative 'superbugs'. This report details the structure-activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.

  10. Permeability barrier of Gram-negative cell envelopes and approaches to bypass it

    DOE PAGES

    Zgurskaya, Helen I.; López, Cesar A.; Gnanakaran, Sandrasegaram

    2015-09-18

    Gram-negative bacteria are intrinsically resistant to many antibiotics. Species that have acquired multidrug resistance and cause infections that are effectively untreatable present a serious threat to public health. The problem is broadly recognized and tackled at both the fundamental and applied levels. This article summarizes current advances in understanding the molecular bases of the low permeability barrier of Gram-negative pathogens, which is the major obstacle in discovery and development of antibiotics effective against such pathogens. Gaps in knowledge and specific strategies to break this barrier and to achieve potent activities against difficult Gram-negative bacteria are also discussed.

  11. A bivalent cationic dye enabling selective photo-inactivation against Gram-negative bacteria.

    PubMed

    Li, Ke; Zhang, Yang-Yang; Jiang, Guo-Yu; Hou, Yuan-Jun; Zhang, Bao-Wen; Zhou, Qian-Xiong; Wang, Xue-Song

    2015-05-07

    A piperazine-modified Crystal Violet was found to be able to selectively inactivate Gram-negative bacteria upon visible light irradiation but left Gram-positive bacteria less damaged, which can serve as a blueprint for the development of novel narrow-spectrum agents to replenish the current arsenal of photodynamic antimicrobial chemotherapy (PACT).

  12. Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria.

    PubMed

    Pontel, Lucas B; Soncini, Fernando C

    2009-07-01

    Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP. A DeltacueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Deltacus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.

  13. Resistance-Nodulation-Division Multidrug Efflux Pumps in Gram-Negative Bacteria: Role in Virulence.

    PubMed

    Fernando, Dinesh M; Kumar, Ayush

    2013-03-18

    Resistance-Nodulation-Division (RND) efflux pumps are one of the most important determinants of multidrug resistance (MDR) in Gram-negative bacteria. With an ever increasing number of Gram-negative clinical isolates exhibiting MDR phenotypes as a result of the activity of RND pumps, it is clear that the design of novel effective clinical strategies against such pathogens must be grounded in a better understanding of these pumps, including their physiological roles. To this end, recent evidence suggests that RND pumps play an important role in the virulence of Gram-negative pathogens. In this review, we discuss the important role RND efflux pumps play in different facets of virulence including colonization, evasion of host defense mechanisms, and biofilm formation. These studies provide key insights that may ultimately be applied towards strategies used in the design of effective therapeutics against MDR Gram negative bacterial pathogens.

  14. Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal Gram-positive and Gram-negative pathogenic bacteria.

    PubMed

    Prasanth, Chandra Sekhar; Karunakaran, Suneesh C; Paul, Albish K; Kussovski, Vesselin; Mantareva, Vanya; Ramaiah, Danaboyina; Selvaraj, Leslie; Angelov, Ivan; Avramov, Latchezar; Nandakumar, Krishnankutty; Subhash, Narayanan

    2014-01-01

    The Gram-negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic-resistant bacteria, antimicrobial Photodynamic therapy (aPDT) seems to be a plausible alternative. In this work, photosensitization was performed on Gram-positive and Gram-negative bacteria in pure culture using new-age cationic porphyrins, namely mesoimidazolium-substituted porphyrin derivative (ImP) and pyridinium-substituted porphyrin derivative (PyP). The photophysical properties of both the sensitizers including absorption, fluorescence emission, quantum yields of the triplet excited states and singlet oxygen generation efficiencies were evaluated in the context of aPDT application. The studied porphyrins exhibited high ability to accumulate into bacterial cells with complete penetration into early stage biofilms. As compared with ImP, PyP was found to be more effective for photoinactivation of bacterial strains associated with periodontitis, without any signs of dark toxicity, owing to its high photocytotoxicity.

  15. Incidence of Carbapenem-Resistant Gram Negatives in Italian Transplant Recipients: A Nationwide Surveillance Study

    PubMed Central

    Lanini, Simone; Costa, Alessandro Nanni; Puro, Vincenzo; Procaccio, Francesco; Grossi, Paolo Antonio; Vespasiano, Francesca; Ricci, Andrea; Vesconi, Sergio; Ison, Michael G.; Carmeli, Yehuda; Ippolito, Giuseppe

    2015-01-01

    Background Bacterial infections remain a challenge to solid organ transplantation. Due to the alarming spread of carbapenem-resistant gram negative bacteria, these organisms have been frequently recognized as cause of severe infections in solid organ transplant recipients. Methods and Findings Between 15 May and 30 September 2012 we enrolled 887 solid organ transplant recipients in Italy with the aim to describe the epidemiology of gram negative bacteria spreading, to explore potential risk factors and to assess the effect of early isolation of gram negative bacteria on recipients’ mortality during the first 90 days after transplantation. During the study period 185 clinical isolates of gram negative bacteria were reported, for an incidence of 2.39 per 1000 recipient-days. Positive cultures for gram negative bacteria occurred early after transplantation (median time 26 days; incidence rate 4.33, 1.67 and 1.14 per 1,000 recipient-days in the first, second and third month after SOT, respectively). Forty-nine of these clinical isolates were due to carbapenem-resistant gram negative bacteria (26.5%; incidence 0.63 per 1000 recipient-days). Carbapenems resistance was particularly frequent among Klebsiella spp. isolates (49.1%). Recipients with longer hospital stay and those who received either heart or lung graft were at the highest risk of testing positive for any gram negative bacteria. Moreover recipients with longer hospital stay, lung recipients and those admitted to hospital for more than 48h before transplantation had the highest probability to have culture(s) positive for carbapenem-resistant gram negative bacteria. Forty-four organ recipients died (0.57 per 1000 recipient-days) during the study period. Recipients with at least one positive culture for carbapenem-resistant gram negative bacteria had a 10.23-fold higher mortality rate than those who did not. Conclusion The isolation of gram-negative bacteria is most frequent among recipient with hospital stays

  16. A Universal Culture Medium for Screening Polymyxin-Resistant Gram-Negative Isolates

    PubMed Central

    Jayol, Aurélie; Poirel, Laurent

    2016-01-01

    The colistin-containing SuperPolymyxin medium was developed for screening polymyxin-resistant Gram-negative bacteria. It was evaluated with 88 polymyxin-susceptible or polymyxin-resistant cultured Gram-negative isolates. Its sensitivity and specificity of detection were ca. 100%. The SuperPolymyxin medium is the first screening medium that is able to detect intrinsic and acquired polymyxin-resistant bacteria. PMID:26984971

  17. Rational Design of a Plasmid Origin That Replicates Efficiently in Both Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    Bryksin, Anton V.; Matsumura, Ichiro

    2010-01-01

    Background Most plasmids replicate only within a particular genus or family. Methodology/Principal Findings Here we describe an engineered high copy number expression vector, pBAV1K-T5, that produces varying quantities of active reporter proteins in Escherichia coli, Acinetobacter baylyi ADP1, Agrobacterium tumefaciens, (all Gram-negative), Streptococcus pneumoniae, Leifsonia shinshuensis, Peanibacillus sp. S18-36 and Bacillus subtilis (Gram-positive). Conclusions/Significance Our results demonstrate the efficiency of pBAV1K-T5 replication in different bacterial species, thereby facilitating the study of proteins that don't fold well in E. coli and pathogens not amenable to existing genetic tools. PMID:20949038

  18. Amplifiable DNA from Gram-negative and Gram-positive bacteria by a low strength pulsed electric field method

    PubMed Central

    Vitzthum, Frank; Geiger, Georg; Bisswanger, Hans; Elkine, Bentsian; Brunner, Herwig; Bernhagen, Jürgen

    2000-01-01

    An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of <60 V/cm. Pulses had an exponential decay waveform with a time constant of 3.4 µs. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications. PMID:10734214

  19. Reproducible discrimination between gram-positive and gram-negative bacteria using surface enhanced Raman spectroscopy with infrared excitation.

    PubMed

    Prucek, Robert; Ranc, Václav; Kvítek, Libor; Panáček, Aleš; Zbořil, Radek; Kolář, Milan

    2012-06-21

    The on time diagnostics of bacterial diseases is one of the essential steps in the foregoing treatment of such pathogens. Here we sought to present an easy to use and robust method for the discrimination between Gram-positive (Enterococcus faecalis and Streptococcus pyogenes) and Gram-negative (Acinetobacter baumannii and Klebsiella pneumoniae) bacterial genera based on surface enhanced Raman scattering (SERS) spectroscopy. The robustness of our approach lies in the novel method for the production of the SER substrate based on silver nanoparticles and their subsequent re-crystallization in solutions containing high concentrations of chloride ions. The method presented here could be an interesting alternative both to commonly used histochemical approaches and commercial SERS substrates.

  20. 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.

  1. 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.

  2. Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.

    PubMed

    Lupetti, A; Barnini, S; Morici, P; Ghelardi, E; Nibbering, P H; Campa, M

    2013-04-01

    The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

  3. 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

    2015-09-09

    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.

  4. Gram staining for the treatment of peritonsillar abscess.

    PubMed

    Takenaka, Yukinori; Takeda, Kazuya; Yoshii, Tadashi; Hashimoto, Michiko; Inohara, Hidenori

    2012-01-01

    Objective. To examine whether Gram staining can influence the choice of antibiotic for the treatment of peritonsillar abscess. Methods. Between 2005 and 2009, a total of 57 cases of peritonsillar abscess were analyzed with regard to cultured bacteria and Gram staining. Results. Only aerobes were cultured in 16% of cases, and only anaerobes were cultured in 51% of cases. Mixed growth of aerobes and anaerobes was observed in 21% of cases. The cultured bacteria were mainly aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. Phagocytosis of bacteria on Gram staining was observed in 9 cases. The bacteria cultured from these cases were aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. The sensitivity of Gram staining for the Gram-positive cocci and Gram-negative rods was 90% and 64%, respectively. The specificity of Gram staining for the Gram-positive cocci and Gram-negative rods was 62% and 76%, respectively. Most of the Gram-positive cocci were sensitive to penicillin, but some of anaerobic Gram-negative rods were resistant to penicillin. Conclusion. When Gram staining shows only Gram-positive cocci, penicillin is the treatment of choice. In other cases, antibiotics effective for the penicillin-resistant organisms should be used.

  5. Increase in Antibiotic-Resistant Gram-Negative Bacterial Infections in Febrile Neutropenic Children

    PubMed Central

    2016-01-01

    Background The incidence of bacteremia caused by Gram-negative bacteria has increased recently in febrile neutropenic patients with the increase of antibiotic-resistant Gram-negative bacterial infections. This study aimed to identify the distribution of causative bacteria and the proportion of antibiotic-resistant bacteria in bacteremia diagnosed in febrile neutropenic children. Materials and Methods The medical records of febrile neutropenic children diagnosed with bacteremia between 2010 and 2014 were retrospectively reviewed. The causative bacteria and proportion of antibiotic-resistant bacteria were investigated and compared yearly during the study period. The clinical impact of antibiotic-resistant bacterial infections was also determined. Results A total of 336 bacteremia episodes were identified. During the entire study period, 181 (53.9%) and 155 (46.1%) episodes were caused by Gram-negative and Gram-positive bacteria, respectively. Viridans streptococci (25.9%), Klebsiella spp. (16.7%), and Escherichia coli (16.4%) were the most frequent causative bacteria. The overall distribution of causative bacteria was not significantly different annually. Antibiotic-resistant bacteria were identified in 85 (25.3%) episodes, and the proportion of antibiotic-resistant bacteria was not significantly different annually. Extended-spectrum β-lactamase-producing E. coli and Klebsiella spp. were most common among antibiotic-resistant Gram-negative bacteria, and they accounted for 30.6% (n = 34) of the identified E. coli and K. pneumoniae. Methicillin-resistant coagulase-negative staphylococci were most common among antibiotic-resistant Gram-positive bacteria, and it accounted for 88.5% (n = 23) of the identified coagulase-negative staphylococci. Antibiotic-resistant bacterial infections, especially antibiotic-resistant Gram-negative bacterial infections, caused significantly higher mortality due to bacteremia compared with non-antibiotic-resistant bacterial infections (P <0

  6. Colistin: an antibiotic and its role in multiresistant Gram-negative infections.

    PubMed

    Loho, Tonny; Dharmayanti, Anti

    2015-04-01

    Increasing number of infection cases caused by multiresistant Gram-negative bacteria or multidrug resistant organism (MDRO) has become a major problem worldwide since there have been a lot of resistance to many classes of antibiotics. Mutant isolates such as fluoroquinolone-resistant and -lactamase-resistant bacteria have been commonly found, particularly in intensive care unit (ICU). During the last two decades, there has been no study of developing antibiotics in search of discovering new type of antibiotics; meanwhile, the resistance of Gram-negative bacteria or MDRO to antibiotics is increasing. Colistin or polymyxin E is an old antibiotic, which has been used since 1959 for treating infection caused by Gram-negative MDRO. It was revealed that colistin has side effects of nephrotoxicity and neurotoxicity; therefore, the use of this antibiotic was stopped and it was replaced by other antibiotics which were effective and were considered safer at that time. There is an increasing number of infections with multi-resistant Gram-negative (MDRO) against the available antibiotics and the availability of alternative antibiotics has not been satisfying; therefore, microbiologists are searching back to the old option, which has been proven to be effective against multi-resistant Gram-negative bacteria, the old antibiotic that has been long forgotten, i.e. colistin, as an alternative treatment against Gram-negative MDRO. It is expected that colistin may have essential and reliable role as future antibiotics for treatment of multi-resistant Gram-negative infections and as an alternative of antibiotics that have been available so far.

  7. Breaking barriers: expansion of the use of endolysins as novel antibacterials against Gram-negative bacteria.

    PubMed

    Briers, Yves; Lavigne, Rob

    2015-01-01

    The emergence and spread of antibiotic-resistant bacteria drives the search for novel classes of antibiotics to replenish our armamentarium against bacterial infections. This is particularly critical for Gram-negative pathogens, which are intrinsically resistant to many existing classes of antibiotics due to the presence of a protective outer membrane. In addition, the antibiotics development pipeline is mainly oriented to Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus. A promising novel class of antibacterials is endolysins. These enzymes encoded by bacterial viruses hydrolyze the peptidoglycan layer with high efficiency, resulting in abrupt osmotic lysis and cell death. Their potential as novel antibacterials to treat Gram-positive bacteria has been extensively demonstrated; however, the Gram-negative outer membrane has presented a formidable barrier for the use of endolysins against Gram-negatives until recently. This review reports on the most recent advances in the development of endolysins to kill Gram-negative species with a special focus on endolysin-engineered Artilysins(®).

  8. Nanoemulsion Therapy for Burn Wounds is Effective as a Topical Antimicrobial Against Gram Negative and Gram Positive Bacteria

    PubMed Central

    Dolgachev, Vladislav A.; Ciotti, Susan M.; Eisma, Rone; Gracon, Stephen; Wilkinson, J. Erby; Baker, James R.; Hemmila, Mark R.

    2014-01-01

    Objective The aim of this study is to investigate the antimicrobial efficacy of two different nanoemulsion formulations against Gram positive and Gram negative bacteria in an in vivo rodent scald burn model. Methods Male Sprague-Dawley rats were anesthetized and received a partial-thickness scald burn. Eight hours following burn injury the wound was inoculated with 1x108 colony forming units of Pseudomonas aeruginosa or Staphylococcus aureus. Treatment groups consisted of two different nanoemulsion formulations (NB-201, NB-402), nanoemulsion vehicle (NE vehicle), or saline. Topical application of the treatment was performed at 16 and 24 hours after burn injury. Animals were euthanized 32 hours after burn injury and skin samples 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. Results Both nanoemulsion formulations (NB-201, NB 402) significantly reduced burn wound infections with either Pseudomonas aeruginosa or Staphylococcus aureus, and decreased median bacterial counts at least 3 logs as compared to animals with saline applications (p<0.0001). NB-201 and NB-402 also decreased dermal neutrophil recruitment and sequestration into the wound as measured by myeloperoxidase assay and histopathology (p<0.05). In addition, there was a reduction in the pro-inflammatory dermal cytokines (IL-1β, IL-6 and TNF-α) and the neutrophil chemoattractants CXCL1 and CXCL2. By histology examination, both NB-201 and NB-402 appeared to suppress burn wound progression 72 hours after injury. Conclusions Topically applied NB-201 and NB-402 are effective in decreasing Gram positive and negative bacteria growth in burn wounds, reducing inflammation and abrogating burn wound progression. PMID:26182074

  9. Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance.

    PubMed

    Stokes, Jonathan M; MacNair, Craig R; Ilyas, Bushra; French, Shawn; Côté, Jean-Philippe; Bouwman, Catrien; Farha, Maya A; Sieron, Arthur O; Whitfield, Chris; Coombes, Brian K; Brown, Eric D

    2017-03-06

    The increasing use of polymyxins(1) in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defence against multidrug-resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine-as is mediated by Mcr-1 (ref. 2)-which reduce the affinity of polymyxins for lipopolysaccharide(3). Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections(4). The difficulty in eradicating Gram-negative bacteria is largely due to their highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics(5). Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer-membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine(6) as an effective perturbant of the Gram-negative outer membrane through its interaction with lipopolysaccharide. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin-resistant bacteria in vitro and in vivo. Overall, pentamidine and its structural analogues represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.

  10. Molecular studies neglect apparently gram-negative populations in the human gut microbiota.

    PubMed

    Hugon, Perrine; Lagier, Jean-Christophe; Robert, Catherine; Lepolard, Catherine; Papazian, Laurent; Musso, Didier; Vialettes, Bernard; Raoult, Didier

    2013-10-01

    Studying the relationships between gut microbiota, human health, and diseases is a major challenge that generates contradictory results. Most studies draw conclusions about the gut repertoire using a single biased metagenomics approach. We analyzed 16 different stool samples collected from healthy subjects who were from different areas, had metabolic disorders, were immunocompromised, or were treated with antibiotics at the time of the stool collection. The analyses performed included Gram staining, flow cytometry, transmission electron microscopy (TEM), quantitative real-time PCR (qPCR) of the Bacteroidetes and Firmicutes phyla, and pyrosequencing of the 16S rRNA gene amplicons targeting the V6 region. We quantified 10(10) prokaryotes per gram of feces, which is less than was previously described. The Mann-Whitney test revealed that Gram-negative proportions of the prokaryotes obtained by Gram staining, TEM, and pyrosequencing differed according to the analysis used, with Gram-negative prokaryotes yielding median percentages of 70.6%, 31.0%, and 16.4%, respectively. A comparison of TEM and pyrosequencing analyses highlighted a difference of 14.6% in the identification of Gram-negative prokaryotes, and a Spearman test showed a tendency toward correlation, albeit not significant, in the Gram-negative/Gram-positive prokaryote ratio (ρ = 0.3282, P = 0.2146). In contrast, when comparing the qPCR and pyrosequencing results, a significant correlation was found for the Bacteroidetes/Firmicutes ratio (ρ = 0.6057, P = 0.0130). Our study showed that the entire diversity of the human gut microbiota remains unknown because different techniques generate extremely different results. We found that to assess the overall composition of bacterial communities, multiple techniques must be combined. The biases that exist for each technique may be useful in exploring the major discrepancies in molecular studies.

  11. Growth ability of Gram negative bacteria in free-living amoebae.

    PubMed

    Zeybek, Zuhal; Binay, Ali Rıza

    2014-11-01

    When bacteria and free-living amoebae (FLAs) live both in natural waters and man-made aquatic systems, they constantly interact with each other. Some bacteria can survive and grow within FLAs. Therefore, it has recently been thought that FLAs play an important role in spreading pathogenic bacteria in aquatic systems. In this study we investigated the intracellular growing ability of 7 different Gram-negative bacteria (Pseudomonas fluorescens, Pseudomonas putida, Pasteurella pneumotropica, Aeromonas salmonicida, Legionella pneumophila serogroup 1, L. pneumophila serogroup 3, L. pneumophila serogroup 6) in four different FLA isolates (A1-A4). Among these, four bacterial isolates (P. fluorescens, P.putida, P.pneumotropica, A.salmonicida) and two free-living amoebae isolates (A3, A4) were isolated from the tap water in our city (Istanbul). It was found that 4 different Gram-negative bacteria could grow in A1, 2 different Gram-negative bacteria could grow in A2, 4 different Gram-negative bacteria could grow in A3, 1 Gram-negative bacterium could grow in A4. In conclusion, we think that this ability of growth could vary according to the characteristics of both bacteria and FLA isolates. Also, other factors such as environmental temperature, bacterial concentration, and extended incubation period may play a role in these interactions. This situation can be clarified with future studies.

  12. Design, synthesis and biological evaluation of monobactams as antibacterial agents against gram-negative bacteria.

    PubMed

    Fu, Hai-Gen; Hu, Xin-Xin; Li, Cong-Ran; Li, Ying-Hong; Wang, Yan-Xiang; Jiang, Jian-Dong; Bi, Chong-Wen; Tang, Sheng; You, Xue-Fu; Song, Dan-Qing

    2016-03-03

    A series of monobactam derivatives were prepared and evaluated for their antibacterial activities against susceptible and resistant Gram-negative strains, taking Aztreonam and BAL30072 as the leads. Six conjugates (12a-f) bearing PIH-like siderophore moieties were created to enhance the bactericidal activities against Gram-negative bacteria based on Trojan Horse strategy, and all of them displayed potencies against susceptible Gram-negative strains with MIC ≤ 8 μg/mL. SAR revealed that the polar substituents on the oxime side chain were beneficial for activities against resistant Gram-negative bacteria. Compounds 19c and 33a-b exhibited the promising potencies against ESBLs-producing E. coli and Klebsiella pneumoniae with MICs ranging from 2 μg/mL to 8 μg/mL. These results offered powerful information for further strategic optimization in search of the antibacterial candidates against MDR Gram-negative bacteria.

  13. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    PubMed

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  14. In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

    PubMed Central

    Miyasaki, K T; Bodeau, A L; Ganz, T; Selsted, M E; Lehrer, R I

    1990-01-01

    Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria. Images PMID:2254020

  15. Functionalized magnetic iron oxide (Fe3O4) nanoparticles for capturing gram-positive and gram-negative bacteria.

    PubMed

    Reddy, P Muralidhar; Chang, Kai-Chih; Liu, Zhen-Jun; Chen, Cheng-Tung; Ho, Yen-Peng

    2014-08-01

    The development of nanotechnology in biology and medicine has raised the need for conjugation of nanoparticles (NPs) to biomolecules. In this study, magnetic and functionalized magnetic iron oxide nanoparticles were synthesized and used as affinity probes to capture Gram-positive/negative bacteria. The morphology and properties of the magnetic NPs were examined by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. Furthermore, this study investigated the interaction between functionalized magnetic nanoparticles and Gram positive/negative bacteria. The positively and negatively charged magnetic nanoparticles include functionalities of Fe3O4, SiO2, TiO2, ZrO2, poly ethyleneimine (PEI) and poly acrylic acid. Their capture efficiencies for bacteria were investigated based on factors such as zeta potential, concentration and pH value. PEI particles carry a positive charge over a range of pH values from 3 to 10, and the particles were found to be an excellent candidate for capturing bacteria over such pH range. Since the binding force is mainly electrostatic, the architecture and orientation of the functional groups on the NP surface are not critical. Finally the captured bacteria were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The minimum detection limit was 10(4) CFU/mL and the analysis time was reduced to be less than 1 hour. In addition, the detection limit could be reduced to an extremely low concentration of 50 CFU/mL when captured bacteria were cultivated.

  16. Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II

    PubMed Central

    Cochrane, Stephen A.; Findlay, Brandon; Bakhtiary, Alireza; Acedo, Jeella Z.; Rodriguez-Lopez, Eva M.; Mercier, Pascal; Vederas, John C.

    2016-01-01

    Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1 exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1 in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1–lipid II complex. These results suggest that TriA1 kills Gram-negative bacteria by a mechanism of action using a lipid-II–binding motif. PMID:27688760

  17. [Antimicrobial therapy in severe infections with multidrug-resistant Gram-negative bacterias].

    PubMed

    Duszyńska, Wiesława

    2010-01-01

    Multidrug-resistant Gram-negative bacteria pose a serious and rapidly emerging threat to patients in healthcare settings, and are especially prevalent and problematic in intensive therapy units. Recently, the emergence of pandrug-resistance in Gram-negative bacteria poses additional concerns. This review examines the clinical impact and epidemiology of multidrug-resistant Gram-negative bacteria as a cause of increased morbidity and mortality among ITU patients. Beta-lactamases, cephalosporinases and carbapenemases play the most important role in resistance to antibiotics. Despite the tendency to increased resistance, carbapenems administered by continuous infusion remain the most effective drugs in severe sepsis. Drug concentration monitoring, albeit rarely used in practice, is necessary to ensure an effective therapeutic effect.

  18. Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II.

    PubMed

    Cochrane, Stephen A; Findlay, Brandon; Bakhtiary, Alireza; Acedo, Jeella Z; Rodriguez-Lopez, Eva M; Mercier, Pascal; Vederas, John C

    2016-10-11

    Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1 exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1 in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1-lipid II complex. These results suggest that TriA1 kills Gram-negative bacteria by a mechanism of action using a lipid-II-binding motif.

  19. Bacteriocins active against multi-resistant gram negative bacteria implicated in nosocomial infections.

    PubMed

    Ghodhbane, Hanen; Elaidi, Sabrine; Sabatier, Jean-Marc; Achour, Sami; Benhmida, Jeannette; Regaya, Imed

    2015-01-01

    Multiresistant Gram-negative bacteria are the prime mover of nosocomial infections. Some are naturally resistant to antibiotics, their genetic makes them insensitive to certain families of antibiotics and they transmit these resistors to their offspring. Moreover, when bacteria are subjected to antibiotics, they eventually develop resistance against drugs to which they were previously sensitive. In recent years, many bacteriocins active against gram-negative bacteria have been identified proving their efficacy in treating infections. While further investigation remains necessary before the possibilities for bacteriocins in clinical practice can be described more fully, this review provides an overview of bacteriocins acting on the most common infectious gram negative bacteria (Klebsiella, Acinetobacter, Pseudomonas aeruginosa and E. coli).

  20. Physical methods to quantify small antibiotic molecules uptake into Gram-negative bacteria.

    PubMed

    Winterhalter, Mathias; Ceccarelli, Matteo

    2015-09-01

    The development of antibiotics against Gram-negative bacteria is a challenge: any active compound must cross the outer cell envelope composed of a hydrophilic highly charged lipopolysaccharide layer followed by a tight hydrophobic layer containing water filled gates called porins to reach the hydrophilic periplasmic space and depending on the target with the further need to cross the hydrophobic inner membrane. In addition to a possible rapid enzymatic deactivation efflux pumps shuffle compounds back outside. The resulting low permeability of cell envelope requires high dose and leads therefore to toxicity problems. Despite its relevance the permeability barrier in Gram-negative bacteria is not well understood partially caused by the lack of appropriate direct assays. Here we give a brief introduction on current available techniques to quantify passive diffusion of small hydrophilic molecules into Gram-negative bacteria.

  1. [Estimation of abundance dynamics of gram-negative bacteria in soil].

    PubMed

    Polianskaia, L M; Ivanov, K E; Guzev, V S; Zviagintsev, D G

    2008-01-01

    Bacterial succession in soil was studied for two variants of initiation (moistening and moistening with addition of glucose). To determine the numbers of viable gram-negative bacteria, the modified nalidixic acid method was applied. The numbers of gram-negative bacteria revealed by this method were 2 to 3.5 times higher than those determined by the traditional method. In a developing community, the highest total bacterial numbers were observed on day 7; afterwards their numbers decreased and stabilized at a level exceeding four- to fivefold the initial one. In both experimental variants, the highest numbers of viable gram-negative bacteria were revealed on day 15 (75-85% of the total bacterial numbers). Morphology of these bacteria suggests their classification as cytophagas (chitinophagas) utilizing chitin from the dead fungal mycelium.

  2. Emerging issues in gram-negative bacterial resistance: an update for the practicing clinician.

    PubMed

    Vasoo, Shawn; Barreto, Jason N; Tosh, Pritish K

    2015-03-01

    The rapid and global spread of antimicrobial-resistant organisms in recent years has been unprecedented. Although resistant gram-positive infections have been concerning to clinicians, the increasing incidence of antibiotic-resistant gram-negative infections has become the most pressing issue in bacterial resistance. Indiscriminate antimicrobial use in humans and animals coupled with increased global connectivity facilitated the transmission of gram-negative infections harboring extended-spectrum β-lactamases in the 1990s. Carbapenemase-producing Enterobacteriaceae, such as those containing Klebsiella pneumoniae carbapenemases and New Delhi metallo-β-lactamases, have been the latest scourge since the late 1990s to 2000s. Besides β-lactam resistance, these gram-negative infections are often resistant to multiple drug classes, including fluoroquinolones, which are commonly used to treat community-onset infections. In certain geographic locales, these pathogens, which have been typically associated with health care-associated infections, are disseminating into the community, posing a significant dilemma for clinicians treating community-onset infections. In this Concise Review, we summarize emerging trends in antimicrobial resistance. We also review the current knowledge on the detection, treatment, and prevention of infection with these organisms, with a focus on the carbapenemase-producing gram-negative bacilli. Finally, we discuss emerging therapies and areas that need further research and effort to stem the spread of antimicrobial resistance.

  3. Antimicrobial Resistance in the Intensive Care Unit: A Focus on Gram-Negative Bacterial Infections.

    PubMed

    MacVane, Shawn H

    2017-01-01

    Bacterial infections are a frequent cause of hospitalization, and nosocomial infections are an increasingly common condition, particularly within the acute/critical care setting. Infection control practices and new antimicrobial development have primarily focused on gram-positive bacteria; however, in recent years, the incidence of infections caused by gram-negative bacteria has risen considerably in intensive care units. Infections caused by multidrug-resistant (MDR) gram-negative organisms are associated with high morbidity and mortality, with significant direct and indirect costs resulting from prolonged hospitalizations due to antibiotic treatment failures. Of particular concern is the increasing prevalence of antimicrobial resistance to β-lactam antibiotics (including carbapenems) among Pseudomonas aeruginosa and Acinetobacter baumannii and, recently, among pathogens of the Enterobacteriaceae family. Treatment options for infections caused by these pathogens are limited. Antimicrobial stewardship programs focus on optimizing the appropriate use of currently available antimicrobial agents with the goals of improving outcomes for patients with infections caused by MDR gram-negative organisms, slowing the progression of antimicrobial resistance, and reducing hospital costs. Newly approved treatment options are available, such as β-lactam/β-lactamase inhibitor combinations, which significantly extend the armamentarium against MDR gram-negative bacteria.

  4. Biocompatible Fe3O4 increases the efficacy of amoxicillin delivery against Gram-positive and Gram-negative bacteria.

    PubMed

    Grumezescu, Alexandru Mihai; Gestal, Monica Cartelle; Holban, Alina Maria; Grumezescu, Valentina; Vasile, Bogdan Stefan; Mogoantă, Laurențiu; Iordache, Florin; Bleotu, Coralia; Mogoșanu, George Dan

    2014-04-22

    This paper reports the synthesis and characterization of amoxicillin- functionalized magnetite nanostructures (Fe3O4@AMO), revealing and discussing several biomedical applications of these nanomaterials. Our results proved that 10 nm Fe3O4@AMO nanoparticles does not alter the normal cell cycle progression of cultured diploid cells, and an in vivo murine model confirms that the nanostructures disperse through the host body and tend to localize in particular sites and organs. The nanoparticles were found clustered especially in the lungs, kidneys and spleen, next to the blood vessels at this level, while being totally absent in the brain and liver, suggesting that they are circulated through the blood flow and have low toxicity. Fe3O4@AMO has the ability to be easily circulated through the body and optimizations may be done so these nanostructures cluster to a specific target region. Functionalized magnetite nanostructures proved a great antimicrobial effect, being active against both the Gram positive pathogen S. aureus and the Gram negative pathogen E. coli. The fabricated nanostructures significantly reduced the minimum inhibitory concentration (MIC) of the active drug. This result has a great practical relevance, since the functionalized nanostructures may be used for decreasing the therapeutic doses which usually manifest great severe side effects, when administrated in high doses. Fe3O4@AMO represents also a suitable approach for the development of new alternative strategies for improving the activity of therapeutic agents by targeted delivery and controlled release.

  5. Nanoparticle targeting of Gram-positive and Gram-negative bacteria for magnetic-based separations of bacterial pathogens

    NASA Astrophysics Data System (ADS)

    Lu, Hoang D.; Yang, Shirley S.; Wilson, Brian K.; McManus, Simon A.; Chen, Christopher V. H.-H.; Prud'homme, Robert K.

    2017-02-01

    Antimicrobial resistance is a healthcare problem of increasing significance, and there is increasing interest in developing new tools to address bacterial infections. Bacteria-targeting nanoparticles hold promise to improve drug efficacy, compliance, and safety. In addition, nanoparticles can also be used for novel applications, such as bacterial imaging or bioseperations. We here present the use of a scalable block-copolymer-directed self-assembly process, Flash NanoPrecipitation, to form zinc(II)-bis(dipicolylamine) modified nanoparticles that bind to both Gram-positive and Gram-negative bacteria with specificity. Particles have tunable surface ligand densities that change particle avidity and binding efficacy. A variety of materials can be encapsulated into the core of the particles, such as optical dyes or iron oxide colloids, to produce imageable and magnetically active bacterial targeting constructs. As a proof-of-concept, these particles are used to bind and separate bacteria from solution in a magnetic column. Magnetic manipulation and separation would translate to a platform for pathogen identification or removal. These magnetic and targeted nanoparticles enable new methods to address bacterial infections.

  6. Unraveling the Differences between Gram-Positive and Gram-Negative Probiotics in Modulating Protective Immunity to Enteric Infections

    PubMed Central

    Kandasamy, Sukumar; Vlasova, Anastasia N.; Fischer, David D.; Chattha, Kuldeep S.; Shao, Lulu; Kumar, Anand; Langel, Stephanie N.; Rauf, Abdul; Huang, Huang-Chi; Rajashekara, Gireesh; Saif, Linda J.

    2017-01-01

    The role of intestinal microbiota and probiotics in prevention and treatment of infectious diseases, including diarrheal diseases in children and animal models, is increasingly recognized. Intestinal commensals play a major role in development of the immune system in neonates and in shaping host immune responses to pathogens. Lactobacilli spp. and Escherichia coli Nissle 1917 are two probiotics that are commonly used in children to treat various medical conditions including human rotavirus diarrhea and inflammatory bowel disease. Although the health benefits of probiotics have been confirmed, the specific effects of these established Gram-positive (G+) and Gram-negative (G−) probiotics in modulating immunity against pathogens and disease are largely undefined. In this review, we discuss the differences between G+ and G− probiotics/commensals in modulating the dynamics of selected infectious diseases and host immunity. These probiotics modulate the pathogenesis of infectious diseases and protective immunity against pathogens in a species- and strain-specific manner. Collectively, it appears that the selected G− probiotic is more effective than the various tested G+ probiotics in enhancing protective immunity against rotavirus in the gnotobiotic piglet model.

  7. 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.

  8. Comparative in vitro activity of gemifloxacin against gram-positive and gram-negative clinical isolates in Argentina.

    PubMed

    Lopez, H; Stepanik, D; Vilches, V; Scarano, S; Sarachian, B; Mikaelian, G; Finlay, J; Sucari, A

    2001-08-01

    The in vitro activity of gemifloxacin against 1,000 clinical isolates of 147 Streptococcus pneumoniae (115, penicilin susceptible; 26, intermediate penicillin-resistant and 6, penicillin-resistant), 127 Hemophilus influenzae (109, beta lactamasa non-producer; 18, beta lactamase producers), 95 Streptococcus pyogenes (6, azytromycin-resistant), 84 Moraxella catarrhalis (79, beta lactamase producers), 110 Staphilococcus aureus (89, methicillin-susceptible; 21, methicilin-resistant), 98 Eenterococcus faecalis and 339 Enterobacteriacea, (recovered from patients with respiratory tract infection; skin and soft tissue infection and urinary tract infection), was compared with the activities of four fluorquinolones and five other antimicrobial agents. Of the quinolones tested, gemifloxacin was the most potent against Streptococcus pneumoniae, including penicillin intermediate and resistant strains. Mic(90) values obtained for gemifloxacin, ciprofloxacin, ofloxacin, levofloxacin and trvafloxacin were 0.03, 2, 2, 1 and 0.25 mg/L respectively. Gemifloxacin was 16 fold more potent than ciprofloxacin against methicillin-susceptible Staphylococcus aureus and 32 fold more potent than ciprofloxacin against Streptococcus pyogenes. When tested against Hemophilus influenzae, Moraxella catarrhalis and Enterobacteriaceae, all the quinolones showed similar activity. Our results demonstrate that gemifloxacin has similar activity than the other quinolones tested against Gram-negative organisms and is considerably more potent against Gram-positive organisms.

  9. Stronger T cell immunogenicity of ovalbumin expressed intracellularly in Gram-negative than in Gram-positive bacteria.

    PubMed

    Martner, Anna; Ostman, Sofia; Lundin, Samuel; Rask, Carola; Björnsson, Viktor; Telemo, Esbjörn; Collins, L Vincent; Axelsson, Lars; Wold, Agnes E

    2013-01-01

    This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G-) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4(+) T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G- bacteria and may be relevant for the use of bacterial carriers in vaccine development.

  10. 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

  11. 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.

  12. Impact of fluoroquinolone resistance in Gram-negative bloodstream infections on healthcare utilization.

    PubMed

    Brigmon, M M; Bookstaver, P Brandon; Kohn, J; Albrecht, H; Al-Hasan, M N

    2015-09-01

    There has been a concerning increase in fluoroquinolone resistance among Gram-negative bloodstream isolates. This retrospective cohort study examines the implications of fluoroquinolone resistance on use of healthcare resources in patients with Gram-negative bloodstream infections (BSI). Hospitalized adults with first episodes of community-onset Gram-negative BSI from 2010 to 2012 at Palmetto Health Hospitals in Columbia, SC, USA were identified. Multivariate linear regression was used to examine risk factors for prolonged hospital length of stay (HLOS) in survivors of Gram-negative BSI. Among 474 unique patients, 384 (81%) and 90 (19%) had BSI due to fluoroquinolone-susceptible (FQ-S) and fluoroquinolone non-susceptible (FQ-NS) Gram-negative bacilli, respectively. The FQ-NS bloodstream isolates, particularly Escherichia coli, were more likely than FQ-S isolates to be multi-drug resistant (56% versus 6%, p < 0.001). Compared with patients with BSI due to FQ-S bloodstream isolates, those with FQ-NS isolates were more likely to receive inappropriate empirical antimicrobial therapy (26% versus 3%, p < 0.001), have longer mean HLOS (11.6 versus 9.3 days, p 0.03) and treatment duration with intravenous antibiotics during hospitalization (9.1 versus 7.1 days, p 0.001), and use outpatient intravenous antibiotics at hospital discharge (15% versus 8%, p 0.05). After adjustments in the multivariate model, inappropriate empirical antimicrobial therapy was an independent risk factor for prolonged HLOS in survivors of Gram-negative BSI (parameter estimate 3.65 days, 95% CI 0.43-6.86). Multi-drug resistance among FQ-NS bloodstream isolates limits both empirical and definitive antimicrobial treatment options and poses excessive burdens on the healthcare system.

  13. Infections Caused by Resistant Gram-Negative Bacteria: Epidemiology and Management.

    PubMed

    Kaye, Keith S; Pogue, Jason M

    2015-10-01

    Infections caused by resistant gram-negative bacteria are becoming increasingly prevalent and now constitute a serious threat to public health worldwide because they are difficult to treat and are associated with high morbidity and mortality rates. In the United States, there has been a steady increase since 2000 in rates of extended-spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii, particularly among hospitalized patients with intraabdominal infections, urinary tract infections, ventilator-associated pneumonia, and bacteremia. Colonization with resistant gram-negative bacteria is common among residents in long-term care facilities (particularly those residents with an indwelling device), and these facilities are considered important originating sources of such strains for hospitals. Antibiotic resistance is associated with a substantial clinical and economic burden, including increased mortality, greater hospital and antibiotic costs, and longer stays in hospitals and intensive care units. Control of resistant gram-negative infections requires a comprehensive approach, including strategies for risk factor identification, detection and identification of resistant organisms, and implementation of infection-control and prevention strategies. In treating resistant gram-negative infections, a review of surveillance data and hospital-specific antibiograms, including resistance patterns within local institutions, and consideration of patient characteristics are helpful in guiding the choice of empiric therapy. Although only a few agents are available with activity against resistant gram-negative organisms, two recently released β-lactam/β-lactamase inhibitor combinations - ceftolozane/tazobactam and ceftazidime/avibactam - have promising activity against these organisms. In this article, we review the epidemiology, risk factors, and

  14. Inhibitory effect of short cationic homopeptides against Gram-negative bacteria.

    PubMed

    Carvajal-Rondanelli, Patricio; Aróstica, Mónica; Marshall, Sergio Hernan; Albericio, Fernando; Álvarez, Claudio Andrés; Ojeda, Claudia; Aguilar, Luis Felipe; Guzmán, Fanny

    2016-06-01

    Previous work demonstrated that Lys homopeptides with an odd number of residues (9, 11 and 13) were capable of inhibiting the growth of Gram-positive bacteria in a broader spectrum and more efficiently than those with an even number of Lys residues or Arg homopeptides of the same size. Indeed, all Gram-positive bacteria tested were totally inhibited by 11-residue Lys homopeptides. In the present work, a wide variety of Gram-negative bacteria were used to evaluate the inhibitory activity of chemically synthesized homopeptides of L-Lys and L-Arg ranging from 7 to 14 residues. Gram-negative bacteria were comparatively more resistant than Gram-positive bacteria to Lys homopeptides with an odd number of residues, but exhibited a similar inhibition pattern than on Gram-positive bacteria. CD spectra for the odd-numbered Lys homopeptides in anionic lipid dimyristoylphosphatidylglycerol, and Escherichia coli membrane extract increased polyproline II content, as compared to those measured in phosphate buffer solution. Lys and Arg homopeptides were covalently linked to rhodamine to visualize the peptide interactions with E. coli cells using confocal laser scanning microscopy. Analysis of Z-stack images showed that Arg homopeptides indeed appear to be localized intracellularly, while the Lys homopeptide is localized exclusively on the plasma membrane. Moreover, these Lys homopeptides induced membrane disruption since the Sytox fluorophore was able to bind to the DNA in E. coli cultures.

  15. Susceptibility of Multidrug-Resistant Gram-Negative Urine Isolates to Oral Antibiotics

    PubMed Central

    Zucchi, Paola C.; Chen, Alice; Raux, Brian R.; Kirby, James E.; McCoy, Christopher; Eliopoulos, George M.

    2016-01-01

    Increasing resistance among Gram-negative uropathogens limits treatment options, and susceptibility data for multidrug-resistant isolates are limited. We assessed the activity of five oral agents against 91 multidrug-resistant Gram-negative urine isolates that were collected from emergency department/hospitalized patients. Fosfomycin and nitrofurantoin were most active (>75% susceptibility). Susceptibilities to sulfamethoxazole-trimethoprim, ciprofloxacin, and ampicillin were ≤40%; empirical use of these agents likely provides inadequate coverage in areas with a high prevalence of multidrug-resistant uropathogens. PMID:26883704

  16. Fluorogenic substrates for differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria.

    PubMed Central

    Kämpfer, P; Kulies, I; Dott, W

    1992-01-01

    A total of 803 strains of gram-negative nonfermentative and oxidase-positive fermentative bacteria (38 taxa) were investigated for their ability to hydrolyze 53 different fluorogenic 4-methylumbelliferyl- and beta-naphthylamide-linked substrates within 6 h of incubation. The hydrolysis of 16 fluorogenic substrates showed high separation index values among the tested taxa, was reproducible, and showed good agreement with data in the literature. In combination with other biochemical tests (like carbon substrate utilization tests and classical biochemical tests), hydrolysis profiles can improve the differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria. PMID:1624555

  17. Intrinsic, adaptive and acquired antimicrobial resistance in Gram-negative bacteria.

    PubMed

    Arzanlou, Mohsen; Chai, Wern Chern; Venter, Henrietta

    2017-02-28

    Gram-negative bacteria are responsible for a large proportion of antimicrobial-resistant infections in humans and animals. Among this class of bacteria are also some of the most successful environmental organisms. Part of this success is their adaptability to a variety of different niches, their intrinsic resistance to antimicrobial drugs and their ability to rapidly acquire resistance mechanisms. These mechanisms of resistance are not exclusive and the interplay of several mechanisms causes high levels of resistance. In this review, we explore the molecular mechanisms underlying resistance in Gram-negative organisms and how these different mechanisms enable them to survive many different stress conditions.

  18. Antibacterial activities of Emblica officinalis and Coriandrum sativum against Gram negative urinary pathogens.

    PubMed

    Saeed, Sabahat; Tariq, Perween

    2007-01-01

    Present investigation is focused on antibacterial potential of aqueous infusions and aqueous decoctions of Emblica officinalis (amla) and Coriandrum sativum (coriander) against 345 bacterial isolates belonging to 6 different genera of Gram negative bacterial population isolated from urine specimens by employing well diffusion technique. Aqueous infusion and decoction of Emblica officinalis exhibited potent antibacterial activity against Escherichia coli (270), Klebsiella pneumoniae (51), K. ozaenae (3), Proteus mirabilis (5), Pseudomonas aeruginosa (10), Salmonella typhi (1), S. paratyphi A (2), S. paratyphi B (1) and Serratia marcescens (2) but did not show any antibacterial activity against Gram negative urinary pathogens.

  19. Evolution of β-lactams resistance in Gram-negative bacteria in Tunisia.

    PubMed

    Chouchani, Chedly; Marrakchi, Rim; El Salabi, Allaaeddin

    2011-08-01

    Antimicrobial resistance is a major health problem worldwide, but marked variations in the resistance profiles of bacterial pathogens are found between countries and in different patient settings. In Tunisia, the strikingly high prevalence of resistance of bacteria to penicillins and cephalorosporins drugs including fourth generation in clinical isolates of Gram negative bacteria has been reported. During 30 years, the emerging problem of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates is substantial, and some unique enzymes have been found. Recently, evidence that Gram-negative bacteria are resistant to nearly all available antimicrobial agents, including carbapenems, have emerged.

  20. An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria

    PubMed Central

    Cox, Georgina; Koteva, Kalinka; Wright, Gerard D.

    2014-01-01

    Objectives An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. Methods E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. Results The anthracycline Antibiotic 301A1 was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure–activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A1 abolishes activity. Conclusions Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives. PMID:24627312

  1. 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

  2. Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed Gram-positive and Gram-negative bacteria.

    PubMed

    Beran, O; Potměšil, R; Holub, M

    2011-05-01

    Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognise conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. Here, we compared the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed Gram-negative and Gram-positive human pathogens Neisseria meningitidis, Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of Gram-positive bacteria, whereas higher concentrations of Gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase compared with low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, Gram-negative pathogens induced higher cytokine levels than Gram-positive ones at same concentrations. These results indicate that Gram-positive and Gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not in cytokine expression.

  3. Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

    PubMed

    Zdybicka-Barabas, Agnieszka; Mak, Pawel; Klys, Anna; Skrzypiec, Krzysztof; Mendyk, Ewaryst; Fiołka, Marta J; Cytryńska, Małgorzata

    2012-11-01

    Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

  4. Strategies for the use of bacteriocins in Gram-negative bacteria: relevance in food microbiology.

    PubMed

    Prudêncio, Cláudia Vieira; Dos Santos, Miriam Teresinha; Vanetti, Maria Cristina Dantas

    2015-09-01

    Bacteriocins are ribosomally synthesized peptides that have bacteriostatic or bactericidal effects on other bacteria. The use of bacteriocins has emerged as an important strategy to increase food security and to minimize the incidence of foodborne diseases, due to its minimal impact on the nutritional and sensory properties of food products. Gram-negative bacteria are naturally resistant to the action of bacteriocins produced by Gram-positive bacteria, which are widely explored in foods. However, these microorganisms can be sensitized by mild treatments, such as the use of chelating agents, by treatment with plant essential oils or by physical treatments such as heating, freezing or high pressure processing. This sensitization is important in food microbiology, because most pathogens that cause foodborne diseases are Gram-negative bacteria. However, the effectiveness of these treatments is influenced by several factors, such as pH, temperature, the composition of the food and target microbiota. In this review, we comment on the main methods used for the sensitization of Gram-negative bacteria, especially Salmonella, to improve the action of bacteriocins produced by Gram-positive bacteria.

  5. Performance of Gram staining on blood cultures flagged negative by an automated blood culture system.

    PubMed

    Peretz, A; Isakovich, N; Pastukh, N; Koifman, A; Glyatman, T; Brodsky, D

    2015-08-01

    Blood is one of the most important specimens sent to a microbiology laboratory for culture. Most blood cultures are incubated for 5-7 days, except in cases where there is a suspicion of infection caused by microorganisms that proliferate slowly, or infections expressed by a small number of bacteria in the bloodstream. Therefore, at the end of incubation, misidentification of positive cultures and false-negative results are a real possibility. The aim of this work was to perform a confirmation by Gram staining of the lack of any microorganisms in blood cultures that were identified as negative by the BACTEC™ FX system at the end of incubation. All bottles defined as negative by the BACTEC FX system were Gram-stained using an automatic device and inoculated on solid growth media. In our work, 15 cultures that were defined as negative by the BACTEC FX system at the end of the incubation were found to contain microorganisms when Gram-stained. The main characteristic of most bacteria and fungi growing in the culture bottles that were defined as negative was slow growth. This finding raises a problematic issue concerning the need to perform Gram staining of all blood cultures, which could overload the routine laboratory work, especially laboratories serving large medical centers and receiving a large number of blood cultures.

  6. Gram-Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria

    PubMed Central

    Li, Zusheng; Clarke, Anthony J.; Beveridge, Terry J.

    1998-01-01

    Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected. Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity. PMID:9765585

  7. Pyridone Methylsulfone Hydroxamate LpxC Inhibitors for the Treatment of Serious Gram-Negative Infections

    SciTech Connect

    Montgomery, Justin I.; Brown, Matthew F.; Reilly, Usa; Price, Loren M.; Abramite, Joseph A.; Arcari, Joel; Barham, Rose; Che, Ye; Chen, Jinshan Michael; Chung, Seung Won; Collantes, Elizabeth M.; Desbonnet, Charlene; Doroski, Matthew; Doty, Jonathan; Engtrakul, Juntyma J.; Harris, Thomas M.; Huband, Michael; Knafels, John D.; Leach, Karen L.; Liu, Shenping; Marfat, Anthony; McAllister, Laura; McElroy, Eric; Menard, Carol A.; Mitton-Fry, Mark; Mullins, Lisa; Noe, Mark C.; O’Donnell, John; Oliver, Robert; Penzien, Joseph; Plummer, Mark; Shanmugasundaram, Veerabahu; Thoma, Christy; Tomaras, Andrew P.; Uccello, Daniel P.; Vaz, Alfin; Wishka, Donn G.

    2012-11-09

    The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.

  8. Persistence of associated gram-negative bacteria in experimental actinomycotic lesions in mice.

    PubMed Central

    Jordan, H V; Kelly, D M

    1983-01-01

    Mixed actinomycotic infections were established in a susceptible weanling mouse model by using combinations of Actinomyces israelii and Eikenella corrodens or A. israelii and Actinobacillus actinomycetemcomitans. Acute lesions caused by either of the gram-negative organisms alone were resolved within a few weeks; however, these organisms persisted up to 3 months in chronic lesions in combination with A. israelii. PMID:6341251

  9. Control of emergence of multi-resistant gram-negative bacilli by exclusive use of amikacin.

    PubMed

    Ruiz-Palacios, G M; Ponce de Leon, S; Sifuentes, J; Ponce de Leon, S; Calva, J J; Huazano, F; Ontiveros, C; Ojeda, F; Bobadilla, M

    1986-06-30

    Results of a three-year prospective study of amikacin as the only aminoglycoside used at the Instituto Nacional de la Nutrición "Salvador Zubirán" are presented. During the initial three-month baseline period, resistance to amikacin, gentamicin, and tobramycin among 870 gram-negative bacterial isolates was 3.2 percent, 17.4 percent, and 11.2 percent, respectively. In this period, the overall consumption of aminoglycosides was 69 percent for gentamicin, 30.5 percent for amikacin, and 0.5 percent for tobramycin. In the following period of exclusive amikacin use, sensitivity patterns of 9,344 gram-negative strains isolated over three years were recorded. During this period, amikacin constituted 99.3 percent of all aminoglycosides used. The percentage of gentamicin-resistant gram-negative strains declined to 7.4 percent (p less than 0.0001), whereas the percentage of amikacin-resistant strains did not change significantly. Quarterly trend analysis of aminoglycoside-resistant strains also demonstrated a significant decrease in gentamicin resistance (p less than 0.005) and an overall steady state of amikacin resistance. It is concluded that the exclusive use of amikacin was not accompanied by a significant increase in amikacin resistance during a three-year period, and may even lead to a decrease in resistance to gentamicin and tobramycin among most gram-negative organisms.

  10. Chloramphenicol – A Potent Armament Against Multi-Drug Resistant (MDR) Gram Negative Bacilli?

    PubMed Central

    2016-01-01

    Introduction Multidrug-resistant gram-negative bacteria cause infections which are hard to treat and cause high morbidity and mortality. Due to limited therapeutic options there is a renewed interest upon older antimicrobials which had fallen into disuse as a result of toxic side effects. One such antibiotic is chloramphenicol which was sidelined due to reports linking its use with the development of aplastic anaemia. Aim A study was conducted to evaluate the susceptibility of chloramphenicol in light of the emerging problem of multi-drug resistant gram negative bacteria (MDR GNB). Materials and Methods A total of 483 MDR GNB of the 650 consecutive Gram Negative Bacteria isolated from various clinical samples of patients admitted at a tertiary care hospital in Jaipur between January-June 2014 were screened for chloramphenicol susceptibility by the disc diffusion method as per CLSI guidelines. Results The MDR GNB isolates were obtained from 217 (45%) urine, 163 (34%) from respiratory samples, 52(11%) from pus, 42 (9%) from blood and 9 (2%) from body fluids. A 68% of the MDR GNB isolates were found to be sensitive to chloramphenicol. Conclusion Clinicians should always check for the local susceptibility of Gram-negative bacteria to chloramphenicol. This antibiotic has a potential to play a role in the therapeutic management of infections due to MDR GNB pathogens. PMID:27042458

  11. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M; Nichols, Wright W; Malouin, François

    2016-02-01

    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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria

    PubMed Central

    2013-01-01

    Background The emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics. Results Initial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli. Conclusions Combining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy. PMID:24069959

  17. Effectiveness of oral antibiotics for definitive therapy of Gram-negative bloodstream infections.

    PubMed

    Kutob, Leila F; Justo, Julie Ann; Bookstaver, P Brandon; Kohn, Joseph; Albrecht, Helmut; Al-Hasan, Majdi N

    2016-11-01

    There is paucity of data evaluating intravenous-to-oral antibiotic switch options for Gram-negative bloodstream infections (BSIs). This retrospective cohort study examined the effectiveness of oral antibiotics for definitive treatment of Gram-negative BSI. Patients with Gram-negative BSI hospitalised for <14 days at Palmetto Health Hospitals in Columbia, SC, from 1 January 2010 through 31 December 2013 and discharged on oral antibiotics were included in this study. The cohort was stratified into three groups based on bioavailability of oral antibiotics prescribed (high, ≥95%; moderate, 75-94%; and low, <75%). Kaplan-Meier analysis and multivariate Cox proportional hazards regression were used to examine treatment failure. Among the 362 patients, high, moderate and low bioavailability oral antibiotics were prescribed to 106, 179 and 77 patients, respectively, for definitive therapy of Gram-negative BSI. Mean patient age was 63 years, 217 (59.9%) were women and 254 (70.2%) had a urinary source of infection. Treatment failure rates were 2%, 12% and 14% in patients receiving oral antibiotics with high, moderate and low bioavailability, respectively (P = 0.02). Risk of treatment failure in the multivariate Cox model was higher in patients receiving antibiotics with moderate [adjusted hazard ratio (aHR) = 5.9, 95% CI 1.6-38.5; P = 0.005] and low bioavailability (aHR = 7.7, 95% CI 1.9-51.5; P = 0.003) compared with those receiving oral antimicrobial agents with high bioavailability. These data demonstrate the effectiveness of oral antibiotics with high bioavailability for definitive therapy of Gram-negative BSI. Risk of treatment failure increases as bioavailability of the oral regimen declines.

  18. Rapid testing using the Verigene Gram-negative blood culture nucleic acid test in combination with antimicrobial stewardship intervention against Gram-negative bacteremia.

    PubMed

    Bork, Jacqueline T; Leekha, Surbhi; Heil, Emily L; Zhao, LiCheng; Badamas, Rilwan; Johnson, J Kristie

    2015-03-01

    Rapid identification of microorganisms and antimicrobial resistance is paramount for targeted treatment in serious bloodstream infections (BSI). The Verigene Gram-negative blood culture nucleic acid test (BC-GN) is a multiplex, automated molecular diagnostic test for identification of eight Gram-negative (GN) organisms and resistance markers from blood culture with a turnaround time of approximately 2 h. Clinical isolates from adult patients at the University Maryland Medical Center with GN bacteremia from 1 January 2012 to 30 June 2012 were included in this study. Blood culture bottles were spiked with clinical isolates, allowed to incubate, and processed by BC-GN. A diagnostic evaluation was performed. In addition, a theoretical evaluation of time to effective and optimal antibiotic was performed, comparing actual antibiotic administration times from chart review ("control") to theoretical administration times based on BC-GN reporting and antimicrobial stewardship team (AST) review ("intervention"). For organisms detected by the assay, BC-GN correctly identified 95.6% (131/137), with a sensitivity of 97.1% (95% confidence interval [CI], 90.7 to 98.4%) and a specificity of 99.5% (95% CI, 98.8 to 99.8%). CTX-M and OXA resistance determinants were both detected. Allowing 12 h from Gram stain for antibiotic implementation, the intervention group had a significantly shorter duration to both effective (3.3 versus 7.0 h; P < 0.01) and optimal (23.5 versus 41.8 h; P < 0.01) antibiotic therapy. BC-GN with AST intervention can potentially decrease time to both effective and optimal antibiotic therapy in GN BSI.

  19. Rapid Testing Using the Verigene Gram-Negative Blood Culture Nucleic Acid Test in Combination with Antimicrobial Stewardship Intervention against Gram-Negative Bacteremia

    PubMed Central

    Leekha, Surbhi; Heil, Emily L.; Zhao, LiCheng; Badamas, Rilwan; Johnson, J. Kristie

    2014-01-01

    Rapid identification of microorganisms and antimicrobial resistance is paramount for targeted treatment in serious bloodstream infections (BSI). The Verigene Gram-negative blood culture nucleic acid test (BC-GN) is a multiplex, automated molecular diagnostic test for identification of eight Gram-negative (GN) organisms and resistance markers from blood culture with a turnaround time of approximately 2 h. Clinical isolates from adult patients at the University Maryland Medical Center with GN bacteremia from 1 January 2012 to 30 June 2012 were included in this study. Blood culture bottles were spiked with clinical isolates, allowed to incubate, and processed by BC-GN. A diagnostic evaluation was performed. In addition, a theoretical evaluation of time to effective and optimal antibiotic was performed, comparing actual antibiotic administration times from chart review (“control”) to theoretical administration times based on BC-GN reporting and antimicrobial stewardship team (AST) review (“intervention”). For organisms detected by the assay, BC-GN correctly identified 95.6% (131/137), with a sensitivity of 97.1% (95% confidence interval [CI], 90.7 to 98.4%) and a specificity of 99.5% (95% CI, 98.8 to 99.8%). CTX-M and OXA resistance determinants were both detected. Allowing 12 h from Gram stain for antibiotic implementation, the intervention group had a significantly shorter duration to both effective (3.3 versus 7.0 h; P < 0.01) and optimal (23.5 versus 41.8 h; P < 0.01) antibiotic therapy. BC-GN with AST intervention can potentially decrease time to both effective and optimal antibiotic therapy in GN BSI. PMID:25547353

  20. Minimum inhibitory concentrations of herbal essential oils and monolaurin for gram-positive and gram-negative bacteria.

    PubMed

    Preuss, Harry G; Echard, Bobby; Enig, Mary; Brook, Itzhak; Elliott, Thomas B

    2005-04-01

    New, safe antimicrobial agents are needed to prevent and overcome severe bacterial, viral, and fungal infections. Based on our previous experience and that of others, we postulated that herbal essential oils, such as those of origanum, and monolaurin offer such possibilities. We examined in vitro the cidal and/or static effects of oil of origanum, several other essential oils, and monolaurin on Staphylococcus aureus, Bacillus anthracis Sterne, Escherichia coli, Klebsiella pneumoniae, Helicobacter pylori, and Mycobacterium terrae. Origanum proved cidal to all tested organisms with the exception of B. anthracis Sterne in which it was static. Monolaurin was cidal to S. aureus and M. terrae but not to E. coli and K. pneumoniae. Unlike the other two gram-negative organisms, H. pylori were extremely sensitive to monolaurin. Similar to origanum, monolaurin was static to B. anthracis Sterne. Because of their longstanding safety record, origanum and/or monolaurin, alone or combined with antibiotics, might prove useful in the prevention and treatment of severe bacterial infections, especially those that are difficult to treat and/or are antibiotic resistant.

  1. Prediction of Fluoroquinolone Resistance in Gram-Negative Bacteria Causing Bloodstream Infections.

    PubMed

    Dan, Seejil; Shah, Ansal; Justo, Julie Ann; Bookstaver, P Brandon; Kohn, Joseph; Albrecht, Helmut; Al-Hasan, Majdi N

    2016-04-01

    Increasing rates of fluoroquinolone resistance (FQ-R) have limited empirical treatment options for Gram-negative infections, particularly in patients with severe beta-lactam allergy. This case-control study aims to develop a clinical risk score to predict the probability of FQ-R in Gram-negative bloodstream isolates. Adult patients with Gram-negative bloodstream infections (BSI) hospitalized at Palmetto Health System in Columbia, South Carolina, from 2010 to 2013 were identified. Multivariate logistic regression was used to identify independent risk factors for FQ-R. Point allocation in the fluoroquinolone resistance score (FQRS) was based on regression coefficients. Model discrimination was assessed by the area under receiver operating characteristic curve (AUC). Among 824 patients with Gram-negative BSI, 143 (17%) had BSI due to fluoroquinolone-nonsusceptible Gram-negative bacilli. Independent risk factors for FQ-R and point allocation in FQRS included male sex (adjusted odds ratio [aOR], 1.97; 95% confidence intervals [CI], 1.36 to 2.98; 1 point), diabetes mellitus (aOR, 1.54; 95% CI, 1.03 to 2.28; 1 point), residence at a skilled nursing facility (aOR, 2.28; 95% CI, 1.42 to 3.63; 2 points), outpatient procedure within 30 days (aOR, 3.68; 95% CI, 1.96 to 6.78; 3 points), prior fluoroquinolone use within 90 days (aOR, 7.87; 95% CI, 4.53 to 13.74; 5 points), or prior fluoroquinolone use within 91 to 180 days of BSI (aOR, 2.77; 95% CI, 1.17 to 6.16; 3 points). The AUC for both final logistic regression and FQRS models was 0.73. Patients with an FQRS of 0, 3, 5, or 8 had predicted probabilities of FQ-R of 6%, 22%, 39%, or 69%, respectively. The estimation of patient-specific risk of antimicrobial resistance using FQRS may improve empirical antimicrobial therapy and fluoroquinolone utilization in Gram-negative BSI.

  2. Prediction of Fluoroquinolone Resistance in Gram-Negative Bacteria Causing Bloodstream Infections

    PubMed Central

    Dan, Seejil; Shah, Ansal; Justo, Julie Ann; Bookstaver, P. Brandon; Kohn, Joseph; Albrecht, Helmut

    2016-01-01

    Increasing rates of fluoroquinolone resistance (FQ-R) have limited empirical treatment options for Gram-negative infections, particularly in patients with severe beta-lactam allergy. This case-control study aims to develop a clinical risk score to predict the probability of FQ-R in Gram-negative bloodstream isolates. Adult patients with Gram-negative bloodstream infections (BSI) hospitalized at Palmetto Health System in Columbia, South Carolina, from 2010 to 2013 were identified. Multivariate logistic regression was used to identify independent risk factors for FQ-R. Point allocation in the fluoroquinolone resistance score (FQRS) was based on regression coefficients. Model discrimination was assessed by the area under receiver operating characteristic curve (AUC). Among 824 patients with Gram-negative BSI, 143 (17%) had BSI due to fluoroquinolone-nonsusceptible Gram-negative bacilli. Independent risk factors for FQ-R and point allocation in FQRS included male sex (adjusted odds ratio [aOR], 1.97; 95% confidence intervals [CI], 1.36 to 2.98; 1 point), diabetes mellitus (aOR, 1.54; 95% CI, 1.03 to 2.28; 1 point), residence at a skilled nursing facility (aOR, 2.28; 95% CI, 1.42 to 3.63; 2 points), outpatient procedure within 30 days (aOR, 3.68; 95% CI, 1.96 to 6.78; 3 points), prior fluoroquinolone use within 90 days (aOR, 7.87; 95% CI, 4.53 to 13.74; 5 points), or prior fluoroquinolone use within 91 to 180 days of BSI (aOR, 2.77; 95% CI, 1.17 to 6.16; 3 points). The AUC for both final logistic regression and FQRS models was 0.73. Patients with an FQRS of 0, 3, 5, or 8 had predicted probabilities of FQ-R of 6%, 22%, 39%, or 69%, respectively. The estimation of patient-specific risk of antimicrobial resistance using FQRS may improve empirical antimicrobial therapy and fluoroquinolone utilization in Gram-negative BSI. PMID:26833166

  3. TLR4-mediated podosome loss discriminates gram-negative from gram-positive bacteria in their capacity to induce dendritic cell migration and maturation.

    PubMed

    van Helden, Suzanne F G; van den Dries, Koen; Oud, Machteld M; Raymakers, Reinier A P; Netea, Mihai G; van Leeuwen, Frank N; Figdor, Carl G

    2010-02-01

    Chronic infections are caused by microorganisms that display effective immune evasion mechanisms. Dendritic cell (DC)-dependent T cell-mediated adaptive immunity is one of the mechanisms that have evolved to prevent the occurrence of chronic bacterial infections. In turn, bacterial pathogens have developed strategies to evade immune recognition. In this study, we show that gram-negative and gram-positive bacteria differ in their ability to activate DCs and that gram-negative bacteria are far more effective inducers of DC maturation. Moreover, we observed that only gram-negative bacteria can induce loss of adhesive podosome structures in DCs, a response necessary for the induction of effective DC migration. We demonstrate that the ability of gram-negative bacteria to trigger podosome turnover and induce DC migration reflects their capacity to selectively activate TLR4. Examining mice defective in TLR4 signaling, we show that this DC maturation and migration are mainly Toll/IL-1 receptor domain-containing adaptor-inducing IFNbeta-dependent. Furthermore, we show that these processes depend on the production of PGs by these DCs, suggesting a direct link between TLR4-mediated signaling and arachidonic metabolism. These findings demonstrate that gram-positive and gram-negative bacteria profoundly differ in their capacity to activate DCs. We propose that this inability of gram-positive bacteria to induce DC maturation and migration is part of the armamentarium necessary for avoiding the induction of an effective cellular immune response and may explain the frequent involvement of these pathogens in chronic infections.

  4. Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in gram-positive and gram-negative bacteria.

    PubMed

    Hankins, Jessica V; Madsen, James A; Giles, David K; Brodbelt, Jennifer S; Trent, M Stephen

    2012-05-29

    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.

  5. Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses.

    PubMed

    Coton, Monika; Delbés-Paus, Céline; Irlinger, Françoise; Desmasures, Nathalie; Le Fleche, Anne; Stahl, Valérie; Montel, Marie-Christine; Coton, Emmanuel

    2012-02-01

    The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.

  6. An O2-sensing stressosome from a Gram-negative bacterium.

    PubMed

    Jia, Xin; Wang, Jian-Bo; Rivera, Shannon; Duong, Duc; Weinert, Emily E

    2016-08-04

    Bacteria have evolved numerous pathways to sense and respond to changing environmental conditions, including, within Gram-positive bacteria, the stressosome complex that regulates transcription of general stress response genes. However, the signalling molecules recognized by Gram-positive stressosomes have yet to be identified, hindering our understanding of the signal transduction mechanism within the complex. Furthermore, an analogous pathway has yet to be described in Gram-negative bacteria. Here we characterize a putative stressosome from the Gram-negative bacterium Vibrio brasiliensis. The sensor protein RsbR binds haem and exhibits ligand-dependent control of the stressosome complex activity. Oxygen binding to the haem decreases activity, while ferrous RsbR results in increased activity, suggesting that the V. brasiliensis stressosome may be activated when the bacterium enters anaerobic growth conditions. The findings provide a model system for investigating ligand-dependent signalling within stressosome complexes, as well as insights into potential pathways controlled by oxygen-dependent signalling within Vibrio species.

  7. An O2-sensing stressosome from a Gram-negative bacterium

    PubMed Central

    Jia, Xin; Wang, Jian-bo; Rivera, Shannon; Duong, Duc; Weinert, Emily E.

    2016-01-01

    Bacteria have evolved numerous pathways to sense and respond to changing environmental conditions, including, within Gram-positive bacteria, the stressosome complex that regulates transcription of general stress response genes. However, the signalling molecules recognized by Gram-positive stressosomes have yet to be identified, hindering our understanding of the signal transduction mechanism within the complex. Furthermore, an analogous pathway has yet to be described in Gram-negative bacteria. Here we characterize a putative stressosome from the Gram-negative bacterium Vibrio brasiliensis. The sensor protein RsbR binds haem and exhibits ligand-dependent control of the stressosome complex activity. Oxygen binding to the haem decreases activity, while ferrous RsbR results in increased activity, suggesting that the V. brasiliensis stressosome may be activated when the bacterium enters anaerobic growth conditions. The findings provide a model system for investigating ligand-dependent signalling within stressosome complexes, as well as insights into potential pathways controlled by oxygen-dependent signalling within Vibrio species. PMID:27488264

  8. Rationalizing the permeation of polar antibiotics into Gram-negative bacteria.

    PubMed

    Scorciapino, Mariano Andrea; Acosta-Gutierrez, Silvia; Benkerrou, Dehbia; D'Agostino, Tommaso; Malloci, Giuliano; Samanta, Susruta; Bodrenko, Igor; Ceccarelli, Matteo

    2017-03-22

    The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI

  9. Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

    NASA Astrophysics Data System (ADS)

    Scorciapino, Mariano Andrea; Acosta-Gutierrez, Silvia; Benkerrou, Dehbia; D’Agostino, Tommaso; Malloci, Giuliano; Samanta, Susruta; Bodrenko, Igor; Ceccarelli, Matteo

    2017-03-01

    The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI

  10. Susceptibility of Gram-negative bacteria to isepamicin: a systematic review.

    PubMed

    Falagas, Matthew E; Karageorgopoulos, Drosos E; Georgantzi, Georgia G; Sun, Chunguang; Wang, Rui; Rafailidis, Petros I

    2012-02-01

    We sought to review the potential role of isepamicin against infections with contemporary Gram-negative bacteria. We searched PubMed and Scopus databases to identify relevant microbiological and clinical studies published between 2000 and 2010, and we retrieved 11 and three studies, respectively. A total of 4901 isolates were examined in the in vitro studies. Isepamicin had higher in vitro activity compared with amikacin in four studies, was as active as amikacin in six studies and in the remaining study both were inactive. Regarding specifically the studies that included multidrug-resistant bacteria, isepamicin appeared superior to amikacin in two studies, as active as amikacin in one study and both did not exhibit activity in one study. In the clinical studies, isepamicin was as active as amikacin for the treatment of 55 children with urinary tract infections. In conclusion, isepamicin might be active in vitro against Gram-negative bacteria with resistance to amikacin and other aminoglycosides.

  11. Carbapenemase-producing Gram-negative bacteria: current epidemics, antimicrobial susceptibility and treatment options.

    PubMed

    Jean, Shio-Shin; Lee, Wen-Sen; Lam, Carlos; Hsu, Chin-Wang; Chen, Ray-Jade; Hsueh, Po-Ren

    2015-01-01

    Carbapenemases, with versatile hydrolytic capacity against β-lactams, are now an important cause of resistance of Gram-negative bacteria. The genes encoding for the acquired carbapenemases are associated with a high potential for dissemination. In addition, infections due to Gram-negative bacteria with acquired carbapenemase production would lead to high clinical mortality rates. Of the acquired carbapenemases, Klebsiella pneumoniae carbapenemase (Ambler class A), Verona integron-encoded metallo-β-lactamase (Ambler class B), New Delhi metallo-β-lactamase (Ambler class B) and many OXA enzymes (OXA-23-like, OXA-24-like, OXA-48-like, OXA-58-like, class D) are considered to be responsible for the worldwide resistance epidemics. As compared with monotherapy with colistin or tigecycline, combination therapy has been shown to effectively lower case-fatality rates. However, development of new antibiotics is crucial in the present pandrug-resistant era.

  12. Trojan Horse Antibiotics—A Novel Way to Circumvent Gram-Negative Bacterial Resistance?

    PubMed Central

    Tillotson, Glenn S.

    2016-01-01

    Antibiotic resistance has been emerged as a major global health problem. In particular, gram-negative species pose a significant clinical challenge as bacteria develop or acquire more resistance mechanisms. Often, these bacteria possess multiple resistance mechanisms, thus nullifying most of the major classes of drugs. Novel approaches to this issue are urgently required. However, the challenges of developing new agents are immense. Introducing novel agents is fraught with hurdles, thus adapting known antibiotic classes by altering their chemical structure could be a way forward. A chemical addition to existing antibiotics known as a siderophore could be a solution to the gram-negative resistance issue. Siderophore molecules rely on the bacterial innate need for iron ions and thus can utilize a Trojan Horse approach to gain access to the bacterial cell. The current approaches to using this potential method are reviewed. PMID:27773991

  13. Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria

    PubMed Central

    Abergel, Chantal; Monchois, Vincent; Byrne, Deborah; Chenivesse, Sabine; Lembo, Frédérique; Lazzaroni, Jean-Claude; Claverie, Jean-Michel

    2007-01-01

    Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates. PMID:17405861

  14. Antibiotic-non-antibiotic combinations for combating extremely drug-resistant Gram-negative 'superbugs'.

    PubMed

    Schneider, Elena K; Reyes-Ortega, Felisa; Velkov, Tony; Li, Jian

    2017-02-28

    The emergence of antimicrobial resistance of Gram-negative pathogens has become a worldwide crisis. The status quo for combating resistance is to employ synergistic combinations of antibiotics. Faced with this fast-approaching post-antibiotic era, it is critical that we devise strategies to prolong and maximize the clinical efficacy of existing antibiotics. Unfortunately, reports of extremely drug-resistant (XDR) Gram-negative pathogens have become more common. Combining antibiotics such as polymyxin B or the broad-spectrum tetracycline and minocycline with various FDA-approved non-antibiotic drugs have emerged as a novel combination strategy against otherwise untreatable XDR pathogens. This review surveys the available literature on the potential benefits of employing antibiotic-non-antibiotic drug combination therapy. The apex of this review highlights the clinical utility of this novel therapeutic strategy for combating infections caused by 'superbugs'.

  15. New plate medium for screening and presumptive identification of gram-negative urinary tract pathogens.

    PubMed Central

    Thaller, M C; Berlutti, F; Dainelli, B; Pezzi, R

    1988-01-01

    A new selective, differential plating medium to screen the common gram-negative urinary tract pathogens is described. The medium combines adonitol fermentation, phenylalanine deaminase, and beta-glucuronidase tests and allows the indole and cytochrome oxidase tests to be performed directly from the plates. High-level agreement with individual conventional tests was recorded in comparative studies with 504 cultures of gram-negative rods. There was 100% agreement, except for the Providencia spp. indole spot test (61.6% agreement). Adonitol fermentation by Providencia species could not be determined. Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa were identified with a high efficiency (100, 85.7, 83.5, and 100% agreement, respectively) without further testing. There was 96% overall agreement for the 267 infected urine samples tested. PMID:3366875

  16. Epidemiology of gentamicin-resistant, gram-negative bacillary colonization in a spinal cord injury unit.

    PubMed Central

    Shlaes, D M; Currie, C A; Rotter, G; Eanes, M; Floyd, R

    1983-01-01

    A prospective epidemiological survey of a spinal cord injury unit for gentamicin-resistant, gram-negative bacilli was undertaken. The initial survey of the unit suggested a low level of cross-infection involving Pseudomonas aeruginosa and Providencia stuartii. However, a longitudinal study of new admissions revealed that only 13 of 52 nosocomial acquisitions could be considered to be due to cross colonization. Comparison of data on antibiotic use did not suggest selective pressure for resistant endogenous flora. Nosocomial acquisition was directly related to the length of the hospital stay. Antibiotic susceptibility testing of gentamicin-resistant, gram-negative bacilli showed only minor differences between nosocomial isolates and those present during the initial survey. Of the usual antimicrobial agents, amikacin, carbenicillin, and cefoxitin were the most active against all organisms, with the exception of Serratia spp. Of the new beta-lactams, ceftazidime and imipemide (N-formimidoyl thienamycin) were most active. PMID:6619279

  17. Quorum-Sensing Signal-Response Systems in Gram-Negative Bacteria

    PubMed Central

    Papenfort, Kai; Bassler, Bonnie

    2016-01-01

    Abstract / Preface Bacteria use quorum sensing to orchestrate gene expression programmes that underlie collective behaviours. Quorum sensing relies on the production, release, detection and group-level response to extracellular signalling molecules, which are called autoinducers. Recent work has discovered new autoinducers in Gram-negative bacteria, shown how these molecules are recognized by cognate receptors, revealed new regulatory components that are embedded in canonical signalling circuits and identified novel regulatory network designs. In this Review we examine how, together, these features of quorum sensing signal–response systems combine to control collective behaviours in Gram-negative bacteria and we discuss the implications for host–microbial associations and antibacterial therapy. PMID:27510864

  18. Construction of p16Slux, a novel vector for improved bioluminescent labeling of gram-negative bacteria.

    PubMed

    Riedel, Christian U; Casey, Pat G; Mulcahy, Heidi; O'Gara, Fergal; Gahan, Cormac G M; Hill, Colin

    2007-11-01

    A novel vector has been constructed for the constitutive luminescent tagging of gram-negative bacteria by site-specific integration into the 16S locus of the bacterial chromosome. A number of gram-negative pathogens were successfully tagged using this vector, and the system was validated during murine infections of living animals.

  19. Gram-negative infections in pediatric and neonatal intensive care units of Latin America.

    PubMed

    Berezin, Eitan N; Solórzano, Fortino

    2014-08-13

    In order to review the epidemiology of Gram-negative infections in the pediatric and neonatal intensive care units (PICUs and NICUs) of Latin America a systematic search of PubMed and targeted search of SciELO was performed to identify relevant articles published since 2005. Independent cohort data indicated that overall infection rates were higher in Latin American PICUs and NICUs versus developed countries (range, 5%-37% vs 6%-15%, respectively). Approximately one third of Latin American patients with an acquired PICU or NICU infection died, and crude mortality was higher among extremely low-birth-weight infants and those with an infection caused by Gram-negative bacteria. In studies reporting > 100 isolates, the frequency of Gram-negative pathogens varied from 31% (Colombia) to 63% (Mexico), with Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli the predominant pathogens in almost all countries, and Acinetobacter spp. and Serratia spp. isolated sporadically. The activity of quinolones and third-generation cephalosporins against P. aeruginosa, Acinetobacter spp., and Enterobacteria was seriously compromised, coincident with a high prevalence of circulating extended-spectrum β-lactamases. Furthermore, we identified two observational studies conducted in Chile and Brazil reporting infections by P. aeruginosa and Acinetobacter baumannii in PICUs, demonstrating resistance to carbapenems, and two outbreaks of carbapenem-resistant K. pneumoniae in Colombia and Brazil. The endemicity of multidrug-resistant Gram-negative infections in Latin American PICUs and NICUs is punctuated by intermittent clonal outbreaks. The problem may be alleviated by ensuring ICUs are less crowded, increasing staffing levels of better-trained health care personnel, and implementing antimicrobial stewardship and surveillance programs.

  20. Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

    DTIC Science & Technology

    2009-08-27

    aeruginosa, Salmonella ty- phimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies en- abled detection of...Antisera against Salmonella typhimurium (recognizing a Antibody Biomarkers for Plague Molecular & Cellular Proteomics 8.5 925 by on A ugust 27, 2009 w w...clinical review of 27 cases. Arch. Intern. Med. 152, 1253–1256 9. Nielsen, K., Smith, P., Yu, W. L., and Halbert, G. (2007) Salmonella enterica serotype

  1. 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%.

  2. 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.

  3. The emerging threat of multidrug-resistant Gram-negative bacteria in urology.

    PubMed

    Zowawi, Hosam M; Harris, Patrick N A; Roberts, Matthew J; Tambyah, Paul A; Schembri, Mark A; Pezzani, M Diletta; Williamson, Deborah A; Paterson, David L

    2015-10-01

    Antibiotic resistance in Gram-negative uropathogens is a major global concern. Worldwide, the prevalence of Enterobacteriaceae that produce extended-spectrum β-lactamase or carbapenemase enzymes continues to increase at alarming rates. Likewise, resistance to other antimicrobial agents including aminoglycosides, sulphonamides and fluoroquinolones is also escalating rapidly. Bacterial resistance has major implications for urological practice, particularly in relation to catheter-associated urinary tract infections (UTIs) and infectious complications following transrectal-ultrasonography-guided biopsy of the prostate or urological surgery. Although some new drugs with activity against Gram-negative bacteria with highly resistant phenotypes will become available in the near future, the existence of a single agent with activity against the great diversity of resistance is unlikely. Responding to the challenges of Gram-negative resistance will require a multifaceted approach including considered use of current antimicrobial agents, improved diagnostics (including the rapid detection of resistance) and surveillance, better adherence to basic measures of infection prevention, development of new antibiotics and research into non-antibiotic treatment and preventive strategies.

  4. Low antibiotic resistance among anaerobic Gram-negative bacteria in periodontitis 5 years following metronidazole therapy.

    PubMed

    Dahlen, G; Preus, H R

    2017-02-01

    The objective of this study was to assess antibiotic susceptibility among predominant Gram-negative anaerobic bacteria isolated from periodontitis patients who 5 years prior had been subject to mechanical therapy with or without adjunctive metronidazole. One pooled sample was taken from the 5 deepest sites of each of 161 patients that completed the 5 year follow-up after therapy. The samples were analyzed by culture. A total number of 85 anaerobic strains were isolated from the predominant subgingival flora of 65/161 patient samples, identified, and tested for antibiotic susceptibility by MIC determination. E-tests against metronidazole, penicillin, amoxicillin, amoxicillin + clavulanic acid and clindamycin were employed. The 73/85 strains were Gram-negative rods (21 Porphyromonas spp., 22 Prevotella/Bacteroides spp., 23 Fusobacterium/Filifactor spp., 3 Campylobacter spp. and 4 Tannerella forsythia). These were all isolated from the treated patients irrespective of therapy procedures (+/-metronidazole) 5 years prior. Three strains (Bifidobacterium spp., Propionibacterium propionicum, Parvimonas micra) showed MIC values for metronidazole over the European Committee on Antimicrobial Susceptibility Testing break point of >4 μg/mL. All Porphyromonas and Tannerella strains were highly susceptible. Metronidazole resistant Gram-negative strains were not found, while a few showed resistance against beta-lactam antibiotics. In this population of 161 patients who had been subject to mechanical periodontal therapy with or without adjunct metronidazole 5 years prior, no cultivable antibiotic resistant anaerobes were found in the predominant subgingival microbiota.

  5. New insight on antimicrobial therapy adjustment strategies for gram-negative bacterial infection

    PubMed Central

    Du, Wei; Chen, Hong; Xiao, Shuzhen; Tang, Wei; Shi, Guochao

    2017-01-01

    Abstract Gram-negative bacterial infections, especially multidrug-resistant (MDR) bacterial infection, are becoming a serious threat to public health. Although it is widely accepted that both appropriate initial empirical therapy and targeted therapy are important, but for patients needing therapy adjustment, few studies have explored whether adjustment strategy based on microbiologic susceptibility test (MST) brings better outcome compared with empirical adjustment. A total of 320 patients with gram-negative bacterial infection (airway, blood, or pleural effusion) were selected and a prospective cohort study was conducted. Baseline characteristics and outcomes (microbiologic, clinical, and economic) were documented during follow-up. MDR and nosocomial infections were common among subjects. Initial therapies consistent with MST could result in reduced in-hospital mortality, treatment failure rate, infection-related death, percentages of patients needing therapy adjustment, and daily hospitalization cost with increased successful treatment rate compared with inconsistent with MST, and microbiologic outcomes were also better with appropriate therapies. For patients needing therapy adjustment, relying on MST gained no significant benefit on mortality, clinical, or microbiologic outcomes compared with depending on clinical experience. But for patients with MDR infection, adjustment relying on MST gained more benefit than non-MDR infection. Appropriate initial therapy significantly improved the prognosis of patients with gram-negative bacterial infections, but improvement was not that obvious for patients needing therapy adjustment which was based on MST compared with clinical experience, and more beneficial effects of adjustment relying on MST were obtained for patients with MDR bacterial infection. PMID:28353572

  6. Weakening Effect of Cell Permeabilizers on Gram-Negative Bacteria Causing Biodeterioration

    PubMed Central

    Alakomi, H.-L.; Paananen, A.; Suihko, M.-L.; Helander, I. M.; Saarela, M.

    2006-01-01

    Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products. PMID:16820461

  7. Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration.

    PubMed

    Alakomi, H-L; Paananen, A; Suihko, M-L; Helander, I M; Saarela, M

    2006-07-01

    Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

  8. Molecular analysis of antimicrobial resistance in gram-negative bacteria isolated from fish farms in Egypt.

    PubMed

    Ishida, Yojiro; Ahmed, Ashraf M; Mahfouz, Nadia B; Kimura, Tomomi; El-Khodery, Sabry A; Moawad, Amgad A; Shimamoto, Tadashi

    2010-06-01

    As little is known about antimicrobial resistance genes in fish farms, this study was conducted to monitor the incidence and prevalence of a wide range of antimicrobial resistance genes in Gram-negative bacteria isolated from water samples taken from fish farms in the northern part of Egypt. Ninety-one out of two hundred seventy-four (33.2%) non-repetitive isolates of Gram-negative bacteria showed multidrug resistance phenotypes and harbored at least one antimicrobial resistance gene. PCR and DNA sequencing results showed that 72 (26.3%) isolates contain tetracycline resistance genes and 19 (6.9%) isolates were positive for class 1 integrons with 12 different gene cassettes. The beta-lactamase-encoding genes were identified in 14 (5.1%) isolates. The plasmid-mediated quinolone resistance genes, qnr and aac(6')-Ib-cr, were identified in 16 (5.8%) and 3 (1.1%) isolates, respectively. Finally, the florphenicol resistance gene, floR, was identified in four (1.5%) isolates. To the best of our knowledge, this is the first report for molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from fish farms in Africa.

  9. Gram-Negative Infections in Adult Intensive Care Units of Latin America and the Caribbean

    PubMed Central

    Luna, Carlos M.; Rodriguez-Noriega, Eduardo; Bavestrello, Luis; Guzmán-Blanco, Manuel

    2014-01-01

    This review summarizes recent epidemiology of Gram-negative infections in selected countries from Latin American and Caribbean adult intensive care units (ICUs). A systematic search of the biomedical literature (PubMed) was performed to identify articles published over the last decade. Where appropriate, data also were collected from the reference list of published articles, health departments of specific countries, and registries. Independent cohort data from all countries (Argentina, Brazil, Chile, Colombia, Cuba, Mexico, Trinidad and Tobago, and Venezuela) signified a high rate of ICU infections (prevalence: Argentina, 24%; Brazil, 57%). Gram-negative pathogens, predominantly Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli, accounted for >50% of ICU infections, which were often complicated by the presence of multidrug-resistant strains and clonal outbreaks. Empirical use of antimicrobial agents was identified as a strong risk factor for resistance development and excessive mortality. Infection control strategies utilizing hygiene measures and antimicrobial stewardship programs reduced the rate of device-associated infections. To mitigate the poor health outcomes associated with infections by multidrug-resistant Gram-negative bacteria, urgent focus must be placed on infection control strategies and local surveillance programs. PMID:25525515

  10. Genotypic Identification of AmpC β-Lactamases Production in Gram-Negative Bacilli Isolates

    PubMed Central

    Wassef, Mona; Behiry, Iman; Younan, Mariam; El Guindy, Nancy; Mostafa, Sally; Abada, Emad

    2014-01-01

    Background: AmpC type β-lactamases are commonly isolated from extended-spectrum Cephalosporin-resistant Gram-negative bacteria. Also, resistance appeared in bacterial species not naturally producing AmpC enzymes. Therefore, a standard test for the detection of the plasmid-mediated AmpC enzyme and new breakpoints for extended spectrum Cephalosporins are urgently necessary. Objectives: To detect plasmid and chromosomal mediated AmpC-β-lactamases in Gram negative bacteria in community and hospital acquired infections. Materials and Methods: 1073 Gram negative clinical isolates were identified by the conventional methods and were screened for AmpC production using Cefoxitin discs. Confirmatory phenotypic identifications were done for the Cefoxitin-resistant isolates using Boronic Acid for combined and double disc synergy tests, Cloxacillin based double disc synergy test, and induction tests. The genotypic identification of plasmid-mediated AmpC was done using multiplex PCR. ESBL production was also screened by discs of Ceftazidime and Cefotaxime with and without Clavulanic Acid (10 μg). Results: The AmpC-producing isolates among all identified Gram negative bacilli were 5.8% (62/1073) as detected by screening disc diffusion methods, where 72% were positive for AmpC by combined disc method (Cefotetan and Boronic Acid), 56.5% were positive by each of Boronic Acid and Cloxacillin double disc synergy tests, 35.5% were positive by the induction test, and 25.8% were plasmid-mediated AmpC β-lactamase producers by the multiplex PCR. Plasmid-mediated AmpC genes retrieved, belonged to the families (MOX, FOX, EBC and CIT). ESBL producers were found in 26 (41.9%) isolates, 15 (57%) of which also produced AmpC. Isolates caused hospital acquired infections were (53/62); of which (39/62) were AmpC producers. While only (8/62) of the isolates caused community-acquired infections, were AmpC producers, and (1.6%) (1/62) were non AmpC producer. Conclusions: The AmpC

  11. Survival and detection of coliforms, Enterobacteriaceae, and gram-negative bacteria in Greek yogurt.

    PubMed

    Hervert, C J; Martin, N H; Boor, K J; Wiedmann, M

    2017-02-01

    Despite the widespread use of coliforms as indicator bacteria, increasing evidence suggests that the Enterobacteriaceae (EB) and total gram-negative groups more accurately reflect the hygienic status of high-temperature, short-time pasteurized milk and processing environments. If introduced into milk as postpasteurization contamination, these bacteria may grow to high levels and produce a wide range of sensory-related defects. However, limited information is available on the use and survival of bacterial hygiene indicators in dairy products outside of pasteurized fluid milk and cheese. The goal of this study was to (1) provide information on the survival of a diverse set of bacterial hygiene indicators in the low pH environment of Greek yogurt, (2) compare traditional and alternative detection methods for their ability to detect bacterial hygiene indicators in Greek yogurt, and (3) offer insight into optimal hygiene indicator groups for use in low-pH fermented dairy products. To this end, we screened 64 bacterial isolates, representing 24 dairy-relevant genera, for survival and detection in Greek yogurt using 5 testing methods. Before testing, isolates were inoculated into plain, 0% fat Greek yogurt (pH 4.35 to 4.65), followed by a 12-h hold period at 4 ± 1°C. Yogurts were subsequently tested using Coliform Petrifilm (3M, St. Paul, MN) to detect coliforms; Enterobacteriaceae Petrifilm (3M), violet red bile glucose agar and the D-Count (bioMérieux, Marcy-l'Étoile, France) to detect EB; and crystal violet tetrazolium agar (CVTA) to detect total gram-negative bacteria. Overall, the non-EB gram-negative isolates showed significantly larger log reductions 12 h after inoculation into Greek yogurt (based on bacterial numbers recovered on CVTA) compared with the coliform and noncoliform EB isolates tested. The methods evaluated varied in their ability to detect different microbial hygiene indicators in Greek yogurt. Crystal violet tetrazolium agar detected the highest

  12. Hormone fatty acid modifications: gram negative bacteria and vertebrates demonstrate common structure and function.

    PubMed

    Tizzano, Marco; Sbarbati, Andrea

    2006-01-01

    Bacteria are known to regulate diverse physiological processes through a mechanism called quorum sensing (QS). Prokaryotes communicate by extracellular signalling compounds, i.e. autoinducers (acyl homoserine lactone, AHL of Gram negative bacteria) or pheromones (post-translationally modified peptides of Gram positive bacteria), which activate genetic pathways when they reach a sufficient concentration (QS). A large number of Gram-negative quorum-sensing systems studied so far utilize N-acyl homoserine lactones as signal molecules. In vertebrates small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. GH release is stimulated by hypothalamic GH-releasing hormone (GHRH) and ghrelin (endogenous ligand of the GHS-receptor, GHS-R). Ghrelin is a 28-amino acid peptide, in which the serine-3 (Ser3) is n-octanoylated, and this modification is essential for ghrelin's activity. Ghrelin is the first known case of a peptide hormone modified by a fatty acid. The major active form of ghrelin is a 28-amino acid peptide with octanoylated Ser3; one of the more represented bacterial autoinducers is the N-Octanoyl-DL-homoserine lactone (C8-HL) molecule. The authors hypothesize that Gram-negative bacteria and vertebrates have a functional similarity in the search of food and an important structural homology of AHL and ghrelin for the highly conserved Serine-acylated motive in both molecules. Our suggestions could help one to understand the convergent origin and the biologic meaning of the Serine-acylated group in these organisms, a biologic meaning very important due to the high conservation in two kingdoms which are so different.

  13. 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

  14. Preparation of Membrane Models of Gram-Negative Bacteria and Their Interaction with Antimicrobial Peptides Studied by CD and NMR.

    PubMed

    Hicks, Rickey

    2017-01-01

    The antibiotic activity of antimicrobial peptides is generally derived via some type of disruption of the cell membrane(s). The most common models used to mimic the properties of bacterial membranes consist of mixtures of various zwitterionic and anionic phospholipids. This approach works reasonably well for Gram-positive bacteria. However, since the membranes of Gram-negative bacteria contain lipopolysaccharides, as well as zwitterionic and anionic phospholipids, a more complex model is required to simulate the outer membrane of Gram-negative bacteria. Herein we present a protocol for the preparation of models of the outer membranes of the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. This protocol can be used to prepare models of other Gram-negative bacteria provided the strain-specific lipopolysaccharides are available.

  15. Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay.

    PubMed

    Ledeboer, Nathan A; Lopansri, Bert K; Dhiman, Neelam; Cavagnolo, Robert; Carroll, Karen C; Granato, Paul; Thomson, Richard; Butler-Wu, Susan M; Berger, Heather; Samuel, Linoj; Pancholi, Preeti; Swyers, Lettie; Hansen, Glen T; Tran, Nam K; Polage, Christopher R; Thomson, Kenneth S; Hanson, Nancy D; Winegar, Richard; Buchan, Blake W

    2015-08-01

    Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; blaCTX-M, 98.9%; blaKPC, 100%; blaNDM, 96.2%; blaOXA, 94.3%; blaVIM, 100%; and blaIMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths

  16. Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay

    PubMed Central

    Ledeboer, Nathan A.; Lopansri, Bert K.; Dhiman, Neelam; Cavagnolo, Robert; Carroll, Karen C.; Granato, Paul; Thomson, Richard; Butler-Wu, Susan M.; Berger, Heather; Samuel, Linoj; Pancholi, Preeti; Swyers, Lettie; Hansen, Glen T.; Tran, Nam K.; Polage, Christopher R.; Thomson, Kenneth S.; Hanson, Nancy D.; Winegar, Richard

    2015-01-01

    Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; blaCTX-M, 98.9%; blaKPC, 100%; blaNDM, 96.2%; blaOXA, 94.3%; blaVIM, 100%; and blaIMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths

  17. 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.

  18. Di-N-Methylation of Anti-Gram-Positive Aminoglycoside-Derived Membrane Disruptors Improves Antimicrobial Potency and Broadens Spectrum to Gram-Negative Bacteria.

    PubMed

    Benhamou, Raphael I; Shaul, Pazit; Herzog, Ido M; Fridman, Micha

    2015-11-09

    The effect of di-N-methylation of bacterial membrane disruptors derived from aminoglycosides (AGs) on antimicrobial activity is reported. Di-N-methylation of cationic amphiphiles derived from several diversely structured AGs resulted in a significant increase in hydrophobicity compared to the parent compounds that improved their interactions with membrane lipids. The modification led to an enhancement in antibacterial activity and a broader antimicrobial spectrum. While the parent compounds were either modestly active or inactive against Gram-negative pathogens, the corresponding di-N-methylated compounds were potent against the tested Gram-negative as well as Gram-positive bacterial strains. The reported modification offers a robust strategy for the development of broad-spectrum membrane-disrupting antibiotics for topical use.

  19. In Vitro Antibacterial Activity of Several Plant Extracts and Oils against Some Gram-Negative Bacteria

    PubMed Central

    Al-Mariri, Ayman; Safi, Mazen

    2014-01-01

    Background: Medicinal plants are considered new resources for producing agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the antibacterial activity of 28 plant extracts and oils against four Gram-negative bacterial species. Methods: Experimental, in vitro, evaluation of the activities of 28 plant extracts and oils as well as some antibiotics against E. coli O157:H7, Yersinia enterocolitica O9, Proteus spp., and Klebsiella pneumoniae was performed. The activity against 15 isolates of each bacterium was determined by disc diffusion method at a concentration of 5%. Microdilution susceptibility assay was used in order to determine the minimal inhibitory concentrations (MICs) of the plant extracts, oils, and antibiotics. Results: Among the evaluated herbs, only Origanum syriacum L., Thymus syriacus Boiss., Syzygium aromaticum L., Juniperus foetidissima Wild, Allium sativum L., Myristica fragrans Houtt, and Cinnamomum zeylanicum L. essential oils and Laurus nobilis L. plant extract showed anti-bacterial activity. The MIC50 values of these products against the Gram-negative organisms varied from 1.5 (Proteus spp. and K. pneumoniae( and 6.25 µl/ml (Yersinia enterocolitica O9 ) to 12.5 µl/ml (E. coli O:157). Conclusion: Among the studied essential oils, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. essential oils were the most effective. Moreover, Cephalosporin and Ciprofloxacin were the most effective antibiotics against almost all the studied bacteria. Therefore, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. could act as bactericidal agents against Gram-negative bacteria. PMID:24453392

  20. Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

    PubMed Central

    Henderson, Jeremy C.; O'Brien, John P.; Brodbelt, Jennifer S.; Trent, M. Stephen

    2013-01-01

    Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate

  1. Emergence of multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh.

    PubMed

    Islam, M A; Talukdar, P K; Hoque, A; Huq, M; Nabi, A; Ahmed, D; Talukder, K A; Pietroni, M A C; Hays, J P; Cravioto, A; Endtz, H P

    2012-10-01

    The main objective of this study was to investigate the prevalence of bla (NDM-1) in Gram-negative bacteria in Bangladesh. In October 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratories, 1,816 consecutive clinical samples were tested for imipenem-resistant Gram-negative organisms. Imipenem-resistant isolates were tested for the bla (NDM-1) gene. Among 403 isolates, 14 (3.5 %) were positive for bla (NDM-1), and the predominant species were Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. All bla (NDM-1)-positive isolates were resistant to multiple antibiotics. Among β-lactamase genes, bla (CTX-M-1-group) was detected in ten isolates (eight bla (CTX-M-15)), bla (OXA-1-group) in six, bla (TEM) in nine, bla (SHV) in seven, and bla (VIM) and bla (CMY) in two isolates each. The 16S rRNA methylase gene, armA, was detected in five K. pneumoniae isolates and in one E. coli isolate. rmtB and rmtC were detected in a Citrobacter freundii and two K. pneumoniae isolates, respectively. qnr genes were detected in two K. pneumoniae isolates (one qnrB and one qnrS) and in an E. coli isolate (qnrA). Transferable plasmids (60-100 MDa) carrying bla (NDM-1) were detected in 7 of the 11 plasmid-containing isolates. Pulsed-field gel electrophoresis (PFGE) analysis grouped K. pneumoniae isolates into three clusters, while E. coli isolates differed significantly from each other. This study reports that approximately 3.5 % of Gram-negative clinical isolates in Bangladesh are NDM-1-producing.

  2. Surveillance and correlation of antibiotic prescription and resistance of Gram-negative bacteria in Singaporean hospitals.

    PubMed

    Hsu, Li-Yang; Tan, Thean-Yen; Tam, Vincent H; Kwa, Andrea; Fisher, Dale Andrew; Koh, Tse-Hsien

    2010-03-01

    A surveillance study was performed in four Singapore public hospitals from 2006 to 2008 to determine the correlation between antibiotic prescription and Gram-negative bacterial antimicrobial resistance. Targeted organisms included ceftriaxone- and ciprofloxacin-resistant Escherichia coli and Klebsiella pneumoniae, as well as imipenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. Antibiotic prescription data were collated in the WHO anatomical therapeutic chemical (ATC)/defined daily dose (DDD) format, while antibiotic resistance was expressed as incidence density adjusted for total inpatient-days every quarter. Individual trends were determined by linear regression, while possible associations between antibiotic prescription and resistance were evaluated via cross-correlation analysis. Results over 3 years indicated significantly rising incidence densities of ceftriaxone- and ciprofloxacin-resistant E. coli and imipenem-resistant Acinetobacter spp. (blood isolates only). Antimicrobial-resistant Klebsiella pneumoniae rates declined. The prescription rates of piperacillin-tazobactam, ertapenem, meropenem, ciprofloxacin, and levofloxacin increased significantly, while imipenem and moxifloxacin prescription decreased. Cross-correlation analysis demonstrated possible associations between prescription of fluoroquinolones and ciprofloxacin-resistant E. coli (R(2) = 0.46), fluoroquinolones and ceftriaxone-resistant E. coli (R(2) = 0.47), and carbapenems and imipenem-resistant Acinetobacter spp. (R(2) = 0.48), all at zero time lag. Changes in meropenem prescription were associated with a similar trend in imipenem-resistant Acinetobacter blood isolates after a 3-month time lag. No correlation was found between cephalosporin use and resistance. In conclusion, our data demonstrated correlation between prescription of and Gram-negative bacterial resistance to several, but not all, key antimicrobial agents in Singapore hospitals. In areas where Gram-negative bacterial

  3. Role of Gram-Negative Bacteria and Their Endotoxins in Rat Death after Heat Stress,

    DTIC Science & Technology

    1981-02-26

    PERIOD COVERED S Role of Gram-Negative Bacteria and their Endotoxins in Rat Death after Heat Stress tle 6. PERFORMING ORG. REPORT NUMBER M 4/81 S7...AUTHOR(e) . CONTRACT OR GRANT NUMBER(&) D. A. DuBose, K. Basamania, L. Maglione , and J. Rowlands 9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM...Block 20, ,if diffrent from Rejlgir )ll’..;’ J.- : m NIA :’" EB I ] 18. SUPPLEMENTARY NOTES A N/A IS. KEY WORDS (Continue on reverse side if necessary

  4. Prostate abscess: MRSA spreading its influence into Gram-negative territory: case report and literature review.

    PubMed

    Deshpande, Aartee; Haleblian, George; Rapose, Alwyn

    2013-03-25

    Prostate abscess is a rare complication of an ascending urinary tract infection (UTI). Its incidence has reduced secondary to routine and early use of antibiotics for treatment of UTIs. Prostate abscess has been reported in patients with uncontrolled diabetes, prolonged indwelling urinary catheters, prostate biopsy or other instrumentation of lower urinary tract. Prostate abscess is most commonly associated with Gram-negative bacteria. Staphylococcus aureus is rarely implicated and has been reported in patients with underlying risk factors like long-term or uncontrolled diabetes, intravenous drug abuse or bacteraemia. We present a rare case of prostate abscess due to methicillin resistant S aureus without obvious risk factors.

  5. Adhesins involved in attachment to abiotic surfaces by Gram-negative bacteria

    PubMed Central

    Berne, Cécile; Ducret, Adrien; Hardy, Gail G; Brun, Yves V.

    2015-01-01

    During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending upon the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or non-specific adhesion under various environmental conditions. This chapter will review the recent advances in our understanding of the secretion, assembly and regulation of the bacterial adhesins during biofilm formation with a particular emphasis on the fimbrial, non-fimbrial and discrete polysaccharide adhesins in Gram-negative bacteria. PMID:26350310

  6. The Outer Membrane of Gram-negative Bacteria: Lipid A Isolation and Characterization

    PubMed Central

    Needham, Brittany D.; Brodbelt, Jennifer S.; Trent, M. Stephen

    2016-01-01

    Summary The isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) are important methodologies utilized to gain understanding of the Gram-negative cell envelope. Here, we describe protocols often employed by our laboratory for small- and large-scale isolation of lipid A from bacterial cells. Additionally, we describe various methodologies including isolation of radiolabeled lipid A, thin layer chromatography, and various mass spectrometry methods. Tandem mass spectrometry is an integral tool for the structural characterization of lipid A molecules, and both coventional collision induced dissociation (CID) and new ultraviolet photodissociation (UVPD) methods are described. PMID:23299739

  7. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

    PubMed Central

    Schwechheimer, Carmen; Kuehn, Meta J.

    2017-01-01

    Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles. PMID:26373371

  8. Complete Genome Sequence of Acidaminococcus intestini RYC-MR95, a Gram-Negative Bacterium from the Phylum Firmicutes

    PubMed Central

    D'Auria, Giuseppe; Galán, Juan-Carlos; Rodríguez-Alcayna, Manuel; Moya, Andrés; Baquero, Fernando; Latorre, Amparo

    2011-01-01

    Acidaminococcus intestini belongs to the family Acidaminococcaceae, order Selenomonadales, class Negativicutes, phylum Firmicutes. Negativicutes show the double-membrane system of Gram-negative bacteria, although their chromosomal backbone is closely related to that of Gram-positive bacteria of the phylum Firmicutes. The complete genome of a clinical A. intestini strain is here presented. PMID:22123762

  9. Potentiation of photoinactivation of Gram-positive and Gram-negative bacteria mediated by six phenothiazinium dyes by addition of azide ion.

    PubMed

    Kasimova, Kamola R; Sadasivam, Magesh; Landi, Giacomo; Sarna, Tadeusz; Hamblin, Michael R

    2014-11-01

    Antimicrobial photodynamic inactivation (APDI) using phenothiazinium dyes is mediated by reactive oxygen species consisting of a combination of singlet oxygen (quenched by azide), hydroxyl radicals and other reactive oxygen species. We recently showed that addition of sodium azide paradoxically potentiated APDI of Gram-positive and Gram-negative bacteria using methylene blue as the photosensitizer, and this was due to electron transfer to the dye triplet state from azide anion, producing azidyl radical. Here we compare this effect using six different homologous phenothiazinium dyes: methylene blue, toluidine blue O, new methylene blue, dimethylmethylene blue, azure A, and azure B. We found both significant potentiation (up to 2 logs) and also significant inhibition (>3 logs) of killing by adding 10 mM azide depending on Gram classification, washing the dye from the cells, and dye structure. Killing of E. coli was potentiated with all 6 dyes after a wash, while S. aureus killing was only potentiated by MB and TBO with a wash and DMMB with no wash. More lipophilic dyes (higher log P value, such as DMMB) were more likely to show potentiation. We conclude that the Type I photochemical mechanism (potentiation with azide) likely depends on the microenvironment, i.e. higher binding of dye to bacteria. Bacterial dye-binding is thought to be higher with Gram-negative compared to Gram-positive bacteria, when unbound dye has been washed away, and with more lipophilic dyes.

  10. Gram staining.

    PubMed

    Coico, Richard

    2005-10-01

    Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  11. Gram staining.

    PubMed

    Coico, R

    2001-05-01

    Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  12. In vitro activity of tigecycline and comparators against Gram-positive and Gram-negative isolates collected from the Middle East and Africa between 2004 and 2011.

    PubMed

    Kanj, Souha S; Whitelaw, Andrew; Dowzicky, Michael J

    2014-02-01

    The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) was established in 2004 to monitor longitudinal changes in bacterial susceptibility to numerous antimicrobial agents, specifically tigecycline. In this study, susceptibility among Gram-positive and Gram-negative isolates between 2004 and 2011 from the Middle East and Africa was examined. Antimicrobial susceptibilities were determined using Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, and minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. US Food and Drug Administration (FDA)-approved breakpoints were used for tigecycline. In total, 2967 Gram-positive and 6322 Gram-negative isolates were examined from 33 participating centres. All Staphylococcus aureus isolates, including meticillin-resistant S. aureus, were susceptible to tigecycline, linezolid and vancomycin. Vancomycin, linezolid, tigecycline and levofloxacin were highly active (>97.6% susceptibility) against Streptococcus pneumoniae, including penicillin-non-susceptible strains. All Enterococcus faecium isolates were susceptible to tigecycline and linezolid, including 32 vancomycin-resistant isolates. Extended-spectrum β-lactamases were produced by 16.6% of Escherichia coli and 32.9% of Klebsiella pneumoniae. More than 95% of E. coli and Enterobacter spp. were susceptible to amikacin, tigecycline, imipenem and meropenem. The most active agents against Pseudomonas aeruginosa and Acinetobacter baumannii were amikacin (88.0% susceptible) and minocycline (64.2% susceptible), respectively; the MIC90 (MIC required to inhibit 90% of the isolates) of tigecycline against A. baumannii was low at 2mg/L. Tigecycline and carbapenem agents were highly active against most Gram-negative pathogens. Tigecycline, linezolid and vancomycin showed good activity against most Gram-positive pathogens from the Middle East and Africa.

  13. Circulating Inflammatory Mediators during Start of Fever in Differential Diagnosis of Gram-Negative and Gram-Positive Infections in Leukopenic Rats

    PubMed Central

    Tavares, Eva; Maldonado, Rosario; Ojeda, Maria L.; Miñano, Francisco J.

    2005-01-01

    Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections. PMID:16148175

  14. 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

  15. Neisseria weaveri sp. nov., formerly CDC group M-5, a gram-negative bacterium associated with dog bite wounds.

    PubMed Central

    Andersen, B M; Steigerwalt, A G; O'Connor, S P; Hollis, D G; Weyant, R S; Weaver, R E; Brenner, D J

    1993-01-01

    CDC group M-5 is a rod-shaped, gram-negative, nonmotile bacterium associated with dog bite wounds. DNA-DNA relatedness and biochemical and growth characteristics were studied for 54 strains from the collection at the Centers for Disease Control and Prevention. One typical M-5 strain, 8142, was further studied by 16S rRNA sequencing. DNA from 40 of 53 strains showed 82 to 100% relatedness (hydroxyapatite method) to labeled DNA from strain 8142. The guanine-plus-cytosine (G + C) content in 8 of the 41 highly related M-5 strains was 50.5 to 52 mol%. These 41 strains were oxidase and catalase positive, nonfermentative, nitrite positive, nitrate negative, weakly phenylalanine deaminase positive, aerobic, and alpha-hemolytic (sheep blood). DNA from the 13 remaining strains showed only 7 to 46% DNA relatedness to strain 8142. These 13 non-M-5 strains differed from the M-5 strains in G + C content, growth characteristics, and biochemical profiles. DNA from M-5 strain 8142 was most closely related to DNA from groups EF-4b (47%) and EF-4a (45%). 16S rRNA sequence analysis placed M-5 strain 8142 in the Neisseriaceae cluster of the beta-3 subgroup of the class Proteobacteria. It was most homologous (98.4 to 98.8%) to Neisseria animalis, Neisseria flavescens, Neisseria canis, and Neisseria elongata. All data are consistent with M-5 being a new species of Neisseria, for which we propose the name Neisseria weaveri. PMID:8408570

  16. 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.

  17. 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

  18. Optimizing Antibiotic Dosing Strategies for the Treatment of Gram-negative Infections in the Era of Resistance.

    PubMed

    Monogue, Marguerite L; Kuti, Joseph L; Nicolau, David P

    2016-01-01

    Gram-negative organisms are an increasing source of concern within the healthcare setting due to their common presence as a cause of infection and emerging resistance to current therapies. However, current antimicrobial dosing recommendations may be insufficient for the treatment of gram-negative infections. Applying knowledge of an antibiotic's pharmacokinetic/pharmacodynamic profile when designing a dosing regimen leads to a greater likelihood of achieving optimal exposure, including against gram-negative pathogens with higher MICs. Additionally, administering antibiotics directly to the site of infection, such as via aerosolization for pneumonia, is another method to achieve optimized drug exposure at the site of infection. Incorporating these treatment strategies into clinical practice will assist antimicrobial stewardship programs in successfully treating gram-negative infections.

  19. A general system for studying protein-protein interactions in gram-negative bacteria

    SciTech Connect

    Pelletier, Dale A.; Hurst, G. B.; Foote, Linda J.; Lankford, Patricia K.; McKeown, Cathy K.; Lu, Tse-Yuan S.; Schmoyer, Denise D.; Shah, Manesh B.; Hervey IV, W. J.; McDonald, W. Hayes; Hooker, Brian S.; Cannon, William R.; Daly, Don S.; Gilmore, Jason M.; Wiley, H. S.; Auberry, Deanna L.; Wang, Yisong; Larimer, Frank; Kennel, S. J.; Doktycz, M. J.; Morrell-Falvey, Jennifer; Owens, Elizabeth T.; Buchanan, M. V.

    2008-08-01

    One of the most promising of the emerging methods for large-scale studies of interactions among proteins is co-isolation of an affinity-tagged protein and its interaction partners, followed by mass spectrometric identification of the co-purifying proteins. We describe a methodology for systematically identifying the proteins that interact with affinity-tagged “bait” proteins expressed from a medium copy plasmid, which are based on a broad host range (pBBR1MCS5) vector backbone that has been modified to incorporate the Gateway DEST plasmid multiple cloning region. This construct was designed to facilitate expression of fusion proteins bearing an affinity tag, across a range of Gram negative bacterial hosts. We demonstrate the performance of this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results from the RNA polymerase complex from these two species compared favorably with those for both plasmid- and chromosomally-encoded affinity-tagged fusion proteins expressed in a model organism, E. coli.

  20. Prevalence and emergence of carbapenemases-producing Gram-negative bacteria in Mediterranean basin.

    PubMed

    Mathlouthi, Najla; Al-Bayssari, Charbel; Bakour, Sofiane; Rolain, Jean Marc; Chouchani, Chedly

    2017-02-01

    The emergence and the global spread of carbapenemases concern to health services worldwide. Their celestial rise among Gram-negative bacilli has challenged both the scientific and pharmaceutical sectors. Indeed, infections caused by these bacteria have limited treatment options and have been associated with high mortality and morbidity rates. Carbapenemase producers are mainly identified among Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii and still mostly in hospital settings and rarely in the community. They are closely related to KPC, VIM, IMP, NDM, and OXA-48 types. The encoding genes are mostly plasmid located and associated with various mobile genetic elements. The Mediterranean area is of interest due to a great diversity and population mixing. The prevalence of carbapenemases is particularly high and variant among countries, partially depending on the population exchange relationship between the regions and the possible reservoirs of each carbapenemase. This review described the epidemiology of carbapenemases in this region of the world highlighting the worrisome situation and the need to screen and detect these enzymes to prevent and control their dissemination especially as it is clear that very few novel antibiotics will be introduced in the next few years, making the dissemination of carbapenem-resistant Gram-negative bacteria of crucial importance worldwide.

  1. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria

    PubMed Central

    Roier, Sandro; Zingl, Franz G.; Cakar, Fatih; Durakovic, Sanel; Kohl, Paul; Eichmann, Thomas O.; Klug, Lisa; Gadermaier, Bernhard; Weinzerl, Katharina; Prassl, Ruth; Lass, Achim; Daum, Günther; Reidl, Joachim; Feldman, Mario F.; Schild, Stefan

    2016-01-01

    Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo. PMID:26806181

  2. A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria

    SciTech Connect

    Pelletier, Dale A; Auberry, Deanna L; Buchanan, Michelle V; Cannon, Bill; Daly, Don S.; Doktycz, Mitchel John; Foote, Linda J; Hervey, IV, William Judson; Hooker, Brian; Hurst, Gregory {Greg} B; Kennel, Steve J; Lankford, Patricia K; Larimer, Frank W; Lu, Tse-Yuan S; McDonald, W Hayes; McKeown, Catherine K; Morrell-Falvey, Jennifer L; Owens, Elizabeth T; Schmoyer, Denise D; Shah, Manesh B; Wiley, Steven; Wang, Yisong; Gilmore, Jason

    2008-01-01

    Abstract One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged bait proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

  3. The Multidrug-Resistant Gram-negative Superbugs Threat Require Intelligent Use of the Last Weapon.

    PubMed

    Deris, Zakuan Zainy

    2015-09-01

    The global emergence and dissemination of multidrug-resistant Gram-negative superbugs, particularly carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae, lead to the limited effectiveness of antibiotics for treating nosocomial infections. In most cases, polymyxins are the last resort therapy, and these antibiotics must be used intelligently to prolong their efficacy in clinical practice. Polymyxin B and colistin (polymyxin E) were introduced prior to modern drug regulation, and the majority of the 'old' drug information is unreliable. Recent pharmacokinetic data do not support the renal dose adjustment of intravenous (IV) polymyxin B as suggested by the manufacturer, and this drug must be scaled by the total body weight. Whereas IV colistin is formulated as an inactive prodrug, colistin methanesulfonate (CMS) has different pharmacokinetic profiles than polymyxin B. To achieve maximum efficacy, CMS should be administered as a loading dose scaled to body weight and a maintenance dose according to the renal profiles. Polymyxin combination therapy is suggested due to a sub-therapeutic plasma concentration in a significant proportion of patients and a high incidence of polymyxin hetero-resistance among Gram-negative superbugs. In conclusion, polymyxins must be reserved as a last resort and should be wisely used when truly indicated.

  4. Probing the Penetration of Antimicrobial Polymyxin Lipopeptides into Gram-Negative Bacteria

    PubMed Central

    2015-01-01

    The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative ‘superbugs’ has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains. PMID:24635310

  5. [Detection of endotoxins of Gram-negative bacteria on the basis of electromagnetic radiation frequency spectrum].

    PubMed

    Likhoded, V G; Kuleshova, N V; Sergieva, N V; Konev, Iu V; Trubnikova, I A; Sudzhian, E V

    2007-01-01

    Method of Gram-negative bacteria endotoxins detection on the basis of their own spectrum of electromagnetic radiation frequency was developed. Frequency spectrum typical for chemotype Re glycolipid, which is a part of lypopolysaccharides in the majority of Gram-negative bacteria, was used. Two devices--"Mini- Expert-DT" (manufactured by IMEDIS, Moscow) and "Bicom" (manufactured by Regumed, Germany)--were used as generators of electromagnetic radiation. Detection of endotoxin using these devices was performed by electropuncture vegetative resonance test. Immunoenzyme reaction with antibodies to chemotype Re glycolipid was used during analysis of preparations for assessment of resonance-frequency method specificity. The study showed that resonance-frequency method can detect lypopolysaccharides of different enterobacteria in quantities up to 0.1 pg as well as bacteria which contain lypopolysaccharides. At the same time, this method does not detect such bacteria as Staphylococcus aureus, Bifidobacterium spp., Lactobacillus spp., and Candida albicans. The method does not require preliminary processing of blood samples and can be used for diagnostics of endotoxinemia, and detection of endotoxins in blood samples or injection solutions.

  6. Cutting edge: natural DNA repetitive extragenic sequences from gram-negative pathogens strongly stimulate TLR9.

    PubMed

    Magnusson, Mattias; Tobes, Raquel; Sancho, Jaime; Pareja, Eduardo

    2007-07-01

    Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-alpha production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9(tm1Aki) knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.

  7. Gram-Negative Bacteria That Produce Carbapenemases Causing Death Attributed to Recent Foreign Hospitalization

    PubMed Central

    Ahmed-Bentley, Jasmine; Chandran, A. Uma; Joffe, A. Mark; French, Desiree; Peirano, Gisele

    2013-01-01

    Overseas travel, as a risk factor for the acquisition of infections due to antimicrobial-resistant organisms, has recently been linked to carbapenemase-producing Gram-negative bacteria. Multiresistant Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii strains were isolated from a wound of a Canadian patient with a recent history of hospitalization in India. This resulted in the initiation of outbreak management that included surveillance cultures. Epidemiological and molecular investigations showed that NDM-1-producing K. pneumoniae ST16 and OXA-23-producing A. baumannii ST10 strains were transmitted to 5 other patients, resulting in the colonization of 4 patients and the death of 1 patient due to septic shock caused by the OXA-23-producing A. baumannii strain. The high rate of false positivity of the screening cultures resulted in additional workloads and increased costs for infection control and clinical laboratory work. We believe that this is the first report of an infection with carbapenemase-producing Gram-negative bacteria resulting in death attributed to a patient with recent foreign hospitalization. We recommend routine rectal and wound screening for colonization with multiresistant bacteria for patients who have recently been admitted to hospitals outside Canada. PMID:23612195

  8. Broad Specificity Efflux pumps and Their Role in Multidrug Resistance of Gram Negative Bacteria

    PubMed Central

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2013-01-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are producing a worldwide health problem. The continuous dissemination of «multi-drug resistant» (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria, the over-expression of efflux pumps that expel structurally-unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data indicate an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological level, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may open the way to rationally develop an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms. PMID:21707670

  9. Multidrug resistant gram-negative bacteria in clinical isolates from Karachi.

    PubMed

    Saeed, Asma; Khatoon, Hajra; Ansari, Fasihuddin Ahmed

    2009-01-01

    A total of 54 gram-negative bacteria obtained from various pathological labs and hospitals of Karachi were screened for their resistance to ampicillin, chloramphenicol, gentamycin, kanamycin, neomycin, streptomycin and tetracycline antibiotics. Of the 54 bacteria, 50 were resistant to one or more antibiotics. Among the resistant bacteria, 13 out of 28 were found to transfer their resistances by conjugation. This indicates that at least 46% of clinical gram-negative bacteria in Karachi possess various types of transferable R plasmids, such as pAK5, pAK9, pAK10, pAK11, pAK12, pAK13, pAK14, pAK15, pAK16, pAK17, pAK18, pAK19, pAK20 and pAK21. The non-conjugative R plasmids included pMT14 and pZ26. Only pAK15 showed 26% segregation even after 20 consecutive transfers in plain broth (spontaneous segregation) whereas only pAK15 and pAK16 showed any significant loss of their markers in curing by acridine orange. The stability of R plasmids is more dangerous from clinical point of view.

  10. Proteolytic activity and fatal gram-negative sepsis in burned mice: effect of exogenous proteinase inhibition.

    PubMed Central

    Neely, A N; Miller, R G; Holder, I A

    1994-01-01

    Circulating proteolytic activity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed. PMID:8188336

  11. Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

    PubMed Central

    Volzing, Katherine; Borrero, Juan; Sadowsky, Michael J.; Kaznessis, Yiannis N.

    2014-01-01

    We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella. In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis. Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter nisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host’s viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations. PMID:23808914

  12. Nitric oxide synthase-dependent immune response against gram negative bacteria in a crustacean, Litopenaeus vannamei.

    PubMed

    Rodríguez-Ramos, Tania; Carpio, Yamila; Bolívar, Jorge; Gómez, Leonardo; Estrada, Mario Pablo; Pendón, Carlos

    2016-03-01

    Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus -NOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming.

  13. Ciprofloxacin-resistant gram-negative bacilli in the fecal microflora of children.

    PubMed

    Qin, Xuan; Razia, Yasmin; Johnson, James R; Stapp, Jennifer R; Boster, Daniel R; Tsosie, Treva; Smith, Donna L; Braden, Christopher R; Gay, Kathryn; Angulo, Frederick J; Tarr, Phillip I

    2006-10-01

    The extent to which antibiotic-resistant bacteria are excreted by humans who have not been exposed to antibiotics is not known. Children, who rarely receive fluoroquinolones, provide opportunities to assess the frequency of fecal excretion by fluoroquinolone-naïve hosts of fluoroquinolone-resistant gram-negative bacilli. Fresh nondiarrheal stools from children were processed by screening them on agar containing ciprofloxacin to recover ciprofloxacin-resistant gram-negative bacilli. Resistant isolates were identified, and ciprofloxacin MICs were determined. Resistant Escherichia coli isolates were also analyzed for urovirulence-associated loci. Thirteen (2.9%) of 455 stools yielded ciprofloxacin-resistant E. coli (seven children), Stenotrophomonas maltophilia (four children), and Achromobacter xylosoxidans and Enterobacter aerogenes (one child each). Neither the subjects themselves nor members of their households used fluoroquinolones in the 4 weeks preceding collection. Six of the seven resistant E. coli isolates belonged to phylogenetic groups B2 and D, in which extraintestinal pathogenic E. coli bacteria are frequently found. All resistant E. coli isolates contained at least three putative E. coli virulence loci. Most ciprofloxacin-resistant bacteria were resistant to additional antibiotics. Potentially pathogenic bacteria that are resistant to therapeutically important antimicrobial agents are excreted by some humans, despite these persons' lack of exposure to the particular drugs. The sources of these resistant organisms are unknown. This underrecognized reservoir of drug-resistant potential pathogens poses public health challenges.

  14. Capillary electrophoresis for fast detection of heterogeneous population in colistin-resistant Gram-negative bacteria.

    PubMed

    Sautrey, Guillaume; Duval, Raphaël E; Chevalley, Alicia; Fontanay, Stéphane; Clarot, Igor

    2015-10-01

    It has been shown that diverse strains of bacteria can be separated according to their characteristic surface properties by means of CE. We employed here this analytical technique to the study of colistin-resistance in Gram-negative bacteria, which involves the selection of mutants with modified outer membrane composition resulting in changes of surface cell properties. In the same way as with molecular entities, we performed firstly the validation of an ITP-based CE method for three common pathogenic Gram-negative bacteria namely Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Secondly, we compared the electrophoretic profiles of bacterial samples from a colistin-susceptible clinical isolate of K. pneumoniae and from the corresponding colistin-resistant derivative. By a simple CE run taking a few minutes, the coexistence of several bacterial subpopulations in the colistin-resistant derivative was clearly evidenced. This work encourages further research that would allow applications of CE in clinical laboratory for a daily monitoring of bacterial population in cared patients when "last-chance" colistin treatment is initiated against multidrug-resistant bacteria.

  15. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria.

    PubMed

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2012-03-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.

  16. Appropriateness of gram-negative agent use at a tertiary care hospital in the setting of significant antimicrobial resistance.

    PubMed

    Vora, Neil M; Kubin, Christine J; Furuya, E Yoko

    2015-01-01

    Background.  Practicing antimicrobial stewardship in the setting of widespread antimicrobial resistance among gram-negative bacilli, particularly in urban areas, is challenging. Methods.  We conducted a retrospective cross-sectional study at a tertiary care hospital with an established antimicrobial stewardship program in New York, New York to determine appropriateness of use of gram-negative antimicrobials and to identify factors associated with suboptimal antimicrobial use. Adult inpatients who received gram-negative agents on 2 dates, 1 June 2010 or 1 December 2010, were identified through pharmacy records. Clinical data were collected for each patient. Use of gram-negative agents was deemed optimal or suboptimal through chart review and according to hospital guidelines. Data were compared using χ(2) or Fischer's exact test for categorical variables and Student t test or Mann-Whitney U test for continuous variables. Results.  A total of 356 patients were included who received 422 gram-negative agents. Administration was deemed suboptimal in 26% of instances, with the most common reason being spectrum of activity too broad. In multivariable analysis, being in an intensive care unit (adjusted odds ratio [aOR], .49; 95% confidence interval [CI], .29-.84), having an infectious diseases consultation within the previous 7 days (aOR, .52; 95% CI, .28-.98), and having a history of multidrug-resistant gram-negative bacilli within the past year (aOR, .24; 95% CI, .09-.65) were associated with optimal gram-negative agent use. Beta-lactam/beta-lactamase inhibitor combination drug use (aOR, 2.6; 95% CI, 1.35-5.16) was associated with suboptimal use. Conclusions.  Gram-negative agents were used too broadly despite numerous antimicrobial stewardship program activities.

  17. Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

    PubMed Central

    2009-01-01

    Background In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms. Results Bacterial suspension (107 CFU mL-1) treated with different photosensitizers concentrations (0.5, 1.0 and 5.0 μM) were exposed to white light (40 W m-2) for a total light dose of 64.8 J cm-2. The most effective photosensitizers against both bacterial strains were the Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me at 5.0 μM with a light fluence of 64.8 J cm-2, leading to > 7.0 log (> 99,999%) of photoinactivation. The tetracationic porphyrin also proved to be a good photosensitizer against both bacterial strains. Both di-cationic and the monocationic porphyrins were the least effective ones. Conclusion The number of positive charges, the charge distribution in the porphyrins' structure and the meso-substituent groups seem to have different effects on the photoinactivation of both bacteria. As the Tri-Py+-Me-PF porphyrin provides the highest log reduction using lower light doses, this photosensitizer can efficiently photoinactivate a large spectrum of environmental bacteria. The complete inactivation of both bacterial strains with low light fluence (40 W m-2) means that the photodynamic approach can be applied to wastewater treatment under natural

  18. Characterization of carbapenem-resistant Gram-negative bacteria from Tamil Nadu.

    PubMed

    Nachimuthu, Ramesh; Subramani, Ramkumar; Maray, Suresh; Gothandam, K M; Sivamangala, Karthikeyan; Manohar, Prasanth; Bozdogan, Bülent

    2016-10-01

    Carbapenem resistance is disseminating worldwide among Gram-negative bacteria. The aim of this study was to identify carbapenem-resistance level and to determine the mechanism of carbapenem resistance among clinical isolates from two centres in Tamil Nadu. In the present study, a total of 93 Gram-negative isolates, which is found to be resistant to carbapenem by disk diffusion test in two centres, were included. All isolates are identified at species level by 16S rRNA sequencing. Minimal inhibitory concentrations (MICs) of isolates for Meropenem were tested by agar dilution method. Presence of blaOXA, blaNDM, blaVIM, blaIMP and blaKPC genes was tested by PCR in all isolates. Amplicons were sequenced for confirmation of the genes. Among 93 isolates, 48 (%52) were Escherichia coli, 10 (%11) Klebsiella pneumoniae, nine (%10) Pseudomonas aeruginosa. Minimal inhibitory concentration results showed that of 93 suspected carbapenem-resistant isolates, 27 had meropenem MICs ≥ 2 μg/ml. The MIC range, MIC50 and MIC90 were < 0.06 to >128 μg/ml, 0.12 and 16 μg/ml, respectively. Fig. 1 . Among meropenem-resistant isolates, E. coli were the most common (9/48, 22%), followed by K. pneumoniae (7/9, 77%), P. aeruginosa (6/10, 60%), Acinetobacter baumannii (2/2, 100%), Enterobacter hormaechei (2/3, 67%) and one Providencia rettgeri (1/1, 100%). PCR results showed that 16 of 93 carried blaNDM, three oxa181, and one imp4. Among blaNDM carriers, nine were E. coli, four Klebsiella pneumoniae, two E. hormaechei and one P. rettgeri. Three K. pneumoniae were OXA-181 carriers. The only imp4 carrier was P. aeruginosa. A total of seven carbapenem-resistant isolates were negatives by PCR for the genes studied. All carbapenem-resistance gene-positive isolates had meropenem MICs >2 μg/ml. Our results confirm the dissemination of NDM and emergence of OXA-181 beta-lactamase among Gram-negative bacteria in South India. This study showed the emergence of NDM producer in clinical

  19. Aerobic fitness in women and responses to lower body negative pressure

    NASA Technical Reports Server (NTRS)

    Frey, Mary Anne Bassett; Mathes, Karen L.; Hoffler, G. Wyckliffe

    1987-01-01

    The role of tolerance to orthostatic stress in the maintenance of high aerobic fitness in women was investigated by examining the responses of heart rate, stroke volume, cardiac output, Heather index of contractility, arterial pressure, peripheral resistance, change in calf circumference, and thoracic impedance of healthy female subjects to lower body negative pressure (LBNP) applied for 5 min at -50 mm Hg or until a subject became presyncopal. The testing protocol involved a stepwise reduction in pressure and consisted of two parts: an LBNP test in supine position followed by a treadmill test to peak aerobic capacity. Women were found to exhibit the same response pattern to LBNP as was previously reported by Convertino et al. (1984) for men. The results do not support the hypothesis that orthostatic tolerance in women is inversely related to aerobic fitness, as demonstrated by a finding that the peak aerobic capacity of subjects who became presyncopal did not differ from the peak of the tolerant subjects, and that hemodynamic responses to LBNPL were not a function of aerobic capacity.

  20. 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

  1. 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.

  2. Functional synergy of α-helical antimicrobial peptides and traditional antibiotics against Gram-negative and Gram-positive bacteria in vitro and in vivo.

    PubMed

    Feng, Q; Huang, Y; Chen, M; Li, G; Chen, Y

    2015-01-01

    In this study, the antimicrobial activities based on the synergistic effects of traditional antibiotics (imipenem, cefepime, levofloxacin hydrochloride and vancomycin) and antimicrobial peptides (AMPs; PL-5, PL-31, PL-32, PL-18, PL-29 and PL-26), alone or in combination, against three Gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae and Staphylococcus epidermidis) and three Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae) were investigated. In addition, the antimicrobial activity that was based on the synergistic effects of levofloxacin hydrochloride and PL-5 against Staphylococcus aureus in vivo was explored in a mouse infection model. Traditional antibiotics and AMPs showed significant synergistic effects on the antibacterial activities against the different Gram-positive and Gram-negative bacteria in vitro. A strong synergistic effect in the PL-5 and levofloxacin hydrochloride combination against Staphylococcus aureus was observed in the mouse infection model in vivo. The mechanism of synergistic action was due to the different targets of AMPs and traditional antibiotics. The combination of AMPs and traditional antibiotics can dramatically enhance antimicrobial activity and may help prevent or delay the emergence of antibiotic resistance. Thus, this combination therapy could be a promising approach to treat bacterial infections, particularly mixed infections and multi-antibiotic-resistant infections, in the clinics.

  3. 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.

  4. 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.

  5. Alternating electric fields combined with activated carbon for disinfection of Gram negative and Gram positive bacteria in fluidized bed electrode system.

    PubMed

    Racyte, Justina; Bernard, Séverine; Paulitsch-Fuchs, Astrid H; Yntema, Doekle R; Bruning, Harry; Rijnaarts, Huub H M

    2013-10-15

    Strong electric fields for disinfection of wastewaters have been employed already for several decades. An innovative approach combining low strength (7 V/cm) alternating electric fields with a granular activated carbon fluidized bed electrode (FBE) for disinfection was presented recently. For disinfection performance of FBE several pure microbial cultures were tested: Bacillus subtilis, Bacillus subtilis subsp. subtilis, Enterococcus faecalis as representatives from Gram positive bacteria and Erwinia carotovora, Pseudomonas luteola, Pseudomonas fluorescens and Escherichia coli YMc10 as representatives from Gram negative bacteria. The alternating electric field amplitude and shape were kept constant. Only the effect of alternating electric field frequency on disinfection performance was investigated. From the bacteria tested, the Gram negative strains were more susceptible and the Gram positive microorganisms were more resistant to FBE disinfection. The collected data indicate that the efficiency of disinfection is frequency and strain dependent. During 6 h of disinfection, the decrease above 2 Log units was achieved with P. luteola and E. coli at 10 kHz and at dual frequency shift keying (FSK) modulated signal with frequencies of 10 kHz and 140 kHz. FBE technology appears to offer a new way for selective bacterial disinfection, however further optimizations are needed on treatment duration, and energy input, to improve effectiveness.

  6. Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

    DOE PAGES

    Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.; ...

    2014-12-09

    The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutronmore » reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.« less

  7. Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

    SciTech Connect

    Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.; Ciesielski, Filip; Kuzmenko, Ivan; Holt, Stephen A.; Lakey, Jeremy H.

    2014-12-09

    The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

  8. A genomic update on clostridial phylogeny: Gram-negative spore-formers and other misplaced clostridia

    PubMed Central

    Yutin, Natalya; Galperin, Michael Y.

    2014-01-01

    Summary The class Clostridia in the phylum Firmicutes (formerly low-G+C Gram-positive bacteria) includes diverse bacteria of medical, environmental, and biotechnological importance. The Selenomonas-Megasphaera-Sporomusa branch, which unifies members of the Firmicutes with Gram-negative-type cell envelopes, was recently moved from Clostridia to a separate class Negativicutes. However, draft genome sequences of the spore-forming members of the Negativicutes revealed typically clostridial sets of sporulation genes. To address this and other questions in clostridial phylogeny, we have compared a phylogenetic tree for a concatenated set of 50 widespread ribosomal proteins with the trees for beta subunits of the RNA polymerase (RpoB) and DNA gyrase (GyrB) and with the 16S rRNA-based phylogeny. The results obtained by these methods showed remarkable consistency, suggesting that they reflect the true evolutionary history of these bacteria. These data put the Selenomonas-Megasphaera-Sporomusa group back within the Clostridia. They also support placement of Clostridium difficile and its close relatives within the family Peptostreptococcaceae; we suggest resolving the long-standing naming conundrum by renaming it Peptoclostridium difficile. These data also indicate the existence of a group of cellulolytic clostridia that belong to the family Ruminococcaceae. As a tentative solution to resolve the current taxonomical problems, we propose assigning 78 validly described Clostridium species that clearly fall outside the family Clostridiaceae to six new genera: Peptoclostridium, Lachnoclostridium, Ruminiclostridium, Erysipelatoclostridium, Gottschalkia, and Tyzzerella. This work reaffirms that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits, even such key ones as the structure of the cell envelope and Gram-staining pattern. PMID:23834245

  9. Molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Egypt.

    PubMed

    Ahmed, Ashraf M; Shimamoto, Tadashi

    2011-05-01

    The aim of this study was to characterize the genetic basis of multidrug resistance in Gram-negative bacteria isolated from bovine mastitis cases in Egypt. Multidrug resistance phenotypes were found in 34 of 112 (30.4%) Gram-negative bacterial isolates, which harbored at least one antimicrobial resistance gene. The most prevalent multidrug-resistant (MDR) species were Enterobacter cloacae (8 isolates, 7.1%), Klebsiella pneumoniae (7 isolates, 6.3%), Klebsiella oxytoca (7 isolates, 6.3%), Escherichia coli (5 isolates, 4.5%), and Citrobacter freundii (3 isolates, 2.7%). The most commonly observed resistance phenotypes were against ampicillin (97.0%), streptomycin (94.1%), tetracycline (91.2%), trimethoprim-sulfamethoxazole (88.2%), nalidixic acid (85.3%), and chloramphenicol (76.5%). Class 1 integrons were detected in 28 (25.0%) isolates. The gene cassettes within class 1 integrons included those encoding resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17, and dfrA25), aminoglycosides (aadA1, aadA2, aadA5, aadA7, aadA12, aadA22, and aac(3)-Id), chloramphenicol (cmlA), erythromycin (ereA2), and rifampicin (arr-3). Class 2 integrons were identified in 6 isolates (5.4%) with three different profiles. Furthermore, the β-lactamase encoding genes, bla(TEM), bla(SHV), bla(CTX-M), and bla(OXA), the plasmid-mediated quinolone resistance genes, qnr and aac(6)-Ib-cr, and the florfenicol resistance gene, floR, were also identified. To the best of our knowledge, the results identified class 2 integrons, qnr and aac(6)-Ib-cr from cases of mastitis for the first time. This is the first report of molecular characterization for antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Africa.

  10. Structural engineering of a phage lysin that targets Gram-negative pathogens

    SciTech Connect

    Lukacik, Petra; Barnard, Travis J.; Keller, Paul W.; Chaturvedi, Kaveri S.; Seddiki, Nadir; Fairman, James W.; Noinaj, Nicholas; Kirby, Tara L.; Henderson, Jeffrey P.; Steven, Alasdair C.; Hinnebusch, B. Joseph; Buchanan, Susan K.

    2012-11-13

    Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against Gram-positive organisms but not against Gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a {beta}-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and another that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.

  11. Induction of gram-negative bacterial growth by neurochemical containing banana (Musa x paradisiaca) extracts.

    PubMed

    Lyte, M

    1997-09-15

    Bananas contain large quantities of neurochemicals. Extracts from the peel and pulp of bananas in increasing stages of ripening were prepared and evaluated for their ability to modulate the growth of non-pathogenic and pathogenic bacteria. Extracts from the peel, and to a much lesser degree the pulp, increased the growth of Gram-negative bacterial strains Escherichia coli O157:H7, Shigella flexneri, Enterobacter cloacae and Salmonella typhimurium, as well as two non-pathogenic E. coli strains, in direct relation to the content of norepinephrine and dopamine, but not serotonin. The growth of Gram-positive bacteria was not altered by any of the extracts. Supplementation of vehicle and pulp cultures with norepinephrine or dopamine yielded growth equivalent to peel cultures. Total organic analysis of extracts further demonstrated that the differential effects of peel and pulp on bacterial growth was not nutritionally based, but due to norepinephrine and dopamine. These results suggest that neurochemicals contained within foodstuffs may influence the growth of pathogenic and indigenous bacteria through direct neurochemical-bacterial interactions.

  12. Antibacterial Properties of an Oligo-Acyl-Lysyl Hexamer Targeting Gram-Negative Species

    PubMed Central

    Zaknoon, Fadia; Goldberg, Keren; Sarig, Hadar; Epand, Raquel F.; Epand, Richard M.

    2012-01-01

    Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl]5 (α12-5α10). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC90 of 6.2 μM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α12-5α10 from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux. PMID:22751534

  13. 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.

  14. Gram-Negative Marine Bacteria: Structural Features of Lipopolysaccharides and Their Relevance for Economically Important Diseases

    PubMed Central

    Anwar, Muhammad Ayaz; Choi, Sangdun

    2014-01-01

    Gram-negative marine bacteria can thrive in harsh oceanic conditions, partly because of the structural diversity of the cell wall and its components, particularly lipopolysaccharide (LPS). LPS is composed of three main parts, an O-antigen, lipid A, and a core region, all of which display immense structural variations among different bacterial species. These components not only provide cell integrity but also elicit an immune response in the host, which ranges from other marine organisms to humans. Toll-like receptor 4 and its homologs are the dedicated receptors that detect LPS and trigger the immune system to respond, often causing a wide variety of inflammatory diseases and even death. This review describes the structural organization of selected LPSes and their association with economically important diseases in marine organisms. In addition, the potential therapeutic use of LPS as an immune adjuvant in different diseases is highlighted. PMID:24796306

  15. Healthcare-associated Gram-negative bloodstream infections: antibiotic resistance and predictors of mortality.

    PubMed

    Ergönül, Ö; Aydin, M; Azap, A; Başaran, S; Tekin, S; Kaya, Ş; Gülsün, S; Yörük, G; Kurşun, E; Yeşilkaya, A; Şimşek, F; Yılmaz, E; Bilgin, H; Hatipoğlu, Ç; Cabadak, H; Tezer, Y; Togan, T; Karaoğlan, I; İnan, A; Engin, A; Alışkan, H E; Yavuz, S Ş; Erdinç, Ş; Mulazimoglu, L; Azap, Ö; Can, F; Akalın, H; Timurkaynak, F

    2016-12-01

    This article describes the prevalence of antibiotic resistance and predictors of mortality for healthcare-associated (HA) Gram-negative bloodstream infections (GN-BSI). In total, 831 cases of HA GN-BSI from 17 intensive care units in different centres in Turkey were included; the all-cause mortality rate was 44%. Carbapenem resistance in Klebsiella pneumoniae was 38%, and the colistin resistance rate was 6%. Multi-variate analysis showed that age >70 years [odds ratio (OR) 2, 95% confidence interval (CI) 1.22-3.51], central venous catheter use (OR 2.1, 95% CI 1.09-4.07), ventilator-associated pneumonia (OR 1.9, 95% CI 1.1-3.16), carbapenem resistance (OR 1.8, 95% CI 1.11-2.95) and APACHE II score (OR 1.1, 95% CI 1.07-1.13) were significantly associated with mortality.

  16. High dose of tigecycline for extremely resistant Gram-negative pneumonia: yes, we can

    PubMed Central

    2014-01-01

    Few antimicrobials are currently active to treat infections caused by extremely resistant Gram-negative bacilli (ERGNB), which represent a serious global public health concern. Tigecycline, which covers the majority of these ERGNB (with the exception of Pseudomonas aeruginosa), is not currently approved for hospital-acquired pneumonia, and several meta-analyses have suggested an increased risk of death in patients receiving this antibiotic. Other studies suggest that the use of high-dose tigecycline may represent an alternative in daily practice. De Pascale and colleagues report that the clinical cure rate in patients with ventilator-associated pneumonia is significantly higher with a high dose of tigecycline than with the conventional dose, although mortality was unaffected. This high dose is safe; no patients required discontinuation or dose reduction. PMID:25043402

  17. Localization of Somatic Antigen on Gram-Negative Bacteria by Electron Microscopy

    PubMed Central

    Shands, J. W.

    1965-01-01

    Shands, J. W. (University of Florida, Gainesville). Localization of somatic antigen on gram-negative bacteria by electron microscopy. J. Bacteriol. 90:266–270. 1965.—Antisera specific for the somatic antigens of Salmonella typhimurium and Escherichia coli O113 were prepared, and globulins isolated from these antisera were labeled with ferritin. Micrographs of labeled, sectioned bacteria show that somatic antigen is located in considerable quantities on the surface of the bacteria, and, furthermore, that it can extend up to 150 mμ beyond the confines of the cell wall. The arrangement of the ferritin on the bacteria suggests that the antigenic sites are located on fibrillar structures. Images PMID:16562028

  18. Antibiotics and the mechanics of cellular bulging in gram-negative bacteria

    NASA Astrophysics Data System (ADS)

    Daly, K.; Wingreen, Ned S.; Mukhopahyay, Ranjan

    2010-03-01

    For most bacteria, the cell wall, consisting of a cross-linked polymer network, is the primary stress-bearing structure. Due to the high osmotic pressure difference across the cell membrane, the presence of the cell wall is essential for cell stability. Recent experiments have addressed the effect of cell-wall defects induced by antibiotics such as vancomycin, and find that in Gram-negative bacteria, antibiotics can lead to pronounced bulging of the cell membrane and eventually to lysis. Here we address the mechanics of bulging and its relationship to cell-wall defects. We estimate the critical defect size for bulging and discuss the biological implications of our results. We also discuss the relevance of our physical model to blebbing and vesiculation in eukaryotic cells.

  19. Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens

    PubMed Central

    2015-01-01

    Negamycin is a natural product with antibacterial activity against a broad range of Gram-negative pathogens. Recent revelation of its ribosomal binding site and mode of inhibition has reinvigorated efforts to identify improved analogues with clinical potential. Translation-inhibitory potency and antimicrobial activity upon modification of different moieties of negamycin were in line with its observed ribosomal binding conformation, reaffirming stringent structural requirements for activity. However, substitutions on the N6 amine were tolerated and led to N6-(3-aminopropyl)-negamycin (31f), an analogue showing 4-fold improvement in antibacterial activity against key bacterial pathogens. This represents the most potent negamycin derivative to date and may be a stepping stone toward clinical development of this novel antibacterial class. PMID:26288696

  20. DNA/Ag Nanoparticles as Antibacterial Agents against Gram-Negative Bacteria

    PubMed Central

    Takeshima, Tomomi; Tada, Yuya; Sakaguchi, Norihito; Watari, Fumio; Fugetsu, Bunshi

    2015-01-01

    Silver (Ag) nanoparticles were produced using DNA extracted from salmon milt as templates. Particles spherical in shape with an average diameter smaller than 10 nm were obtained. The nanoparticles consisted of Ag as the core with an outermost thin layer of DNA. The DNA/Ag hybrid nanoparticles were immobilized over the surface of cotton based fabrics and their antibacterial efficiency was evaluated using E. coli as the typical Gram-negative bacteria. The antibacterial experiments were performed according to the Antibacterial Standard of Japanese Association for the Functional Evaluation of Textiles. The fabrics modified with DNA/Ag nanoparticles showed a high enough inhibitory and killing efficiency against E. coli at a concentration of Ag ≥ 10 ppm. PMID:28347012

  1. [The identification of nonfermentative gram-negative bacteria. Experiences with 676 apyocyaninogenic strains (author's transl)].

    PubMed

    Berger, U; Piotrowski, H D

    1981-02-01

    During a period of 16 months 1757 strains of nonfermentative gram-negative rods have been isolated from clinical material. Of the, 1205 (69%) were P. aeruginosa, 124 (10%) of which failed to produce pyocyanin. The apyocyaninogenic strains as well as the remaining 552 isolates were differentiated by steps according to a diagnostic scheme developed by us. For identification of species two or three steps were needed. By this procedure, 530 of the 552 strains could be assigned to nineteen species within the genera Pseudomonas, Achromobacter, Alcaligenes, Flavobacterium, Agrobacterium and Acinetobacter. 17 strains could not be identified below the genus level, one strain belonged to CDC-group VE-2 and four strains were not identifiable. 72% of the 552 strains belonged to only four species: Pseudomonas putida, P. maltophilia, Acinetobacter lwoffii and A. anitratus.

  2. Gram-negative bacilli as nontransient flora on the hands of hospital personnel.

    PubMed

    Guenthner, S H; Hendley, J O; Wenzel, R P

    1987-03-01

    The possibility that gram-negative bacilli (GNB) are part of the nontransient flora on hands was examined by using a broth rinse technique to detect low titers of GNB after a hygienic hand wash with soap and water. A total of 100 nurses who had direct patient contact and 40 controls without patient contact had a similar rate of recovery of GNB (46 and 55%, respectively). GNB persisted on the hands of 10 nurses throughout five successive hand washes with soap and water. Hand cultures were obtained daily from 12 nurses before and after a work shift in a surgical intensive care unit. GNB were recovered from 57% of individuals before patient contact and from only 24% after the work shift. Nontransient GNB on the hands of hospital personnel are a potential reservoir for hospital strains, and patient contact is not an obvious source for the acquisition of nontransient GNB.

  3. Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.

    PubMed

    Turner, Robert D; Hurd, Alexander F; Cadby, Ashley; Hobbs, Jamie K; Foster, Simon J

    2013-01-01

    Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.

  4. Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria.

    PubMed

    Benedetti, Ilaria; Nikel, Pablo I; de Lorenzo, Víctor

    2016-03-01

    Engineering of robust microbial cell factories requires the use of dedicated genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We have edited and formatted the ChnR/P chnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The proposed compositional standard was thoroughly verified with a monomeric and superfolder green fluorescent protein (msf•GFP) in Escherichia coli. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Data in this article are related to the research paper "Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes" [1].

  5. Subcellular localization of Gram-negative bacterial proteins using sparse learning.

    PubMed

    Zheng, Zhonglong; Yang, Jie

    2010-04-01

    One of the main challenges faced by biological applications is to predict protein subcellular localization in an automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological system into account. Traditionally such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressive Sensing), we propose a method which performs locality preserving projection with a sparseness criterion such that the feature selection and dimension reduction are merged into one analysis. The proposed sparse method decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse method is quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.

  6. Sanitizing Effect of Ethanol Against Biofilms Formed by Three Gram-Negative Pathogenic Bacteria.

    PubMed

    Park, Han-Saem; Ham, Youngseok; Shin, Keum; Kim, Yeong-Suk; Kim, Tae-Jong

    2015-07-01

    Sanitizing effect of ethanol on a Yersinia enterocolitica biofilm was evaluated in terms of biomass removal and bactericidal activity. We found that 40 % ethanol was most effective for biofilm biomass removal; however, no significant difference was observed in bactericidal activity between treatment with 40 and 70 % ethanol. This unexpected low ethanol concentration requirement for biomass removal was confirmed using biofilms of two additional pathogenic bacteria, Aeromonas hydrophila and Xanthomonas oryzae. Although only three pathogenic Gram-negative bacteria were tested and the biofilm in nature was different from the biofilm in this study, the results in this study suggested the possible re-evaluation of the effective sanitizing ethanol concentration 70 %, which is the concentration commonly employed for sanitization, on bacteria in a biofilm.

  7. T-mod pathway, a reduced sequence for identification of gram-negative urinary tract pathogens.

    PubMed Central

    Berlutti, F; Thaller, M C; Dainelli, B; Pezzi, R

    1989-01-01

    In this paper, we describe a reduced sequence of identification that includes T-mod medium, a selective and differential isolation medium which allows accurate presumptive identification of the most common gram-negative bacteria encountered in urine samples. The present study, performed on bacteria isolated from 1,762 independent urine samples, has shown that a few selected tests (lysine and ornithine decarboxylase, urease and trehalose fermentation tests) improve the identification accuracy of T-mod, making it possible both to identify the less frequent species and to prevent some misidentifications of Klebsiella pneumoniae and Proteus mirabilis. The proposed work flow agreed with conventional identification protocols to a 99.3% extent and allowed identification of 87.4% of the isolates directly from the primary plate, 11.4% after 1 to 3 additional tests, and 1.2% after an identification gallery. PMID:2768451

  8. Antiseptic and antibiotic resistance in Gram-negative bacteria causing urinary tract infection.

    PubMed Central

    Stickler, D J; Thomas, B

    1980-01-01

    A collection of 802 isolates of Gram-negative bacteria causing urinary tract infections was made from general practice, antenatal clinics, and local hospitals. The organisms were tested for their sensitivity to chlorhexidine, cetrimide, glutaraldehyde, phenyl mercuric nitrate, a phenolic formulation, and a proprietary antiseptic containing a mixture of picloxydine, octyl phenoxy polyethoxyethanol, and benzalkonium chloride. Escherichia coli, the major species isolated, proved to be uniformly sensitive to these agents. Approximately 10% of the total number of isolates, however, exhibited a degree of resistance to the cationic agents. These resistant organisms were members of the genera Proteus, Providencia, and Pseudomonas; they were also generally resistant to five, six, or seven antibiotics. It is proposed therefore that an antiseptic policy which involves the intensive use of cationic antiseptics might lead to the selection of a flora of notoriously drug-resistant species. PMID:6769972

  9. [Significance of efflux pumps in multidrug resistance of Gram-negative bacteria].

    PubMed

    Wiercińska, Olga; Chojecka, Agnieszka; Kanclerski, Krzysztof; Rőhm-Rodowald, Ewa; Jakimiak, Bożenna

    2015-01-01

    The phenomenon of multidrug. resistance of bacteria is a serious problem of modern medicine. This resistance largely is a consequence of abuse and improper use of antibacterial substances, especially antibiotics and chemotherapeutics in hospital settings. Multidrug resistance is caused by a number of interacting mechanisms of resistance. Recent studies have indicated that efflux pumps and systems of efflux pumps are an important determinant of this phenomenon. Contribute to this particular RND efflux systems of Gram-negative bacteria, which possess a wide range of substrates such as antibiotics, dyes, detergents, toxins and active substances of disinfectants and antiseptics. These transporters are usually encoded on bacterial chromosomes. Genes encoding efflux pumps' proteins may also be carried on plasmids and other mobile genetic elements. Such pumps are usually specific to a small group of substrates, but as an additional mechanism of resistance may contribute to the multidrug resistance.

  10. FGL chaperone-assembled fimbrial polyadhesins: anti-immune armament of Gram-negative bacterial pathogens.

    PubMed

    Zavialov, Anton; Zav'yalova, Galina; Korpela, Timo; Zav'yalov, Vladimir

    2007-07-01

    This review summarizes the current knowledge on the structure, function, assembly, and biomedical applications of the family of adhesive fimbrial organelles assembled on the surface of Gram-negative pathogens via the FGL chaperone/usher pathway. Recent studies revealed the unique structural and functional properties of these organelles, distinguishing them from a related family, FGS chaperone-assembled adhesive pili. The FGL chaperone-assembled organelles consist of linear polymers of one or two types of protein subunits, each possessing one or two independent adhesive sites specific to different host cell receptors. This structural organization enables these fimbrial organelles to function as polyadhesins. Fimbrial polyadhesins may ensure polyvalent fastening of bacteria to the host cells, aggregating their receptors and triggering subversive signals that allow pathogens to evade immune defense. The FGL chaperone-assembled fimbrial polyadhesins are attractive targets for vaccine and drug design.

  11. Gram-negative marine bacteria: structural features of lipopolysaccharides and their relevance for economically important diseases.

    PubMed

    Anwar, Muhammad Ayaz; Choi, Sangdun

    2014-04-30

    Gram-negative marine bacteria can thrive in harsh oceanic conditions, partly because of the structural diversity of the cell wall and its components, particularly lipopolysaccharide (LPS). LPS is composed of three main parts, an O-antigen, lipid A, and a core region, all of which display immense structural variations among different bacterial species. These components not only provide cell integrity but also elicit an immune response in the host, which ranges from other marine organisms to humans. Toll-like receptor 4 and its homologs are the dedicated receptors that detect LPS and trigger the immune system to respond, often causing a wide variety of inflammatory diseases and even death. This review describes the structural organization of selected LPSes and their association with economically important diseases in marine organisms. In addition, the potential therapeutic use of LPS as an immune adjuvant in different diseases is highlighted.

  12. Multidrug-resistant Gram-negative bacteria: a product of globalization.

    PubMed

    Hawkey, P M

    2015-04-01

    Global trade and mobility of people has increased rapidly over the last 20 years. This has had profound consequences for the evolution and the movement of antibiotic resistance genes. There is increasing exposure of populations all around the world to resistant bacteria arising in the emerging economies. Arguably the most important development of the last two decades in the field of antibiotic resistance is the emergence and spread of extended-spectrum β-lactamases (ESBLs) of the CTX-M group. A consequence of the very high rates of ESBL production among Enterobacteriaceae in Asian countries is that there is a substantial use of carbapenem antibiotics, resulting in the emergence of plasmid-mediated resistance to carbapenems. This article reviews the emergence and spread of multidrug-resistant Gram-negative bacteria, focuses on three particular carbapenemases--imipenem carbapenemases, Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase--and highlights the importance of control of antibiotic use.

  13. Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria.

    PubMed

    Poole, Keith

    2012-05-01

    Bacteria encounter a myriad of potentially growth-compromising conditions in nature and in hosts of pathogenic bacteria. These 'stresses' typically elicit protective and/or adaptive responses that serve to enhance bacterial survivability. Because they impact upon many of the same cellular components and processes that are targeted by antimicrobials, adaptive stress responses can influence antimicrobial susceptibility. In targeting and interfering with key cellular processes, antimicrobials themselves are 'stressors' to which protective stress responses have also evolved. Cellular responses to nutrient limitation (nutrient stress), oxidative and nitrosative stress, cell envelope damage (envelope stress), antimicrobial exposure and other growth-compromising stresses, have all been linked to the development of antimicrobial resistance in Gram-negative bacteria - resulting from the stimulation of protective changes to cell physiology, activation of resistance mechanisms, promotion of resistant lifestyles (biofilms), and induction of resistance mutations.

  14. Development and transmission of antimicrobial resistance among Gram-negative bacteria in animals and their public health impact.

    PubMed

    Mukerji, Shewli; O'Dea, Mark; Barton, Mary; Kirkwood, Roy; Lee, Terence; Abraham, Sam

    2017-02-28

    Gram-negative bacteria are known to cause severe infections in both humans and animals. Antimicrobial resistance (AMR) in Gram-negative bacteria is a major challenge in the treatment of clinical infections globally due to the propensity of these organisms to rapidly develop resistance against antimicrobials in use. In addition, Gram-negative bacteria possess highly efficient mechanisms through which the AMR can be disseminated between pathogenic and commensal bacteria of the same or different species. These unique traits of Gram-negative bacteria have resulted in evolution of Gram-negative bacterial strains demonstrating resistance to multiple classes of antimicrobials. The evergrowing resistance issue has not only resulted in limitation of treatment options but also led to increased treatment costs and mortality rates in humans and animals. With few or no new antimicrobials in production to combat severe life-threatening infections, AMR has been described as the one of the most severe, long-term threats to human health. Aside from overuse and misuse of antimicrobials in humans, another factor that has exacerbated the emergence of AMR in Gram-negative bacteria is the veterinary use of antimicrobials that belong to the same classes considered to be critically important for treating serious life-threatening infections in humans. Despite the fact that development of AMR dates back to before the introduction of antimicrobials, the recent surge in the resistance towards all available critically important antimicrobials has emerged as a major public health issue. This review thus focuses on discussing the development, transmission and public health impact of AMR in Gram-negative bacteria in animals.

  15. N-acyl homoserinelactone-mediated gene regulation in gram-negative bacteria.

    PubMed

    Eberl, L

    1999-12-01

    The view of bacteria as unicellular organisms has strong roots in the tradition of culturing bacteria in liquid media. However, in nature microbial activity is mainly associated with surfaces where bacteria form highly structured and cooperative consortia which are commonly referred to as biofilms. The ability of bacteria to organize structurally and to distribute metabolic activities between the different members of the consortium demands a high degree of coordinated cell-cell interaction. Recent work has established that many bacteria employ sophisticated intercellular communication systems that rely on small signal molecules to control the expression of multiple target genes. In Gram-negative bacteria, the most intensively investigated signal molecules are N-acyl-L-homoserine lactones (AHLs), which are utilized by the bacteria to monitor their own population densities in a process known as 'quorum sensing'. These density-dependent regulatory systems rely on two proteins, an AHL synthase, usually a member of the LuxI family of proteins, and an AHL receptor protein belonging to the LuxR family of transcriptional regulators. At low population densities cells produce a basal level of AHL via the activity of an AHL synthase. As the cell density increases, AHL accumulates in the growth medium. On reaching a critical threshold concentration, the AHL molecule binds to its cognate receptor which in turn leads to the induction/repression of AHL-regulated genes. To date, AHL-dependent quorum sensing circuits have been identified in a wide range of gram-negative bacteria where they regulate various functions including bioluminescence, plasmid conjugal transfer, biofilm formation, motility, antibiotic biosynthesis, and the production of virulence factors in plant and animal pathogens. Moreover, AHL signal molecules appear to play important roles in the ecology of complex consortia as they allow bacterial populations to interact with each other as well as with their

  16. Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry.

    PubMed

    Cataldi, Tommaso R I; Bianco, Giuliana; Fonseca, Juliano; Schmitt-Kopplin, Philippe

    2013-01-01

    Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC-LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC-ESI-qTOF MS has also proved to be suitable for identification of 3O-C(10)HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized.

  17. Intrathecal or intraventricular therapy for post-neurosurgical Gram-negative meningitis: matched cohort study.

    PubMed

    Shofty, B; Neuberger, A; Naffaa, M E; Binawi, T; Babitch, T; Rappaport, Z H; Zaaroor, M; Sviri, G; Paul, M

    2016-01-01

    Gram-negative post-operative meningitis due to carbapenem-resistant bacteria (CR-GNPOM) is a dire complication of neurosurgical procedures. We performed a nested propensity-matched historical cohort study aimed at examining the possible benefit of intrathecal or intraventricular (IT/IV) antibiotic treatment for CR-GNPOM. We included consecutive adults with GNPOM in two centres between 2005 and 2014. Patients receiving combined systemic and IT/IV treatment were matched to patients receiving systemic treatment only. Matching was done based on the propensity of the patients to receive IT/IV treatment. We compared patient groups with 30-day mortality defined as the primary outcome. The cohort included 95 patients with GNPOM. Of them, 37 received IT/IV therapy in addition to systemic treatment (22 with colistin and 15 with amikacin), mostly as initial therapy, through indwelling cerebrospinal fluid drains. Variables associated with IT/IV therapy in the propensity score included no previous neurosurgery, time from admission to meningitis, presence of a urinary catheter and GNPOM caused by carbapenem-resistant Gram-negative bacteria. Following propensity matching, 23 patients given IT/IV therapy and 27 controls were analysed. Mortality was significantly lower with IT/IV therapy: 2/23 (8.7%) versus 9/27 (33.3%), propensity-adjusted OR 0.19, 95% CI 0.04-0.99. Death or neurological deterioration at 30 days, 14-day and in-hospital mortality were lower with IT/IV therapy (OR <0.4 for all) without statistically significant differences. Among patients discharged alive, those receiving IT/IV therapy did not experience more neurological deterioration. Serious adverse events with IT/IV therapy were not documented. Our results support the early use of IT antibiotic treatment for CR-GNPOM when a delivery method is available.

  18. Inhibition of CEA release from epithelial cells by lipid A of Gram-negative bacteria.

    PubMed

    Naghibalhossaini, Fakhraddin; Sayadi, Khatere; Jaberie, Hajar; Bazargani, Abdollah; Eftekhar, Ebrahim; Hosseinzadeh, Massood

    2015-09-01

    A number of bacterial species, both pathogenic and non-pathogenic, use the human CEACAM family members as receptors for internalization into epithelial cells. The GPI-linked CEA and CEACAM6 might play a role in the innate immune defense, protecting the colon from microbial invasion. Previous studies showed that CEA is released from epithelial cells by an endogenous GPI-PLD enzyme. GPI-PLD activity was reported to be inhibited by several synthetic and natural forms of lipid A. We hypothesized that CEA engagement by Gram-negative bacteria might attenuate CEA release from epithelial cells and that this might facilitate bacterial colonization. We tested the hypothesis by examining the effect of Escherichia coli on CEA release from colorectal cancer cells in a co-culture experiment. A subconfluent monolayer culture of colorectal cancer cells (LS-180, Caco-2 and HT29/219) was incubated with E. coli. While there was a significant reduction in CEA secretion from LS-180 and HT29/219 cells, we found only a small reduction of CEA shedding from Caco-2 cells compared to the level from the untreated control cells. Furthermore, lipid A treatment of LS-180 cells inhibited CEA release from the cells in a dosedependent manner. Western blot analysis of total lysates showed that CEA expression levels in cells co-cultured with bacteria did not differ from those in untreated control cells. These results suggest that lipid A of Gram-negative bacteria might play a role in preventing the release of CEA from mucosal surfaces and promote mucosal colonization by bacteria.

  19. Evaluation of the rapid NFT system for identification of gram-negative, nonfermenting rods.

    PubMed Central

    Appelbaum, P C; Leathers, D J

    1984-01-01

    This study evaluated the ability of the Rapid NFT system (API System SA, Montalieu-Vercieu, France) to accurately identify 262 clinically isolated, gram-negative, nonfermentative rods without additional tests. Identifications were classified as correct; low discrimination, with a spectrum of two or more possibilities (additional tests necessary for accurate identification); and incorrect. Correct identification rates were analyzed in two categories: (i) correct to species or biotype for all organism groups except Alcaligenes faecalis-odorans, Moraxella, Pseudomonas testosteroni-alcaligenes-pseudoalcaligenes, and Acinetobacter calcoaceticus biotype haemolyticus-alcaligenes (in this category, the latter four genus-biotype group identifications were taken as correct) and (ii) correct to species or biotype in all cases, including the above four groups. In category i, 87.4% of the strains were correctly identified, with 4.2% low discrimination and 8.4% incorrect. When the criteria of category ii were used, 71.8% of the strains were correctly identified, with 19.9% low discrimination. The Rapid NFT system provided excellent species identification of Pseudomonas and Flavobacterium spp., Bordetella bronchiseptica, and Achromobacter xylosoxidans strains. Within Acinetobacter calcoaceticus, differentiation between biotypes anitratus and lwoffi was satisfactory, but the system did not differentiate between biotypes haemolyticus and alcaligenes. Species resolution within the genera Moraxella and Alcaligenes was incomplete. All Alcaligenes faecalis strains were misidentified and accounted for 50% of misidentifications with the Rapid NFT system; however, these results may reflect taxonomic differences rather than true misidentifications. The Rapid NFT system is easy to inoculate and interpret and represents a worthwhile advance in the identification of gram-negative, nonfermentative rods. PMID:6490857

  20. Understanding Gram-negative Central Line-Associated Blood Stream Infection in a Surgical Trauma ICU.

    PubMed

    Duane, Therese M; Kikhia, Rashid M; Wolfe, Luke G; Ober, Janis; Tessier, Jeffrey M

    2015-08-01

    The purpose of this study was to review central line-associated blood stream infection (CLABSI) data from a surgical trauma intensive care unit to better understand patient risk factors, pathogens, and treatment interventions. We performed a retrospective review of all surgical ICU patients who met the Centers for Disease Control definition for Gram-negative CLABSI from 2006 through 2013. Demographics, pathogens, interventions, and outcomes were evaluated. A total of 40 patients were included with an average age of 49.9 ± 19 years and 72.5 per cent male. The average length of central venous line (CVL) was 11 ± 5.9 days with average time from line placement to positive culture 9.4 ± 6.8 days. Most common organisms were Enterobacter species (37.5%) with 17.8 per cent of all cultured organisms considered multidrug resistant. Piperacillin-tazobactam (67.5%) was the most commonly used antibiotic. Overall mortality rate was 22.5 per cent. A total of 11 patients who developed a recurrence did so at 10.7 ± 8 days and were similar to those without recurrence. Predominant pathogens associated with surgical trauma intensive care unit CLABSI in this study are different from those Gram-negative bacteria associated with published studies in the general hospital population. Further investigation into risk factors for infection and relapse is important to minimize such consequences. Understanding appropriate line placement and use as well as clarifying optimal duration of therapy is integral in improving outcomes.

  1. Characterization of five novel endolysins from Gram-negative infecting bacteriophages.

    PubMed

    Walmagh, Maarten; Boczkowska, Barbara; Grymonprez, Barbara; Briers, Yves; Drulis-Kawa, Zuzanna; Lavigne, Rob

    2013-05-01

    We here characterize five globular endolysins, encoded by a set of Gram-negative infecting bacteriophages: BcepC6gp22 (Burkholderia cepacia phage BcepC6B), P2gp09 (Escherichia coli phage P2), PsP3gp10 (Salmonella enterica phage PsP3), K11gp3.5 and KP32gp15 (Klebsiella pneumoniae phages K11 and KP32, respectively). In silico, BcepC6gp22, P2gp10 and PsP3gp10 are predicted to possess lytic transglycosylase activity, whereas K11gp3.5 and KP32gp15 have putative amidase activity. All five endolysins show muralytic activity on the peptidoglycan of several Gram-negative bacterial species. In vitro, Pseudomonas aeruginosa PAO1 is clearly sensitive for the antibacterial action of the five endolysins in the presence of the outer membrane permeabilizer EDTA: reductions are ranging from 1.89 to 3.08 log units dependent on the endolysin. The predicted transglycosylases BcepC6gp22, P2gp10 and PsP3gp10 have a substantially higher muralytic and in vitro antibacterial activity compared to the predicted amidases K11gp3.5 and KP32gp15, highlighting the impact of the catalytic specificity on endolysin activity. Furthermore, initial data exclude the synergistic lethal effect of a combination of the predicted transglycosylase PsP3gp10 and the predicted amidase K11gp3.5 on PAO1. As these globular endolysins show a lower enzymatic and antibacterial activity, in comparison to modular endolysins, we suggest that the latter should be favored for antibacterial applications.

  2. Ubiquitous and persistent Proteobacteria and other Gram-negative bacteria in drinking water.

    PubMed

    Vaz-Moreira, Ivone; Nunes, Olga C; Manaia, Célia M

    2017-05-15

    Drinking water comprises a complex microbiota, in part shaped by the disinfection and distribution systems. Gram-negative bacteria, mainly members of the phylum Proteobacteria, represent the most frequent bacteria in drinking water, and their ubiquity and physiological versatility raises questions about possible implications in human health. The first step to address this concern is the identification and characterization of such bacteria that is the first objective of this study, aiming at identifying ubiquitous or persistent Gram-negative bacteria, Proteobacteria or members of other phyla, isolated from tap water or from its source. >1000 bacterial isolates were characterized and identified, and a selected group (n=68) was further analyzed for the minimum inhibitory concentrations (MIC) to antibiotics (amoxicillin and gentamicin) and metals (copper and arsenite). Total DNA extracts of tap water were examined for the presence of putatively acquired antibiotic resistance or related genes (intI1, blaTEM, qnrS and sul1). The ubiquitous tap water genera comprised Proteobacteria of the class Alpha- (Blastomonas, Brevundimonas, Methylobacterium, Sphingobium, Sphingomonas), Beta- (Acidovorax, Ralstonia) and Gamma- (Acinetobacter and Pseudomonas). Persistent species were members of genera such as Aeromonas, Enterobacter or Dechloromonas. Ralstonia spp. showed the highest MIC values to gentamicin and Acinetobacter spp. to arsenite. The genes intI1, blaTEM or sul1 were detected, at densities lower than 2.3×10(5)copies/L, 2.4×10(4)copies/L and 4.6×10(2)copies/L, respectively, in most tap water samples. The presence of some bacterial groups, in particular of Beta- or Gammaproteobacteria (e.g. Ralstonia, Acinetobacter, Pseudomonas) in drinking water may deserve attention given their potential as reservoirs or carriers of resistance or as opportunistic pathogens.

  3. Efficacy of surface disinfectant cleaners against emerging highly resistant gram-negative bacteria

    PubMed Central

    2014-01-01

    Background Worldwide, the emergence of multidrug-resistant gram-negative bacteria is a clinical problem. Surface disinfectant cleaners (SDCs) that are effective against these bacteria are needed for use in high risk areas around patients and on multi-touch surfaces. We determined the efficacy of several SDCs against clinically relevant bacterial species with and without common types of multidrug resistance. Methods Bacteria species used were ATCC strains; clinical isolates classified as antibiotic-susceptible; and multi-resistant clinical isolates from Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia marcescens (all OXA-48 and KPC-2); Acinetobacter baumannii (OXA-23); Pseudomonas aeruginosa (VIM-1); and Achromobacter xylosoxidans (ATCC strain). Experiments were carried out according to EN 13727:2012 in quadruplicate under dirty conditions. The five evaluated SDCs were based on alcohol and an amphoteric substance (AAS), an oxygen-releaser (OR), surface-active substances (SAS), or surface-active-substances plus aldehydes (SASA; two formulations). Bactericidal concentrations of SDCs were determined at two different contact times. Efficacy was defined as a log10 ≥ 5 reduction in bacterial cell count. Results SDCs based on AAS, OR, and SAS were effective against all six species irrespective of the degree of multi-resistance. The SASA formulations were effective against the bacteria irrespective of degree of multi-resistance except for one of the four P. aeruginosa isolates (VIM-1). We found no general correlation between SDC efficacy and degree of antibiotic resistance. Conclusions SDCs were generally effective against gram-negative bacteria with and without multidrug resistance. SDCs are therefore suitable for surface disinfection in the immediate proximity of patients. Single bacterial isolates, however, might have reduced susceptibility to selected biocidal agents. PMID:24885029

  4. Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

    PubMed Central

    Kuhnert, P; Heyberger-Meyer, B; Burnens, A P; Nicolet, J; Frey, J

    1997-01-01

    The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance. PMID:9172345

  5. Altered ketone body metabolism during gram-negative sepsis in the rat.

    PubMed

    Lanza-Jacoby, S; Rosato, E; Braccia, G; Tabares, A

    1990-11-01

    To investigate why blood ketone bodies are depressed during sepsis, the production and utilization of ketone bodies was studied in fasted control, fasted, Escherichia coli-treated, fed control, and fed E coli-treated rats. Gram-negative sepsis was induced by intravenous (IV) injection of 8 x 10(7) live colonies of E coli per 100 g body weight. Food was removed from the fasted rats after E coli injection. Fed rats were infused intragastrically with a nutritionally adequate diet for 5 days before inducing sepsis. Twenty-four hours after E coli injection, blood ketone bodies were reduced in fasted septic rats and fed septic rats compared with their respective control rats. Ketogenesis and oxidation of labeled palmitate was not altered in hepatocytes from fasted E coli-treated rats. Yet, ketogenesis declined significantly in hepatocytes from fed E coli-treated rats. Oxidation of labeled palmitate was also significantly reduced in hepatocytes from fed E coli-treated rats. Utilization of ketone bodies as measured by the incorporation of [3-14C]beta-hydroxybutyrate into CO2, increased over threefold in the diaphragm, 12% in the heart, and 19% in the kidneys from the fasted E coli-treated rats. In the fed state, incorporation of [3-14C]beta-hydroxybutyrate into CO2 was elevated fivefold in the heart, fourfold in the diaphragm, and over threefold in the kidneys from the septic rats. These results suggest that in the fasted state, plasma ketone bodies remain low during gram-negative sepsis because peripheral tissues use more ketone bodies and because liver ketogenesis is not increased to compensate for the increased utilization. In the fed state, the reduction in blood ketone bodies appears to be attributed to both impaired ketogenic capacity and increased peripheral utilization.

  6. Silver resistance in Gram-negative bacteria: a dissection of endogenous and exogenous mechanisms

    PubMed Central

    Randall, Christopher P.; Gupta, Arya; Jackson, Nicole; Busse, David; O'Neill, Alex J.

    2015-01-01

    Objectives To gain a more detailed understanding of endogenous (mutational) and exogenous (horizontally acquired) resistance to silver in Gram-negative pathogens, with an emphasis on clarifying the genetic bases for resistance. Methods A suite of microbiological and molecular genetic techniques was employed to select and characterize endogenous and exogenous silver resistance in several Gram-negative species. Results In Escherichia coli, endogenous resistance arose after 6 days of exposure to silver, a consequence of two point mutations that were both necessary and sufficient for the phenotype. These mutations, in ompR and cusS, respectively conferred loss of the OmpC/F porins and derepression of the CusCFBA efflux transporter, both phenotypic changes previously linked to reduced intracellular accumulation of silver. Exogenous resistance involved derepression of the SilCFBA efflux transporter as a consequence of mutation in silS, but was additionally contingent on expression of the periplasmic silver-sequestration protein SilE. Silver resistance could be selected at high frequency (>10−9) from Enterobacteriaceae lacking OmpC/F porins or harbouring the sil operon and both endogenous and exogenous resistance were associated with modest fitness costs in vitro. Conclusions Both endogenous and exogenous silver resistance are dependent on the derepressed expression of closely related efflux transporters and are therefore mechanistically similar phenotypes. The ease with which silver resistance can become selected in some bacterial pathogens in vitro suggests that there would be benefit in improved surveillance for silver-resistant isolates in the clinic, along with greater control over use of silver-containing products, in order to best preserve the clinical utility of silver. PMID:25567964

  7. Evaluation of the Rapidec Carba NP Test Kit for Detection of Carbapenemase-Producing Gram-Negative Bacteria.

    PubMed

    Garg, Atul; Garg, Jaya; Upadhyay, G C; Agarwal, Anurag; Bhattacharjee, Amitabha

    2015-12-01

    Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive ($5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria.

  8. Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria

    SciTech Connect

    Kranz, R.G.; Gennis, R.B.

    1985-02-01

    Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a/sub 1/-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.

  9. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-04-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

  10. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed Central

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-01-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

  11. Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble

    PubMed Central

    2015-01-01

    Background It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. Results In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Conclusions Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg

  12. Lysis of gram-positive and gram-negative bacteria by antibacterial porous polymeric monolith formed in microfluidic biochips for sample preparation.

    PubMed

    Aly, Mohamed Aly Saad; Gauthier, Mario; Yeow, John

    2014-09-01

    Bacterial cell lysis is demonstrated using polymeric microfluidic biochips operating via a hybrid mechanical shearing/contact killing mechanism. These biochips are fabricated from a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by well-controlled, high-throughput laser micromachining. The unreacted double bonds at the surface of X-PMMA provide covalent bonding for the formation of a porous polymeric monolith (PPM), thus contributing to the mechanical stability of the biochip and eliminating the need for surface treatment. The lysis efficiency of these biochips was tested for gram-positive (Enterococcus saccharolyticus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Pseudomonas fluorescens) and confirmed by off-chip PCR without further purification. The influence of the flow rate when pumping the bacterial suspension through the PPM, and of the hydrophobic-hydrophilic balance on the cell lysis efficiency was investigated at a cell concentration of 10(5) CFU/mL. It was shown that the contribution of contact killing to cell lysis was more important than that of mechanical shearing in the PPM. The biochip showed better lysis efficiency than the off-chip chemical, mechanical, and thermal lysis techniques used in this work. The biochip also acts as a filter that isolates cell debris and allows PCR-amplifiable DNA to pass through. The system performs more efficient lysis for gram-negative than for gram-positive bacteria. The biochip does not require chemical/enzymatic reagents, power consumption, or complicated design and fabrication processes, which makes it an attractive on-chip lysis device that can be used in sample preparation for genetics and point-of-care diagnostics. The biochips were reused for 20 lysis cycles without any evidence of physical damage to the PPM, significant performance degradation, or DNA carryover when they were back-flushed between cycles. The biochips efficiently lysed both gram-positive and gram-negative

  13. Gram-negative bacteria facilitate tumor outgrowth and metastasis by promoting lipid synthesis in lung cancer patients

    PubMed Central

    Ye, Maosong; Gu, Xia; Han, Yang

    2016-01-01

    Background Lung cancer is the leading cause of cancer-related death worldwide. Patients with lung cancer are very frequently present with pulmonary infections, in particular with Gram-negative bacteria. Herein, we investigated the effect of the co-presence of Gram-negative bacteria on outgrowth and metastasis of lung cancer cells in clinical patients. Methods Lung cancer cells were isolated from clinical surgical tissues. Heat-inactivated E. coli was used as Gram-negative bacteria. Tumor outgrowth and invasion in vitro was analyzed with MTT assay and Biocoat Matrigel Invasion Chamber. Tumor growth and metastasis in vivo was evaluated in BALB/c nude mice. Lipid synthesis was evidenced by expressions of FASN and ACC1, as well as BODIPY Fluorophores staining. Block lipid synthesis was performed with C75 as a FAS inhibitor and transfection with ACC1 siRNA. Knockdown of TLR4 and TLR9 signaling was achieved by transfection with specific shRNAs and administration of specific antagonists. Results Gram-negative bacteria significantly promoted lung cancer development including growth and metastasis in dose dependent manner. Mechanistically, Gram-negative bacteria activate TLR4 and TLR9 signaling and enhance lipid synthesis in human lung cancer cells. Knockdown of TLR4 and/or TLR9 was able to block Gram-negative bacteria mediated lipid synthesis and lung cancer development. Interference with lipid synthesis efficiently abrogated Gram-negative-bacteria-induced lung cancer development. In lung cancer patients, higher expressions of innate immune receptors, TLR4 and TLR9, were observed in those with Gram-negative infections and associated with the aberrant lipid synthesis that was observed in vitro. Conclusions Pulmonary infections with Gram-negative bacteria lead to aberrant lipid synthesis through TLR4 and TLR9 signaling in lung cancer patients and result in rapid proliferation and metastasis of lung cancer cells. These findings reveal a new mechanism for pulmonary infection

  14. Extended spectrum β-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in Gram-negative bacteria.

    PubMed

    El Salabi, Allaaeddin; Walsh, Timothey R; Chouchani, Chedly

    2013-05-01

    Infectious diseases due to Gram-negative bacteria are a leading cause of morbidity and mortality worldwide. Antimicrobial agents represent one major therapeutic tools implicated to treat these infections. The misuse of antimicrobial agents has resulted in the emergence of resistant strains of Gram-negatives in particular Enterobacteriaceae and non-fermenters; they have an effect not only on a human but on the public health when bacteria use the resistance mechanisms to spread in the hospital environment and to the community outside the hospitals by means of mobile genetic elements. Gram-negative bacteria have become increasingly resistant to antimicrobial agents. They have developed several mechanisms by which they can withstand to antimicrobials, these mechanisms include the production of Extended-spectrum β-lactamases (ESBLs) and carbapenemases, furthermore, Gram-negative bacteria are now capable of spreading such resistance between members of the family Enterobacteriaceae and non-fermenters using mobile genetic elements as vehicles for such resistance mechanisms rendering antibiotics useless. Therefore, addressing the issue of mechanisms of antimicrobial resistance is considered one of most urgent priorities. This review will help to illustrate different resistance mechanisms; ESBLs, carbapenemases encoded by genes carried by mobile genetic elements, which are used by Gram-negative bacteria to escape antimicrobial effect.

  15. False-negative rate of gram-stain microscopy for diagnosis of septic arthritis: suggestions for improvement.

    PubMed

    Stirling, Paul; Faroug, Radwane; Amanat, Suheil; Ahmed, Abdulkhaled; Armstrong, Malcolm; Sharma, Pankaj; Qamruddin, Ahmed

    2014-01-01

    We quantify the false-negative diagnostic rate of septic arthritis using Gram-stain microscopy of synovial fluid and compare this to values reported in the peer-reviewed literature. We propose a method of improving the diagnostic value of Gram-stain microscopy using Lithium Heparin containers that prevent synovial fluid coagulation. Retrospective study of the Manchester Royal Infirmary microbiology database of patients undergoing synovial fluid Gram-stain and culture between December 2003 and March 2012 was undertaken. The initial cohort of 1896 synovial fluid analyses for suspected septic arthritis was reduced to 143 after exclusion criteria were applied. Analysis of our Gram-stain microscopy yielded 111 false-negative results from a cohort size of 143 positive synovial fluid cultures, giving a false-negative rate of 78%. We report a false-negative rate of Gram-stain microscopy for septic arthritis of 78%. Clinicians should therefore avoid the investigation until a statistically significant data set confirms its efficacy. The investigation's value could be improved by using Lithium Heparin containers to collect homogenous synovial fluid samples. Ongoing research aims to establish how much this could reduce the false-negative rate.

  16. Effect of aerobic capacity on Lower Body Negative Pressure (LBNP) tolerance in females

    NASA Technical Reports Server (NTRS)

    Moore, Alan D., Jr.; Fortney, Suzanne M.; Siconolfi, Steven F.

    1993-01-01

    This investigation determined whether a relationship exists in females between: (1) aerobic capacity and Lower Body Negative Pressure (LBNP); and (2) aerobic capacity and change in LBNP tolerance induced by bed rest. Nine females, age 27-47 (34.6 plus or minus 6.0 (Mean plus or minus SD)), completed a treadmill-graded exercise test to establish aerobic capacity. A presyncopal-limited LBNP test was performed prior to and after 13 days of bed rest at a 6 deg head-down tilt. LBNP tolerance was quantified as: (1) the absolute level of negative pressure (NP) tolerated for greater than or equal to 60 sec; and (2) Luft's Cumulative Stress Index (CSI). Aerobic capacity was 33.3 plus or minus 5.0 mL/kg/min and ranged from 25.7 to 38.7. Bed rest was associated with a decrease in NP tolerance (-9.04 1.6 kPa(-67.8 plus or minus 12.0 mmHg) versus -7.7 1.1 kPa(-57.8 plus or minus 8.33 mmHg); p = 0.028) and in CSI (99.4 27.4 kPa min(745.7 plus or minus 205.4 mmHg min) versus 77.0 16.9 kPa min (577.3 plus or minus mmHg min); p = 0.008). The correlation between aerobic capacity and absolute NP or CSI pre-bed rest did not differ significantly from zero (r = -0.56, p = 0.11 for NP; and r = -0.52, p = 0.16 for CSI). Also, no significant correlation was observed between aerobic and pre- to post-rest change for absolute NP tolerance (r = -0.35, p = 0.35) or CSI (r = -0.32, p = 0.40). Therefore, a significant relationship does not exist between aerobic capacity and orthostatic function or change in orthostatic function induced by bed rest.

  17. Novel Aminoglycoside 2″-Phosphotransferase Identified in a Gram-Negative Pathogen

    PubMed Central

    Toth, Marta; Frase, Hilary; Antunes, Nuno T.

    2013-01-01

    Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6′)-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (kcat/Km = 105 to 107 M−1 s−1), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme. PMID:23129050

  18. Clinical Characteristics and Risk Factors of Pyogenic Spondylitis Caused by Gram-Negative Bacteria

    PubMed Central

    Kang, Seung-Ji; Jang, Hee-Chang; Jung, Sook-In; Choe, Pyoeng Gyun; Park, Wan Beom; Kim, Chung-Jong; Song, Kyoung-Ho; Kim, Eu Suk; Kim, Hong Bin; Oh, Myoung-don

    2015-01-01

    Background There are limited data describing the clinical characteristics of pyogenic spondylitis caused by Gram-negative bacteria (GNB). The aim of this study was to investigate the predisposing factors and clinical characteristics of pyogenic spondylitis caused by GNB compared to Gram-positive cocci (GPC). Methods We performed a retrospective review of medical records from patients with culture-confirmed pyogenic spondylitis at four tertiary teaching hospitals over an 8-year period. Results A total of 344 patients with culture-confirmed pyogenic spondylitis were evaluated. There were 62 patients (18.0%) with pyogenic spondylitis caused by GNB and the most common organism was Escherichia coli (n = 35, 10.2%), followed by Pseudomonas aeruginosa (n = 10, 2.9%). Pyogenic spondylitis caused by GNB was more frequently associated with the female gender (64.5 vs. 35.5%, P <0.01), preexisting or synchronous genitourinary tract infection (32.3 vs. 2.1%, P< 0.01), and intra-abdominal infection (12.9 vs. 0.4%, P< 0.01) compared to patients with GPC. Although pyogenic spondylitis caused by GNB presented with severe sepsis more frequently (24.2 vs. 11.3%, P = 0.01), the mortality rate (6.0 vs. 5.2%) and the proportion of patients with residual disability (6.0 vs. 9.0%), defined as grade 3 or 4 (P = 0.78) 3 months after completion of treatment, were not significantly different compared to GPC patients. Conclusion GNB should be considered as the etiologic organism when infectious spondylitis develops in a patient with preexisting or synchronous genitourinary tract and intra-abdominal infection. In addition, the mortality rate and clinical outcomes are not significantly different between pyogenic spondylitis caused by GNB and GPC. PMID:25978839

  19. 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

  20. Myeloid Cell Sirtuin-1 Expression Does Not Alter Host Immune Responses to Gram-Negative Endotoxemia or Gram-Positive Bacterial Infection

    PubMed Central

    Crotty Alexander, Laura E.; Marsh, Brenda J.; Timmer, Anjuli M.; Lin, Ann E.; Zainabadi, Kayvan; Czopik, Agnieszka; Guarente, Leonard; Nizet, Victor

    2013-01-01

    The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington’s disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections. PMID:24386389

  1. A new hybrid bacteriocin, Ent35–MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

    PubMed Central

    Acuña, Leonardo; Picariello, Gianluca; Sesma, Fernando; Morero, Roberto D.; Bellomio, Augusto

    2012-01-01

    Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries. PMID:23650575

  2. 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

  3. Photoinactivation of Gram positive and Gram negative bacteria with the antimicrobial peptide (KLAKLAK)(2) conjugated to the hydrophilic photosensitizer eosin Y.

    PubMed

    Johnson, Gregory A; Muthukrishnan, Nandhini; Pellois, Jean-Philippe

    2013-01-16

    We test the hypothesis that the antimicrobial peptide (KLAKLAK)(2) enhances the photodynamic activity of the photosensitizer eosin Y upon conjugation. The conjugate eosin-(KLAKLAK)(2) was obtained by solid-phase peptide synthesis. Photoinactivation assays were performed against the Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and multidrug resistant Acinetobacter baumannii AYE, as well as the Gram-positive bacteria Staphylococcus aureus , and Staphylococcus epidermidis . Partitioning assays were performed with E. coli and S. aureus . Photohemolysis and photokilling assays were also performed to assess the photodynamic activity of the conjugate toward mammalian cells. Eosin-(KLAKLAK)(2) photoinactivates 99.999% of 10(8) CFU/mL of most bacteria tested at a concentration of 1 μM or below. In contrast, neither eosin Y nor (KLAKLAK)(2) cause any significant photoinactivation under similar conditions. The increase in photodynamic activity of the photosensitizer conferred by the antimicrobial peptide is in part due to the fact that (KLAKLAK)(2) promotes the association of eosin Y to bacteria. Eosin-(KLAKLAK)(2) does not significantly associate with red blood cells or the cultured mammalian cell lines HaCaT, COS-7, and COLO 316. Consequently, little photodamage or photokilling is observed with these cells under conditions for which bacterial photoinactivation is achieved. The peptide (KLAKLAK)(2) therefore significantly enhances the photodynamic activity of eosin Y toward both Gram-positive and Gram-negative bacteria while interacting minimally with human cells. Overall, our results suggest that antimicrobial peptides such as (KLAKLAK)(2) might serve as attractive agents that can target photosensitizers to bacteria specifically.

  4. Carbapenem-Resistant Non-Glucose-Fermenting Gram-Negative Bacilli: the Missing Piece to the Puzzle

    PubMed Central

    Gniadek, Thomas J.; Carroll, Karen C.

    2016-01-01

    The non-glucose-fermenting Gram-negative bacilli Pseudomonas aeruginosa and Acinetobacter baumannii are increasingly acquiring carbapenem resistance. Given their intrinsic antibiotic resistance, this can cause extremely difficult-to-treat infections. Additionally, resistance gene transfer can occur between Gram-negative species, regardless of their ability to ferment glucose. Thus, the acquisition of carbapenemase genes by these organisms increases the risk of carbapenemase spread in general. Ultimately, infection control practitioners and clinical microbiologists need to work together to determine the risk carried by carbapenem-resistant non-glucose-fermenting Gram-negative bacilli (CR-NF) in their institution and what methods should be considered for surveillance and detection of CR-NF. PMID:26912753

  5. The activity of bacteriocins from Carnobacterium maltaromaticum UAL307 against gram-negative bacteria in combination with EDTA treatment.

    PubMed

    Martin-Visscher, Leah A; Yoganathan, Sabesan; Sit, Clarissa S; Lohans, Christopher T; Vederas, John C

    2011-04-01

    Bacteriocins from gram-positive bacteria are potent antimicrobial peptides that inhibit pathogenic and food-spoilage bacteria. They are usually ineffective against gram-negative bacteria because they cannot penetrate the outer membrane (OM). Disruption of the OM of some gram-negative bacteria was reported to sensitize them to certain bacteriocins. This study evaluates the activity of three purified bacteriocins [carnocyclin A (CclA), carnobacteriocin BM1 (CbnBM1) and piscicolin 126 (PisA)] produced by Carnobacterium maltaromaticum UAL307, which has been approved for preservation of food in United States and Canada, against three gram-negative bacteria (Escherichia coli DH5α, Pseudomonas aeruginosa ATCC 14207 and Salmonella Typhimurium ATCC 23564). Their efficacy is compared with bacteriocins of other classes: the lantibiotics nisin A (positive control) and gallidermin, and the cyclic peptide subtilosin A (SubA). In combination with EDTA, CclA inhibited both E. coli and Pseudomonas. PisA inhibited Pseudomonas, but CbnBM1 showed weak activity toward Pseudomonas. In comparison, nisin and gallidermin inhibited the growth of all three strains, whereas SubA was active against E. coli and Pseudomonas only at high concentrations. The results reveal that UAL307 bacteriocins can inhibit gram-negative bacteria if the OM is weakened, and that the different classes of bacteriocins in this study exert unique modes of action toward such bacteria.

  6. Epidemiology of meningitis with a negative CSF Gram stain: under-utilization of available diagnostic tests.

    PubMed

    Nesher, L; Hadi, C M; Salazar, L; Wootton, S H; Garey, K W; Lasco, T; Luce, A M; Hasbun, R

    2016-01-01

    Meningitis with a negative cerebrospinal fluid Gram stain (CSF-GS) poses a diagnostic challenge as more than 50% of patients remain without an aetiology. The introduction of polymerase chain reaction (PCR) and arboviral serologies have increased diagnostic capabilities, yet large scale epidemiological studies evaluating their use in clinical practice are lacking. We conducted a prospective observational study in New Orleans between November 1999 and September 2008 (early era) when PCR was not widely available, and in Houston between November 2008 and June 2013 (modern era), when PCR was commonly used. Patients presenting with meningitis and negative CSF-GS were followed for 4 weeks. All investigations, PCR used, and results were recorded as they became available. In 323 patients enrolled, PCR provided the highest diagnostic yield (24·2%) but was ordered for 128 (39·6%) patients; followed by serology for arboviruses (15%) that was ordered for 100 (31%) of all patients. The yield of blood cultures was (10·3%) and that of CSF cultures was 4%; the yield for all other tests was <10%. Overall, 65% of the patients remained without a diagnosis at 4 weeks: 72·1% in early era vs. 53·4% (P < 0·01) in modern era; this change was attributed to diagnosing more viral pathogens, 8·3% and 26·3% (P < 0·01), respectively. The introduction of PCR and arboviral serologies has improved the yield of diagnosing patients with meningitis and a negative CSF-GS, but both tests are being under-utilized.

  7. 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.

  8. Burden of extensively drug-resistant and pandrug-resistant Gram-negative bacteria at a tertiary-care centre.

    PubMed

    Bhatt, Puneet; Tandel, Kundan; Shete, Vishal; Rathi, K R

    2015-11-01

    The emergence of resistance to multiple antimicrobial agents in Gram-negative bacteria is a significant threat to public health, as it restricts the armamentarium of the clinician against these infections. The aim of this study was to determine the burden of extensively drug-resistant (XDR) and pandrug-resistant (PDR) Gram-negative bacteria at a tertiary-care centre. Antimicrobial susceptibility testing of 1240 clinical isolates of Gram-negative bacteria obtained from various clinical samples during the study period was carried out by the Kirby-Bauer disc diffusion method. Minimum inhibitory concentration of all antibiotics including tigecycline and colistin was determined by Vitek-2 automated susceptibility testing system. Out of 1240 isolates of Gram-negative bacteria, 112 isolates (9%) were resistant to all the antibiotics tested by Kirby-Bauer disc diffusion method. This finding was corroborated by Vitek-2. In addition, Vitek-2 found that 67 isolates were resistant to all antibiotics except tigecycline and colistin. A total of 30 isolates were susceptible to only colistin, and four isolates were susceptible to only tigecycline. It was also found that six isolates (excluding five isolates of Proteus spp.) were resistant to both colistin and tigecycline. Thus, 101 (8.1%) out of 1240 isolates were XDR and 11 isolates (0.9%) were PDR. The findings of this study reveal increased burden of XDR and PDR Gram-negative bacteria in our centre. It also highlights the widespread dissemination of these bacteria in the community. This situation warrants the regular surveillance of antimicrobial resistance of Gram-negative bacteria and implementation of an efficient infection control program.

  9. The Line Blot: An Immunoassay for Monoclonal and Other Antibodies. Its Application to the Serotyping of Gram-Negative Bacteria

    DTIC Science & Technology

    1989-01-01

    Gram-negative J bacteria 12. PERSONAL A1JTIR(S Raoult D, UaschbA 13a. TYPE OF REPOR~T 13b. TIME COVERED 14.. DATE OF REPORT (Year, Month. Day) 15. P...the serotyping of Gram-negative bacteria Didier Raoult 1and Gregory A. Dasch2 Centre National de Reference des Rkckertsioses. C H. U. La Timone...antibodies which recognize heat-sensitive and insensitive epitopes on the typhus rickettsia surface protein antigen is described. A new serotvping assay

  10. Performance of the Verigene Gram-negative blood culture assay for rapid detection of bacteria and resistance determinants.

    PubMed

    Dodémont, Magali; De Mendonça, Ricardo; Nonhoff, Claire; Roisin, Sandrine; Denis, Olivier

    2014-08-01

    Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated.

  11. [Carbapenemases in gram-negative bacteria. Current data and trends of resistance resulting from the work of national reference centres].

    PubMed

    Kaase, M

    2012-11-01

    The spread of carbapenemase-producing gram-negative bacteria is one of the major challenges of the present. Since 2009, the National Reference Laboratory for gram-negative nosocomial pathogens has observed the molecular epidemiology of carbapenemases in Germany. In 2011, 1,454 referred bacterial isolates were tested for the presence of carbapenemases. Carbapenemase was found in 34.4% of Enterobacteriaceae isolates, in 19.9% of Pseudomonas aeruginosa isolates and in 96.3% of Acinetobacter baumannii isolates. The most frequent carbapenemases in Enterobacteriaceae were OXA-48, KPC and VIM-1; in P. aeruginosa it was VIM-2 and in A. baumannii OXA-23.

  12. Epidemiology and Management of Emerging Drug-Resistant Gram-Negative Bacteria: Extended-Spectrum β-Lactamases and Beyond.

    PubMed

    Boyle, Daniel P; Zembower, Teresa R

    2015-11-01

    Worldwide prevalence of antimicrobial resistance is rapidly increasing, primarily a result of antibiotic misuse in the medical community. Resistant infections involving the urinary tract are typically caused by gram-negative bacteria. When treating these infections, clinicians have few effective antimicrobials to choose from and many are associated with significant adverse effects. There are now situations when clinicians are tasked with managing infections from pan-resistant organisms; thus, it is of paramount importance that spread of resistance be controlled. This review discusses common gram-negative resistance classes, highlighting the mechanisms of resistance, risk factors, type of infections, treatment, and outcomes associated with each class.

  13. 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.

  14. Pediatric multicenter evaluation of the Verigene gram-negative blood culture test for rapid detection of inpatient bacteremia involving gram-negative organisms, extended-spectrum beta-lactamases, and carbapenemases.

    PubMed

    Sullivan, K V; Deburger, B; Roundtree, S S; Ventrola, C A; Blecker-Shelly, D L; Mortensen, J E

    2014-07-01

    We evaluated the investigational use only (IUO) version of the rapid Verigene Gram-negative blood culture test (BC-GN), a microarray that detects 9 genus/species targets (Acinetobacter spp., Citrobacter spp., Enterobacter spp., Escherichia coli/Shigella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens) and 6 antimicrobial resistance determinants (blaCTX-M, blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA) directly from positive blood cultures. BC-GN was performed on positive BacT/Alert Pediatric FAN and Bactec Peds Plus blood cultures with Gram-negative organisms at two tertiary pediatric centers. Vitek MS (bioMérieux, Durham, NC) was used to assign gold standard organism identification. The Check MDR CT-102 microarray (Check Points B.V., Wageningen, Netherlands) was used as an alternative method for detecting resistance determinants. In total, 104 organisms were isolated from 97 clinical blood cultures. BC-GN correctly detected 26/26 cultures with Acinetobacter spp., P. aeruginosa, and S. marcescens, 5/6 with Citrobacter spp., 13/14 with Enterobacter spp., 23/24 with E. coli, 2/3 with K. oxytoca, 16/17 with K. pneumoniae, and 0/1 with Proteus spp. BC-GN appropriately reported negative BC-GN results in 8/13 blood cultures that grew organisms that were not represented on the microarray but failed to detect targets in 3/5 cultures that grew multiple Gram-negative organisms. BC-GN detected 5/5 and 1/1 clinical blood cultures with blaCTX-M and blaVIM. All 6 results were corroborated by Check MDR CT-102 microarray testing. The Verigene BC-GN test has the potential to expedite therapeutic decision making in pediatric patients with Gram-negative bacteremia. Sensitivity was satisfactory but may be suboptimal in mixed Gram-negative blood cultures.

  15. Enhancement of Antibacterial Activity of Capped Silver Nanoparticles in Combination with Antibiotics, on Model Gram-Negative and Gram-Positive Bacteria

    PubMed Central

    Kora, Aruna Jyothi; Rastogi, Lori

    2013-01-01

    The nanoparticles used in this study were prepared from AgNO3 using NaBH4 in the presence of capping agents such as citrate, sodium dodecyl sulfate, and polyvinylpyrrolidone. The formed nanoparticles were characterized with UV-Vis, TEM, and XRD. The generation of silver nanoparticles was confirmed from the appearance of yellow colour and an absorption maximum between 399 and 404 nm. The produced nanoparticles were found to be spherical in shape and polydisperse. For citrate, SDS, and PVP capped nanoparticles, the average particle sizes were 38.3 ± 13.5, 19.3 ± 6.0, and 16.0 ± 4.8 nm, respectively. The crystallinity of the nanoparticles in FCC structure is confirmed from the SAED and XRD patterns. Also, the combined antibacterial activity of these differently capped nanoparticles with selected antibiotics (streptomycin, ampicillin, and tetracycline) was evaluated on model Gram-negative and Gram-positive bacteria, employing disc diffusion assay. The activity of the tested antibiotics was enhanced in combination with all the stabilized nanoparticles, against both the Gram classes of bacteria. The combined effects of silver nanoparticles and antibiotics were more prominent with PVP capped nanoparticles as compared to citrate and SDS capped ones. The results of this study demonstrate potential therapeutic applications of silver nanoparticles in combination with antibiotics. PMID:23970844

  16. Enhancement of antibacterial activity of capped silver nanoparticles in combination with antibiotics, on model gram-negative and gram-positive bacteria.

    PubMed

    Kora, Aruna Jyothi; Rastogi, Lori

    2013-01-01

    The nanoparticles used in this study were prepared from AgNO3 using NaBH4 in the presence of capping agents such as citrate, sodium dodecyl sulfate, and polyvinylpyrrolidone. The formed nanoparticles were characterized with UV-Vis, TEM, and XRD. The generation of silver nanoparticles was confirmed from the appearance of yellow colour and an absorption maximum between 399 and 404 nm. The produced nanoparticles were found to be spherical in shape and polydisperse. For citrate, SDS, and PVP capped nanoparticles, the average particle sizes were 38.3 ± 13.5, 19.3 ± 6.0, and 16.0 ± 4.8 nm, respectively. The crystallinity of the nanoparticles in FCC structure is confirmed from the SAED and XRD patterns. Also, the combined antibacterial activity of these differently capped nanoparticles with selected antibiotics (streptomycin, ampicillin, and tetracycline) was evaluated on model Gram-negative and Gram-positive bacteria, employing disc diffusion assay. The activity of the tested antibiotics was enhanced in combination with all the stabilized nanoparticles, against both the Gram classes of bacteria. The combined effects of silver nanoparticles and antibiotics were more prominent with PVP capped nanoparticles as compared to citrate and SDS capped ones. The results of this study demonstrate potential therapeutic applications of silver nanoparticles in combination with antibiotics.

  17. Mechanism of decreased susceptibility for Gram-negative bacteria and synergistic effect with ampicillin of indole-3-carbinol.

    PubMed

    Sung, Woo Sang; Lee, Dong Gun

    2008-09-01

    Indole-3-carbinol (I3C) is a natural compound found in a wide variety of plant food substances including members of the family Cruciferae with antioxidant and potential chemopreventive properties. In a previous study, I3C exhibited broad spectrum antibacterial activities. Particularly, it showed a more potent antibacterial activity against Gram-positive bacteria than Gram-negative bacteria. To elucidate this disparity of antibacterial activity between Gram-positive and Gram-negative bacteria, we investigated the actions of the efflux pumps and the lipopolysaccharide (LPS) barrier of the outer membrane of Gram-negative bacteria. The results showed that the antibacterial activity of I3C was affected by the barrier action of LPS in the outer membrane rather than by the efflux pumps. To assess its potential for combination therapy in treating bacterial infections, we investigated its synergy effects in combination with conventional antibiotics. The results demonstrated that I3C showed considerable synergistic activity in combination with ampicillin against drug-resistant isolates.

  18. Harpins, multifunctional proteins secreted by gram-negative plant-pathogenic bacteria.

    PubMed

    Choi, Min-Seon; Kim, Wooki; Lee, Chanhui; Oh, Chang-Sik

    2013-10-01

    Harpins are glycine-rich and heat-stable proteins that are secreted through type III secretion system in gram-negative plant-pathogenic bacteria. Many studies show that these proteins are mostly targeted to the extracellular space of plant tissues, unlike bacterial effector proteins that act inside the plant cells. Over the two decades since the first harpin of pathogen origin, HrpN of Erwinia amylovora, was reported in 1992 as a cell-free elicitor of hypersensitive response (HR), diverse functional aspects of harpins have been determined. Some harpins were shown to have virulence activity, probably because of their involvement in the translocation of effector proteins into plant cytoplasm. Based on this function, harpins are now considered to be translocators. Their abilities of pore formation in the artificial membrane, binding to lipid components, and oligomerization are consistent with this idea. When harpins are applied to plants directly or expressed in plant cells, these proteins trigger diverse beneficial responses such as induction of defense responses against diverse pathogens and insects and enhancement of plant growth. Therefore, in this review, we will summarize the functions of harpins as virulence factors (or translocators) of bacterial pathogens, elicitors of HR and immune responses, and plant growth enhancers.

  19. Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

    PubMed

    Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E

    2017-03-16

    Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml(-1), as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml(-1) cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera.

  20. Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates.

    PubMed

    Mohamed, Yasmine Fathy; Abou-Shleib, Hamida Moustafa; Khalil, Amal Mohamed; El-Guink, Nadia Mohamed; El-Nakeeb, Moustafa Ahmed

    2016-01-01

    Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.

  1. Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane

    PubMed Central

    Jordan, Lorne D.; Zhou, Yongyao; Smallwood, Chuck R.; Lill, Yoriko; Ritchie, Ken; Yip, Wai Tak; Newton, Salete M.; Klebba, Phillip E.

    2013-01-01

    Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB–ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism. PMID:23798405

  2. Mechanics of membrane bulging during cell-wall disruption in Gram-negative bacteria

    NASA Astrophysics Data System (ADS)

    Daly, Kristopher E.; Huang, Kerwyn Casey; Wingreen, Ned S.; Mukhopadhyay, Ranjan

    2011-04-01

    The bacterial cell wall is a network of sugar strands crosslinked by peptides that serve as the primary structure for bearing osmotic stress. Despite its importance in cellular survival, the robustness of the cell wall to network defects has been relatively unexplored. Treatment of the Gram-negative bacterium Escherichia coli with the antibiotic vancomycin, which disrupts the crosslinking of new material during growth, leads to the development of pronounced bulges and eventually of cell lysis. Here, we model the mechanics of the bulging of the cytoplasmic membrane through pores in the cell wall. We find that the membrane undergoes a transition between a nearly flat state and a spherical bulge at a critical pore radius of ~20 nm. This critical pore size is large compared to the typical distance between neighboring peptides and glycan strands, and hence pore size acts as a constraint on network integrity. We also discuss the general implications of our model to membrane deformations in eukaryotic blebbing and vesiculation in red blood cells.

  3. Antibacterial activities of selected edible plants extracts against multidrug-resistant Gram-negative bacteria

    PubMed Central

    2013-01-01

    Background In response to the propagation of bacteria resistant to many antibiotics also called multi-drug resistant (MDR) bacteria, the discovery of new and more efficient antibacterial agents is primordial. The present study was aimed at evaluating the antibacterial activities of seven Cameroonian dietary plants (Adansonia digitata, Aframomum alboviolaceum, Aframomum polyanthum, Anonidium. mannii, Hibiscus sabdarifa, Ocimum gratissimum and Tamarindus indica). Methods The phytochemical screening of the studied extracts was performed using described methods whilst the liquid broth micro dilution was used for all antimicrobial assays against 27 Gram-negative bacteria. Results The results of the phytochemical tests indicate that all tested extracts contained phenols and triterpenes, other classes of chemicals being selectively present. The studied extracts displayed various degrees of antibacterial activities. The extracts of A. digitata, H. sabdarifa, A. polyanthum, A. alboviolaceum and O. gratissimum showed the best spectra of activity, their inhibitory effects being recorded against 81.48%, 66.66%, 62.96%, 55.55%, and 55.55% of the 27 tested bacteria respectively. The extract of A. polyanthum was very active against E. aerogenes EA294 with the lowest recorded minimal inhibitory concentration (MIC) of 32 μg/ml. Conclusion The results of the present work provide useful baseline information for the potential use of the studied edible plants in the fight against both sensitive and MDR phenotypes. PMID:23837916

  4. β-Lactamase production in key gram-negative pathogen isolates from the Arabian Peninsula.

    PubMed

    Zowawi, Hosam M; Balkhy, Hanan H; Walsh, Timothy R; Paterson, David L

    2013-07-01

    SUMMARY Infections due to Gram-negative bacilli (GNB) are a leading cause of morbidity and mortality worldwide. The extent of antibiotic resistance in GNB in countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Kuwait, Qatar, Oman, and Bahrain, has not been previously reviewed. These countries share a high prevalence of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing GNB, most of which are associated with nosocomial infections. Well-known and widespread β-lactamases genes (such as those for CTX-M-15, OXA-48, and NDM-1) have found their way into isolates from the GCC states. However, less common and unique enzymes have also been identified. These include PER-7, GES-11, and PME-1. Several potential risk factors unique to the GCC states may have contributed to the emergence and spread of β-lactamases, including the unnecessary use of antibiotics and the large population of migrant workers, particularly from the Indian subcontinent. It is clear that active surveillance of antimicrobial resistance in the GCC states is urgently needed to address regional interventions that can contain the antimicrobial resistance issue.

  5. Single daily dose amikacin in paediatric patients with severe gram-negative infections.

    PubMed

    Trujillo, H; Robledo, J; Robledo, C; Espinal, D; Garces, G; Mejia, J; Restrepo, C; Restrepo, F; Mejia de Rodriguez, G I; Tamayo de Guitierrez, M C

    1991-05-01

    Twenty-five children with serious Gram-negative infections were treated in a prospective study with amikacin 20 mg/kg administered in a single daily dose as a 30 min iv infusion for 4 to 12 days. In nine cases the amikacin was combined with beta-lactam antibiotics. Escherichia coli were the most frequent bacteria isolated followed by K. pneumoniae, Providencia and Enterobacter spp. and Pseudomonas aeruginosa with MICs ranging from 1 to 16 mg/l. Mean (+/- S.D.) peak and trough concentrations of days 1 and 4 of therapy ranged from 49 +/- 13.5 to 53.6 +/- 13.4 mg/l and 6 + 1.4 to 7.7 +/- 4.1 mg/l respectively. All patients were clinically and bacteriologically cured. No significant adverse reactions were observed. The results suggest that administration of a single daily dose of 20 mg/kg amikacin should be considered practical and safe in children. Further studies are needed.

  6. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria.

    PubMed

    Li, Xian-Zhi; Plésiat, Patrick; Nikaido, Hiroshi

    2015-04-01

    The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

  7. Australian Group on Antimicrobial Resistance Community-onset Gram-negative Surveillance Program annual report, 2010.

    PubMed

    Turnidge, John D; Gottlieb, Thomas; Mitchell, David H; Coombs, Geoffrey W; Pearson, Julie C; Bell, Jan M

    2013-09-30

    The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2010 survey focussed on community-onset infections, examining isolates from urinary tract infections from patients presenting to outpatient clinics, emergency departments or to community practitioners. Two thousand and ninety-two Escherichia coli, 578 Klebsiella species and 268 Enterobacter species were tested using a commercial automated method (Vitek 2, BioMérieux) and results were analysed using Clinical and Laboratory Standards Institute breakpoints from January 2012. Of the key resistances, non-susceptibility to the third-generation cephalosporin, ceftriaxone, was found in 3.2% of E. coli and 3.2%-4.0% of Klebsiella spp. Non-susceptibility rates to ciprofloxacin were 5.4% for E. coli, 1.0%-2.3% for Klebsiella spp., and 2.5%-6.6% in Enterobacter spp, and resistance rates to piperacillin-tazobactam were 2.8%, 3.2%-6.9%, and 16.8%-18.0% for the same 3 groups respectively. Only 3 strains, 2 Klebsiella spp. and 1 Enterobacter spp, were shown to harbour a carbapenemase (IMP-4).

  8. Hospital-onset Gram-negative Surveillance Program annual report, 2011.

    PubMed

    Turnidge, John D; Gottlieb, Thomas; Mitchell, David H; Coombs, Geoffrey W; Pearson, Julie C; Bell, Jan M

    2014-03-31

    The Australian Group on Antimicrobial Resistance performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2011 survey focussed on hospital-onset infections, examining isolates from all specimens presumed to be causing disease. In 2011, 1,827 Escherichia coli, 537 Klebsiella species and 269 Enterobacter species were tested using a commercial automated method (Vitek 2, BioMérieux) and results were analysed using Clinical and Laboratory Standards Institute breakpoints from January 2012. Of the key resistances, non-susceptibilty to the third-generation cephalosporin, ceftriaxone, was found in 9.6% of E. coli and 9.5%-12.1% of Klebsiella spp. Non-susceptibility rates to ciprofloxacin were 10.6% for E. coli, 0.0%-8.3% for Klebsiella spp. and 0.0%-5.0% in Enterobacter spp. Resistance rates to gentamicin were 8.6%, 2.9%-10.9%, and 0.0%-15.6% for the same 3 groups respectively. Eight strains, 5 Klebsiella spp. and 3 Enterobacter spp. were shown to harbour a carbapenemase (IMP-4).

  9. Community-onset Gram-negative Surveillance Program annual report, 2012.

    PubMed

    Turnidge, John D; Gottlieb, Thomas; Mitchell, David H; Coombs, Geoffrey W; Daly, Denise A; Bell, Jan M

    2014-03-31

    The Australian Group on Antimicrobial Resistance performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2012 survey focussed on community-onset infections, examining isolates from urinary tract infections from patients presenting to outpatient clinics, emergency departments or to community practitioners. In 2012, 2,025 Escherichia coli, 538 Klebsiella species and 239 Enterobacter species were tested using a commercial automated method (Vitek 2, BioMérieux) and results were analysed using Clinical and Laboratory Standards Institute breakpoints from January 2012. Of the key resistances, non-susceptibility to the third-generation cephalosporin, ceftriaxone, was found in 4.2% of E. coli and 4.6%-6.9% of Klebsiella spp. Non-susceptibility rates to ciprofloxacin were 6.9% for E. coli, 0.0%-3.5% for Klebsiella spp. and 0.8%-1.9% in Enterobacter spp, and resistance rates to piperacillin-tazobactam were 1.7%, 0.7%-9.2%, and 8.8%-11.4% for the same 3 groups respectively. Only 1 Enterobacter cloacae was shown to harbour a carbapenemase (IMP-4).

  10. β-Lactamase Production in Key Gram-Negative Pathogen Isolates from the Arabian Peninsula

    PubMed Central

    Balkhy, Hanan H.; Walsh, Timothy R.; Paterson, David L.

    2013-01-01

    SUMMARY Infections due to Gram-negative bacilli (GNB) are a leading cause of morbidity and mortality worldwide. The extent of antibiotic resistance in GNB in countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Kuwait, Qatar, Oman, and Bahrain, has not been previously reviewed. These countries share a high prevalence of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing GNB, most of which are associated with nosocomial infections. Well-known and widespread β-lactamases genes (such as those for CTX-M-15, OXA-48, and NDM-1) have found their way into isolates from the GCC states. However, less common and unique enzymes have also been identified. These include PER-7, GES-11, and PME-1. Several potential risk factors unique to the GCC states may have contributed to the emergence and spread of β-lactamases, including the unnecessary use of antibiotics and the large population of migrant workers, particularly from the Indian subcontinent. It is clear that active surveillance of antimicrobial resistance in the GCC states is urgently needed to address regional interventions that can contain the antimicrobial resistance issue. PMID:23824364

  11. Coprinopsis cinerea intracellular lactonases hydrolyze quorum sensing molecules of Gram-negative bacteria.

    PubMed

    Stöckli, Martina; Lin, Chia-Wei; Sieber, Ramon; Plaza, David F; Ohm, Robin A; Künzler, Markus

    2016-07-27

    Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-β-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle.

  12. Toll-like receptor 4 decoy, TOY, attenuates gram-negative bacterial sepsis.

    PubMed

    Jung, Keehoon; Lee, Jung-Eun; Kim, Hak-Zoo; Kim, Ho Min; Park, Beom Seok; Hwang, Seong-Ik; Lee, Jie-Oh; Kim, Sun Chang; Koh, Gou Young

    2009-10-09

    Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using 'the Hybrid leucine-rich repeats (LRR) technique'. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-kappaB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins.

  13. Development of Quorum-Based Anti-Virulence Therapeutics Targeting Gram-Negative Bacterial Pathogens

    PubMed Central

    Tay, Song Buck; Yew, Wen Shan

    2013-01-01

    Quorum sensing is a cell density-dependent signaling phenomenon used by bacteria for coordination of population-wide phenotypes, such as expression of virulence genes, antibiotic resistance and biofilm formation. Lately, disruption of bacterial communication has emerged as an anti-virulence strategy with enormous therapeutic potential given the increasing incidences of drug resistance in pathogenic bacteria. The quorum quenching therapeutic approach promises a lower risk of resistance development, since interference with virulence generally does not affect the growth and fitness of the bacteria and, hence, does not exert an associated selection pressure for drug-resistant strains. With better understanding of bacterial communication networks and mechanisms, many quorum quenching methods have been developed against various clinically significant bacterial pathogens. In particular, Gram-negative bacteria are an important group of pathogens, because, collectively, they are responsible for the majority of hospital-acquired infections. Here, we discuss the current understanding of existing quorum sensing mechanisms and present important inhibitory strategies that have been developed against this group of pathogenic bacteria. PMID:23939429

  14. Antibacterial effect of ultrafine nanodiamond against gram-negative bacteria Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chatterjee, Anindita; Perevedentseva, Elena; Jani, Mona; Cheng, Chih-Yuan; Ye, Ying-Siou; Chung, Pei-Hua; Cheng, Chia-Liang

    2015-05-01

    We investigate the antibacterial effect of ultrafine nanodiamond particles with an average size of 5 nm against the gram-negative bacteria Escherichia coli (E. coli). UV-visible, Raman spectroscopy, and scanning electron microscopy (SEM) have been employed to elucidate the nature of the interaction. The influence on bacterial growth was monitored by measuring optical densities of E. coli at 600 nm as a function of time in the presence of carboxylated nanodiamond (cND) particles (100 μg/ml) in highly nutritious liquid Luria-Bertani medium. The SEM images prove that cND particles are attached to the bacterial cell wall surface and some portion of the bacterial cell wall undergoes destruction. Due to the change of the protein structure on the bacterial wall, a small Raman shift in the region of 1400 to 1700 cm-1 was observed when E. coli interacted with cNDs. Raman mapping images show strong evidence of cND attachment at the bacterial cell wall surface. Electrotransformation of E. coli with a fluorescent protein markers experiment demonstrated that the interaction mechanisms are different for E. coli treated with cND particles, E. coli by lysozyme treatment, and E. coli that suffer lysis.

  15. Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry

    PubMed Central

    Saint-Ruf, Claude; Crussard, Steve; Franceschi, Christine; Orenga, Sylvain; Ouattara, Jasmine; Ramjeet, Mahendrasingh; Surre, Jérémy; Matic, Ivan

    2016-01-01

    Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility. PMID:27507962

  16. A traceless reversible polymeric colistin prodrug to combat multidrug-resistant (MDR) gram-negative bacteria.

    PubMed

    Zhu, Chongyu; Schneider, Elena K; Wang, Jiping; Kempe, Kristian; Wilson, Paul; Velkov, Tony; Li, Jian; Davis, Thomas P; Whittaker, Michael R; Haddleton, David M

    2017-02-05

    Colistin methanesulfonate (CMS) is the only prodrug of colistin available for clinical use for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Owing to its slow and variable release, an alternative is urgently required to improve effectiveness. Herein we describe a PEGylated colistin prodrug whereby the PEG is attached via a cleavable linker (col-aaPEG) introducing an acetic acid terminated poly (ethylene glycol) methyl ether (aaPEG) onto the Thr residue of colistin. Due to the labile ester containing link, this prodrug is converted back into active colistin in vitro within 24h. Compared to CMS, it showed a similar or better antimicrobial performance against two MDR isolates of Pseudomonas aeruginosa and Acinetobacter baumannii through in vitro disk diffusion, broth dilution and time-kill studies. In a mouse infection model, col-aaPEG displayed acceptable bacterial killing against P. aeruginosa ATCC 27853 and no nephrotoxicity was found after systemic administration, suggesting it to be a potential alternative for CMS.

  17. Forming cross-linked peptidoglycan from synthetic gram-negative Lipid II.

    PubMed

    Lebar, Matthew D; Lupoli, Tania J; Tsukamoto, Hirokazu; May, Janine M; Walker, Suzanne; Kahne, Daniel

    2013-03-27

    The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro. Comparison of PG produced from synthetic canonical E. coli Lipid II with PG isolated from E. coli cells demonstrates that we can produce PG in vitro that resembles native structure. This work provides the tools necessary for reconstituting cell wall synthesis, an essential cellular process and major antibiotic target, in a purified system.

  18. Infection-related hemolysis and susceptibility to Gram-negative bacterial co-infection

    PubMed Central

    Orf, Katharine; Cunnington, Aubrey J.

    2015-01-01

    Increased susceptibility to co-infection with enteric Gram-negative bacteria, particularly non-typhoidal Salmonella, is reported in malaria and Oroya fever (Bartonella bacilliformis infection), and can lead to increased mortality. Accumulating epidemiological evidence indicates a causal association with risk of bacterial co-infection, rather than just co-incidence of common risk factors. Both malaria and Oroya fever are characterized by hemolysis, and observations in humans and animal models suggest that hemolysis causes the susceptibility to bacterial co-infection. Evidence from animal models implicates hemolysis in the impairment of a variety of host defense mechanisms, including macrophage dysfunction, neutrophil dysfunction, and impairment of adaptive immune responses. One mechanism supported by evidence from animal models and human data, is the induction of heme oxygenase-1 in bone marrow, which impairs the ability of developing neutrophils to mount a competent oxidative burst. As a result, dysfunctional neutrophils become a new niche for replication of intracellular bacteria. Here we critically appraise and summarize the key evidence for mechanisms which may contribute to these very specific combinations of co-infections, and propose interventions to ameliorate this risk. PMID:26175727

  19. Resistant gram-negative infections in the outpatient setting in Latin America.

    PubMed

    Salles, M J C; Zurita, J; Mejía, C; Villegas, M V

    2013-12-01

    Latin America has a high rate of community-associated infections caused by multidrug-resistant Enterobacteriaceae relative to other world regions. A review of the literature over the last 10 years indicates that urinary tract infections (UTIs) by Escherichia coli, and intra-abdominal infections (IAIs) by E. coli and Klebsiella pneumoniae, were characterized by high rates of resistance to trimethoprim/sulfamethoxazole, quinolones, and second-generation cephalosporins, and by low levels of resistance to aminoglycosides, nitrofurantoin, and fosfomycin. In addition, preliminary data indicate an increase in IAIs by Enterobacteriaceae producing extended-spectrum β-lactamases, with reduced susceptibilities to third- and fourth-generation cephalosporins. Primary-care physicians in Latin America should recognize the public health threat associated with UTIs and IAIs by resistant Gram-negative bacteria. As the number of therapeutic options become limited, we recommend that antimicrobial prescribing be guided by infection severity, established patient risk factors for multidrug-resistant infections, acquaintance with local antimicrobial susceptibility data, and culture collection.

  20. Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus

    PubMed Central

    2005-01-01

    The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 Å (1 Å=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed β-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339–Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction. PMID:15869470

  1. Synthesis of a polymyxin derivative for photolabeling studies in the gram-negative bacterium Escherichia coli.

    PubMed

    van der Meijden, Benjamin; Robinson, John A

    2015-03-01

    The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics.

  2. The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

    PubMed Central

    Plésiat, Patrick

    2015-01-01

    SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps. PMID:25788514

  3. Enteric Gram-negative bacilli suppress Candida biofilms on Foley urinary catheters.

    PubMed

    Samaranayake, Y H; Bandara, H M H N; Cheung, B P K; Yau, J Y Y; Yeung, S K W; Samaranayake, L P

    2014-01-01

    Mixed Candida-bacterial biofilms in urinary catheters are common in hospitalized patients. (i) The aims of this study were to evaluate, quantitatively and qualitatively, the in vitro development of mono- and dual-species biofilms (MSBs and DSBs) of Candida albicans and two enteric gram-negative bacilli (EGNB; Pseudomonas aeruginosa or Escherichia coli) on Foley catheter (FC) discs, (ii) to determine the biofilm growth in tryptic soy broth or glucose supplemented artificial urine (AU) and (iii) to assess the inhibitory effects of EGNB and their lipopolysaccharides (LPS) on Candida biofilm growth. The growth of MSBs and DSBs on FC discs was monitored by cell counts and SEM. The metabolic activity of LPS-treated Candida biofilms was determined by the XTT reduction assay. Candida albicans and EGNB demonstrated significant inter- and intra-species differences in biofilm growth on FC discs (p < 0.01). Pseudomonas aeruginosa suppressed Candida albicans significantly (p < 0.001) in DSBs. Compared with MSBs, DSB of EGNB in glucose supplemented AU demonstrated robust growth. Escherichia coli and its LPS, significantly suppressed Candida biofilm growth, compared with Pseudomonas aeruginosa and its LPS (p < 0.001). Candida albicans and EGNB colonization in FC is significantly increased in AU with glucose, and variably modified by Escherichia coli, Pseudomonas aeruginosa and their corresponding LPS.

  4. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed

    Mulbry, W W; Karns, J S

    1989-02-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

  5. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies. Images PMID:2541658

  6. Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane.

    PubMed

    Jordan, Lorne D; Zhou, Yongyao; Smallwood, Chuck R; Lill, Yoriko; Ritchie, Ken; Yip, Wai Tak; Newton, Salete M; Klebba, Phillip E

    2013-07-09

    Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.

  7. In vitro antimicrobial activity of five essential oils on multidrug resistant Gram-negative clinical isolates

    PubMed Central

    Sakkas, Hercules; Gousia, Panagiota; Economou, Vangelis; Sakkas, Vassilios; Petsios, Stefanos; Papadopoulou, Chrissanthy

    2016-01-01

    Aim/Background: The emergence of drug-resistant pathogens has drawn attention on medicinal plants for potential antimicrobial properties. The objective of the present study was the investigation of the antimicrobial activity of five plant essential oils on multidrug resistant Gram-negative bacteria. Materials and Methods: Basil, chamomile blue, origanum, thyme, and tea tree oil were tested against clinical isolates of Acinetobacter baumannii (n = 6), Escherichia coli (n = 4), Klebsiella pneumoniae (n = 7), and Pseudomonas aeruginosa (n = 5) using the broth macrodilution method. Results: The tested essential oils produced variable antibacterial effect, while Chamomile blue oil demonstrated no antibacterial activity. Origanum, Thyme, and Basil oils were ineffective on P. aeruginosa isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration values ranged from 0.12% to 1.50% (v/v) for tea tree oil, 0.25-4% (v/v) for origanum and thyme oil, 0.50% to >4% for basil oil and >4% for chamomile blue oil. Compared to literature data on reference strains, the reported MIC values were different by 2SD, denoting less successful antimicrobial activity against multidrug resistant isolates. Conclusions: The antimicrobial activities of the essential oils are influenced by the strain origin (wild, reference, drug sensitive, or resistant) and it should be taken into consideration whenever investigating the plants’ potential for developing new antimicrobials. PMID:27366345

  8. Molecular basis of active copper resistance mechanisms in Gram-negative bacteria.

    PubMed

    Bondarczuk, Kinga; Piotrowska-Seget, Zofia

    2013-12-01

    Copper is a metallic element that is crucial for cell metabolism; however, in extended concentrations, it is toxic for all living organisms. The dual nature of copper has forced organisms, including bacteria, to keep a tight hold on cellular copper content. This challenge has led to the evolution of complex mechanisms that on one hand enable them to deliver the essential element and on the other to protect cells against its toxicity. Such mechanisms have been found in both eukaryotic and prokaryotic cells. In bacteria a number of different systems such as extra- and intracellular sequestration, enzymatic detoxification, and metal removal from the cell enabling them to survive in the presence of high concentration of copper have been identified. Gram-negative bacteria, due to their additional compartment, need to deal with both cytoplasmic and periplasmic copper. Therefore, these bacteria have evolved intricate and precisely regulated systems which interact with each other. In this review the active mechanisms of copper resistance at their molecular level are discussed.

  9. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

    PubMed Central

    McBroom, Amanda J; Kuehn, Meta J

    2007-01-01

    Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism. PMID:17163978

  10. Low-dose polymyxin: an option for therapy of Gram-negative sepsis

    PubMed Central

    Gabor, Franz; Hartmann, Jens

    2016-01-01

    Endotoxins are the major components of the outer membrane of most Gram-negative bacteria and are one of the main targets in inflammatory diseases. The presence of endotoxins in blood can provoke septic shock in case of pronounced immune response. Here we show in vitro inactivation of endotoxins by polymyxin B (PMB). The inflammatory activity of the LPS–PMB complex in blood was examined in vitro in freshly drawn blood samples. Plasma protein binding of PMB was determined by ultracentrifugation using membranes with different molecular cut-offs, and PMB clearance during dialysis was calculated after in vitro experiments using the AV1000S filter. The formed LPS–PMB complex has lower inflammatory activity in blood, which results in highly reduced cytokine secretion. According to in vitro measurements, the appropriate plasma level of PMB for LPS inactivation is between 100 and 200 ng/ml. Furthermore, the combination of cytokine removal by adsorbent treatment with LPS inactivation by PMB dosage leads to strong suppression of inflammatory effects in blood in an in vitro model. Inactivation of endotoxins by low-dose intravenous PMB infusion or infusion into the extracorporeal circuit during blood purification can be applied to overcome the urgent need for endotoxin elimination not only in treatment of sepsis, but also in liver failure. PMID:26993088

  11. Tetracycline improved the efficiency of other antimicrobials against Gram-negative multidrug-resistant bacteria.

    PubMed

    Mawabo, Isabelle K; Noumedem, Jaurès A K; Kuiate, Jules R; Kuete, Victor

    2015-01-01

    Treatment of infectious diseases with antimicrobials constituted a great achievement in the history of medicine. Unfortunately, the emergence of resistant strains of bacteria to all classes of antimicrobials limited their efficacy. The present study was aimed at evaluating the effect of combinations of antibiotics on multi-drug resistant Gram-negative (MDRGN) bacteria. A liquid micro-broth dilution method was used to evaluate the antibacterial activity of 10 different classes of antimicrobials on 20 bacterial strains belonging to six different species. The antimicrobials were associated with phenylalanine β-naphthylamide (PAβN), an efflux pump inhibitor, and with other antimicrobials at their sub-inhibitory concentrations. The effectiveness of each combination was monitored using the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC). Most of the antimicrobials tested showed low antibacterial activity with a MIC value of 128 mg/L on a majority of the bacterial strains, justifying their multidrug-resistant (MDR) profile. Synergistic effects were mostly observed (FIC≤0.5) when ampicillin (AMP), cloxacillin (CLX), erythromycin (ERY), chloramphenicol (CHL), kanamycin (KAN) and streptomycin (STR) were combined with tetracycline (TET) at the sub-inhibitory concentration of MIC/5 or MIC/10. The results of the present work suggest that the association of several antimicrobials with TET could improve the fight against MDRGN bacterial species.

  12. Computational prediction of type III and IV secreted effectors in Gram-negative bacteria

    SciTech Connect

    McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.; Oehmen, Christopher S.; Niemann, George; Cambronne, Eric; Sharp, Danna; Adkins, Joshua N.; Samudrala, Ram; Heffron, Fred

    2011-01-01

    In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

  13. Antibacterial effect of ultrafine nanodiamond against gram-negative bacteria Escherichia coli.

    PubMed

    Chatterjee, Anindita; Perevedentseva, Elena; Jani, Mona; Cheng, Chih-Yuan; Ye, Ying-Siou; Chung, Pei-Hua; Cheng, Chia-Liang

    2015-05-01

    We investigate the antibacterial effect of ultrafine nanodiamond particles with an average size of 5 nm against the gram-negative bacteria Escherichia coli (E. coli). UV-visible, Raman spectroscopy, and scanning electron microscopy (SEM) have been employed to elucidate the nature of the interaction. The influence on bacterial growth was monitored by measuring optical densities of E. coli at 600 nm as a function of time in the presence of carboxylated nanodiamond (cND) particles (100 μg/ml ) in highly nutritious liquid Luria-Bertani medium. The SEM images prove that cND particles are attached to the bacterial cell wall surface and some portion of the bacterial cell wall undergoes destruction. Due to the change of the protein structure on the bacterial wall, a small Raman shift in the region of 1400 to 1700 cm⁻¹ was observed when E. coli interacted with cNDs. Raman mapping images show strong evidence of cND attachment at the bacterial cell wall surface. Electrotransformation of E. coli with a fluorescent protein markers experiment demonstrated that the interaction mechanisms are different for E. coli treated with cND particles, E. coli by lysozyme treatment, and E. coli that suffer lysis.

  14. Plasmid-mediated transfer of the bla(NDM-1) gene in Gram-negative rods.

    PubMed

    Potron, Anaïs; Poirel, Laurent; Nordmann, Patrice

    2011-11-01

    The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase NDM-1 (New Delhi metallo-β-lactamase) producers, mostly in Enterobacteriacae. Five bla(NDM) (-1) -positive plasmids of different incompatibility groups (IncL/M, FII, A/C and two untypeable plasmids) from clinical Enterobacteriaceae were evaluated for conjugation properties and host specificity. Successful conjugative transfers were obtained using all tested enterobacterial species as recipients (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis) and all plasmid types. Conjugation frequencies varied from 1 × 10(-4) to 6 × 10(-8) transconjugants per donor. Higher conjugation rates were obtained for two plasmids at 30 °C compared with that observed at 25 and 37 °C. Carbapenems used as selector did not lead to higher conjugation frequencies. None of the five plasmids was transferable to Acinetobacter baumannii or Pseudomonas aeruginosa by conjugation. This work underlines how efficient the spread of the carbapenemase bla(NDM) (-1) gene could be among Enterobacteriaceae.

  15. Evaluation of brilliance CRE agar for the detection of carbapenem-resistant gram-negative bacteria.

    PubMed

    Bracco, Silvia; Migliavacca, Roberta; Pini, Beatrice; Corbo, Nicoletta; Nucleo, Elisabetta; Brigante, Gioconda; Piazza, Aurora; Micheletti, Piero; Luzzaro, Francesco

    2013-04-01

    The aim of this work was to evaluate the performance of the new chromogenic medium BrillianceTM CREAgar (Thermo Fisher Scientific) for determining the limit of detection of carbapenem-resistant enterobacteria (CRE). A total of 70 clinical isolates were studied. Of these, 30 were well-characterized CRE, including Klebsiella pneumoniae strains producing KPC-, VIM-, and OXA-type enzymes, VIM-positive Enterobacter cloacae and Escherichia coli, NDM-positive E. coli, and enterobacterial isolates characterized by porin loss associated with ESBL production or AmpC hyperproduction. Ten carbapenem-resistant non-fermentative isolates were also included as well as 30 carbapenem-susceptible isolates. Carbapenem-resistant strains were inoculated at three different concentrations onto Brilliance CRE Agar (from 1.5x101 CFU/ml up to 1.5x104 CFU/ml) whereas carbapenem-susceptible isolates were inoculated at a concentration of 1.5x102 CFU/ml. The medium sustained the growth of carbapenem-resistant isolates, showing detection limits from 1.5x101 CFU/ml (in 31/40 cases) to 1.5x104 CFU/ml. No growth was observed with carbapenem-sensitive control strains. Our results indicate that the Brilliance CRE Agar allows the growth of carbapenem-resistant isolates with low detection limits and could represent a useful screening medium for both enterobacteria and non-fermentative Gram-negative strains resistant to carbapenems.

  16. Autotransporter-based cell surface display in Gram-negative bacteria.

    PubMed

    Nicolay, Toon; Vanderleyden, Jos; Spaepen, Stijn

    2015-02-01

    Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

  17. Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

    PubMed Central

    Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S.; Le Brun, Anton P.; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H.

    2016-01-01

    The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion. PMID:27493217

  18. Clinical Response and Outcome in Patients with Multidrug Resistant Gram-negative Infections

    PubMed Central

    Malekolkottab, Masoume; Shojaei, Lida; Khalili, Hossein; Doomanlou, Mahsa

    2017-01-01

    Objective: In this study, frequency and antimicrobial sensitivity pattern of multidrug resistant (MDR) microorganisms were evaluated in a referral teaching hospital in Iran. Methods: Patients with MDR Gram-negative pathogens were followed during the course of hospitalization. Demographic data, baseline diseases, type of biological sample, isolated microorganism, type of infection, antibiotic regimen before the availability of the culture result and change in the antibiotic regimen following receiving the antibiogram results, response to the treatment regimen, and duration of hospitalization and patient's outcome were considered variables for each recruited patient. Findings: In 71% of the patients, antibiotic regimens were changed according to the antibiogram results. A carbapenem alone or plus amikacin or ciprofloxacin were selected regimens for patients with extended-spectrum beta-lactamase (ESBL) infections. For patients with probable carbapenem-resistant Enterobacteriaceae infections, a carbapenem plus colistin was the most common antibiotic regimen. Clinical response was detected in 54.5% of the patients who were treated based on the antibiogram results. Clinical response was higher in the ESBL producers (ESBL-P) than the non-ESBL-P infections (75% vs. 52%). However, this difference was not significant (P = 0.09). Most nonresponders (80%) had sepsis due to Klebsiella species. Finally, 41.9% of the patients were discharged from the hospital and 58.2% died. Conclusion: Same as other countries, infections due MDR microorganisms is increasing in the recent years. This type of resistance caused poor clinical response and high rate mortality in the patients. PMID:28331866

  19. The molecular basis of β-lactamase production in Gram-negative bacteria from Saudi Arabia.

    PubMed

    Yezli, Saber; Shibl, Atef M; Memish, Ziad A

    2015-02-01

    Resistance to β-lactams among Gram-negative bacteria is a worldwide issue. Increased prevalence of extended-spectrum β-lactamase (ESBL)-producers and the dissemination of carbapenem-resistance genes are particularly concerning. ESBL-producing strains are common in the Kingdom of Saudi Arabia, particularly among the Enterobacteriaceae, and carbapenem resistance is on the increase, especially among the non-fermenters. β-lactamase production is a major mechanism of resistance to these agents and although β-lactamase-producing strains have been documented in the Kingdom, relatively few reports characterized the molecular basis of this production. Nevertheless, available data suggest that CTX-M (CTX-M-15 in particular) is the predominant ESBL in the Enterobacteriaceae, with SHV also being prevalent in Klebsiella pneumoniae. Carbapenem resistance in the latter is mainly due to OXA-48 and NDM-1. In Pseudomonas aeruginosa, VEB-like enzymes are the most common ESBLs, and VIM is the prevalent metallo-β-lactamase. OXA-10 extended-spectrum enzymes are also frequent. PER and GES ESBLs have been reported in Acinetobacter baumannii, and oxacillinases (OXA-23 in particular) are the dominant carbapanamases in this species.

  20. Heavy-metal resistance in Gram-negative bacteria isolated from Kongsfjord, Arctic.

    PubMed

    Neethu, C S; Mujeeb Rahiman, K M; Saramma, A V; Mohamed Hatha, A A

    2015-06-01

    Isolation and characterization of heterotrophic Gram-negative bacteria was carried out from the sediment and water samples collected from Kongsfjord, Arctic. In this study, the potential of Arctic bacteria to tolerate heavy metals that are of ecological significance to the Arctic (selenium (Se), mercury (Hg), cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn)) was investigated. Quantitative assay of 130 isolates by means of plate diffusion and tube dilution methods was carried out by incorporation of different concentrations of metals. Growth in Se and Pb at a concentration of 3000 μg/L was significantly lower (P≤0.0001) than at 2000 μg/L. The minimum inhibitory concentration for Cd and Hg was 50 μg/L (P≤0.0001, F=264.23 and P≤0.0001, F=291.08, respectively) even though in the tube dilution test, Hg-containing tubes showed much less growth, revealing its superior toxicity to Cd. Thus, the level of toxicity of heavy metals was found to be in the order of Hg>Cd>Cu>Zn>Pb>Se. Multiple-metal-resistant isolates were investigated for their resistance against antibiotics, and a positive correlation was observed between antibiotic and metal resistance for all the isolates tested. The resistant organisms thus observed might influence the organic and inorganic cycles in the Arctic and affect the ecosystem.

  1. [News of antibiotic resistance among Gram-negative bacilli in Algeria].

    PubMed

    Baba Ahmed-Kazi Tani, Z; Arlet, G

    2014-06-01

    Antibiotic resistance has become a major public health problem in Algeria. Indeed the past decade, we have seen a significant increase in resistance to antibiotics especially in Gram-negative bacilli. Resistance to β-lactams in enterobacteria is dominated by the production of ESBL CTX-M-3 and CTX-M-15. The strains producing these enzymes are often the cause of potentially serious infections in both hospital and community settings. Identified plasmid cephalosporinases are CMY-2, CMY-12 and DHA-1. The isolation of strains of Enterobacteriaceae and Pseudomonas aeruginosa producing carbapenemases is rare in Algeria. Some Enterobacteriaceae producing OXA-48 or VIM-19 have been reported; so far, only VIM-2 has been identified in P. aeruginosa. However, the situation regarding the strains of Acinetobacter baumannii resistant to carbapenemases seems to be more disturbing. The carbapenemase OXA-23 is the most common and seems to be endemic in the north. The carbapenemase NDM-1 has also been identified. Resistance to aminoglycosides is marked by the identification armA gene associated with blaCTX-M genes in strains of Salmonella sp. Several other resistance genes have been identified sporadically in strains of Enterobacteriaceae, P. aeruginosa and A. baumannii. Resistance genes to fluoroquinolones are more recent identification in Algeria. The most common are the Qnr determinants followed by the bifunctional enzyme AAC[6']-Ib-cr. Resistance to sulfonamides and trimethoprim was also reported in Enterobacteriaceae strains in the west of the country.

  2. Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods.

    PubMed

    Schulthess, Bettina; Bloemberg, Guido V; Zbinden, Andrea; Mouttet, Forouhar; Zbinden, Reinhard; Böttger, Erik C; Hombach, Michael

    2016-03-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.

  3. 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

  4. Nasopharyngeal carriage of Klebsiella pneumoniae and other Gram-negative bacilli in pneumonia-prone age groups in Semarang, Indonesia.

    PubMed

    Farida, Helmia; Severin, Juliëtte A; Gasem, M Hussein; Keuter, Monique; van den Broek, Peterhans; Hermans, Peter W M; Wahyono, Hendro; Verbrugh, Henri A

    2013-05-01

    Gram-negative bacilli (GNB) cause many cases of pneumonia in Indonesia. We investigated nasopharyngeal carriage of GNB in Semarang, Indonesia. Klebsiella pneumoniae carriage in adults (15%) was higher than in children (7%) (P = 0.004), while that of other GNB was comparable. Poor food and water hygiene are determinants of carriage of these bacteria.

  5. Immunogenomics for identification of disease resistance genes in pigs: a review focusing on Gram-negative bacilli

    PubMed Central

    2012-01-01

    Over the past years, infectious disease has caused enormous economic loss in pig industry. Among the pathogens, gram negative bacteria not only cause inflammation, but also cause different diseases and make the pigs more susceptible to virus infection. Vaccination, medication and elimination of sick pigs are major strategies of controlling disease. Genetic methods, such as selection of disease resistance in the pig, have not been widely used. Recently, the completion of the porcine whole genome sequencing has provided powerful tools to identify the genome regions that harboring genes controlling disease or immunity. Immunogenomics, which combines DNA variations, transcriptome, immune response, and QTL mapping data to illustrate the interactions between pathogen and host immune system, will be an effective genomics tool for identification of disease resistance genes in pigs. These genes will be potential targets for disease resistance in breeding programs. This paper reviewed the progress of disease resistance study in the pig focusing on Gram-negative bacilli. Major porcine Gram-negative bacilli and diseases, suggested candidate genes/pathways against porcine Gram-negative bacilli, and distributions of QTLs for immune capacity on pig chromosomes were summarized. Some tools for immunogenomics research were described. We conclude that integration of sequencing, whole genome associations, functional genomics studies, and immune response information is necessary to illustrate molecular mechanisms and key genes in disease resistance. PMID:23137309

  6. Direct detection of lipid A on intact Gram-negative bacteria by MALDI-TOF mass spectrometry

    PubMed Central

    Larrouy-Maumus, Gerald; Clements, Abigail; Filloux, Alain; McCarthy, Ronan R.; Mostowy, Serge

    2016-01-01

    The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry. PMID:26656001

  7. Novel touchdown-PCR method for the detection of putrescine producing gram-negative bacteria in food products.

    PubMed

    Wunderlichová, Leona; Buňková, Leona; Koutný, Marek; Valenta, Tomáš; Buňka, František

    2013-06-01

    Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99-91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.

  8. Discovery of a novel class of boron-based antibacterials with activity against gram-negative bacteria.

    PubMed

    Hernandez, Vincent; Crépin, Thibaut; Palencia, Andrés; Cusack, Stephen; Akama, Tsutomu; Baker, Stephen J; Bu, Wei; Feng, Lisa; Freund, Yvonne R; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Mao, Weimin; Rock, Fernando L; Sexton, Holly; Sheoran, Anita; Zhang, Yanchen; Zhang, Yong-Kang; Zhou, Yasheen; Nieman, James A; Anugula, Mahipal Reddy; Keramane, El Mehdi; Savariraj, Kingsley; Reddy, D Shekhar; Sharma, Rashmi; Subedi, Rajendra; Singh, Rajeshwar; O'Leary, Ann; Simon, Nerissa L; De Marsh, Peter L; Mushtaq, Shazad; Warner, Marina; Livermore, David M; Alley, M R K; Plattner, Jacob J

    2013-03-01

    Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

  9. Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples.

    PubMed

    Campana, Eloiza H; Chuster, Stephanie G; da Silva, Isadora R; Paschoal, Raphael P; Bonelli, Raquel R; Moreira, Beatriz M; Picão, Renata C

    2016-12-07

    The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false-positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

  10. Red blood cell distribution width is an independent predictor of mortality in patients with gram-negative bacteremia.

    PubMed

    Ku, Nam Su; Kim, Hye-Won; Oh, Hyung Jung; Kim, Yong Chan; Kim, Min Hyung; Song, Je Eun; Oh, Dong Hyun; Ahn, Jin Young; Kim, Sun Bean; Jeong, Su Jin; Han, Sang Hoon; Kim, Chang Oh; Song, Young Goo; Kim, June Myung; Choi, Jun Yong

    2012-08-01

    Red blood cell distribution width (RDW) is known to be a predictor of severe morbidity and mortality in some chronic diseases such as congestive heart failure. However, to our knowledge, little is known about RDW as a predictor of mortality in patients with Gram-negative bacteremia, a major nosocomial cause of intra-abdominal infections, urinary tract infections, and primary bacteremia. Therefore, we investigated whether RDW is an independent predictor of mortality in patients with Gram-negative bacteremia. Clinical characteristics, laboratory parameters, and outcomes of 161 patients with Gram-negative bacteremia from November 2010 to March 2011 diagnosed at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, were retrospectively analyzed. The main outcome measure was 28-day all-cause mortality. The 28-day mortality rate was significantly higher in the increased RDW group compared with the normal RDW group (P < 0.001). According to multivariate Cox proportional hazard analysis, RDW levels at the onset of bacteremia (per 1% increase, P = 0.036), the Charlson index (per 1-point increase, P < 0.001), and the Sequential Organ Failure Assessment score (per 1-point increase, P = 0.001) were independent risk factors for 28-day mortality. Moreover, the nonsurvivor group had significantly higher RDW levels 72 h after the onset of bacteremia than did the survivor group (P = 0.001). In addition, the area under the curve of RDW at the onset of bacteremia, the 72-h RDW, and the Sequential Organ Failure Assessment score for 28-day mortality were 0.764 (P = 0.001), 0.802 (P < 0.001), and 0.703 (P = 0.008), respectively. Red blood cell distribution width at the onset of bacteremia was an independent predictor of mortality in patients with Gram-negative bacteremia. Also, 72-h RDW could be a predictor for all-cause mortality in patients with Gram-negative bacteremia.

  11. Evaluation of clinical outcomes in patients with Gram-negative bloodstream infections according to cefepime MIC.

    PubMed

    Rhodes, Nathaniel J; Liu, Jiajun; McLaughlin, Milena M; Qi, Chao; Scheetz, Marc H

    2015-06-01

    Predicted and observed failures at higher cefepime MICs have prompted the Clinical and Laboratories Standards Institute (CLSI) to lower the susceptible breakpoint for Enterobacteriaceae to ≤2mg/L, with dose-dependent susceptibility at 4-8mg/L, while the susceptibility breakpoint for nonfermentative organisms remain unchanged at ≥8mg/L. The contribution of increasing cefepime MIC to mortality risk in the setting of aggressive cefepime dosing is not well defined. Patients who were treated with cefepime for Gram-negative blood stream infections (GNBSIs), including both Enterobacteriaceae and nonfermentative organisms, were screened for inclusion in this retrospective cohort study. Demographic and microbiologic variables were collected, including pathogen, cefepime MIC, dosage, and interval. The objective was to define a risk-adjusted mortality breakpoint for cefepime MICs. Secondarily, we looked at time to death and length of stay (LOS) postculture. Ninety-one patients were included in the analysis. Overall, 19 patients died and 72 survived. Classification and Regression Tree analysis identified an inhospital mortality breakpoint at a cefepime MIC between 2 and 4mg/L for patients with a modified Acute Physiology and Chronic Health Evaluation II score ≤16.5 (4.2% versus 25%, respectively). Multivariate logistic regression revealed increased odds of mortality at a cefepime MIC of 4mg/L (adjusted odds ratio [aOR] 6.47; 95% confidence interval [CI] 1.25-33.4) and 64mg/L (aOR 6.54, 95% CI 1.03-41.4). Those with cefepime MICs ≥4mg/L experienced a greater median intensive care unit LOS for survivors (16 versus 2days; P=0.026). Increasing cefepime MIC appears to predict inhospital mortality among patients who received aggressive doses of cefepime for GNBSIs, supporting a clinical breakpoint MIC of 2mg/L.

  12. Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods

    PubMed Central

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

    2015-01-01

    Matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates. PMID:26659214

  13. Use of glycol ethers for selective release of periplasmic proteins from Gram-negative bacteria.

    PubMed

    Allen, Jeffrey R; Patkar, Anant Y; Frank, Timothy C; Donate, Felipe A; Chiu, Yuk Chun; Shields, Jefry E; Gustafson, Mark E

    2007-01-01

    Genetic modification of Gram-negative bacteria to express a desired protein within the cell's periplasmic space, located between the inner cytoplasmic membrane and the outer cell wall, can offer an attractive strategy for commercial production of therapeutic proteins and industrial enzymes. In certain applications, the product expression rate is high, and the ability to isolate the product from the cell mass is greatly enhanced because of the product's compartmentalized location within the cell. Protein release methods that increase the permeability of the outer cell wall for primary recovery, but avoid rupturing the inner cell membrane, reduce contamination of the recovered product with other host cell components and simplify final purification. This article reports representative data for a new release method employing glycol ether solvents. The example involves the use of 2-butoxyethanol (commonly called ethylene glycol n-butyl ether or EB) for selective release of a proprietary biopharmaceutical protein produced in the periplasmic space of Pseudomonas fluorescens. In this example, glycol ether treatment yielded approximately 65% primary recovery with approximately 80% purity on a protein-only basis. Compared with other methods including heat treatment, osmotic shock, and the use of surfactants, the glycol ether treatment yielded significantly reduced concentrations of other host cell proteins, lipopolysaccharide endotoxin, and DNA in the recovered protein solution. The use of glycol ethers also allowed exploitation of temperature-change-induced phase splitting behavior to concentrate the desired product. Heating the aqueous EB extract solution to 55 degrees C formed two liquid phases: a glycol ether-rich phase and an aqueous product phase containing the great majority of the product protein. This phase-splitting step yielded an approximate 10-fold reduction in the volume of the initial product solution and a corresponding increase in the product's concentration.

  14. Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria.

    PubMed

    Lauber, Frédéric; Cornelis, Guy Richard; Renzi, Francesco

    2016-10-25

    Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2 We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane.

  15. T lymphocyte responses to antigens of gram-negative bacteria in pyelonephritis.

    PubMed

    Wilz, S W; Kurnick, J T; Pandolfi, F; Rubin, R H; Warren, H S; Goldstein, R; Kersten, C M; McCluskey, R T

    1993-10-01

    We showed previously that large numbers of T lymphocytes accumulate within a few days in the kidneys of rats with ascending pyelonephritis induced with Escherichia coli or Pseudomonas aeruginosa. CD4+ T cells propagated from the lesions exhibited MHC-restricted proliferative responses to formalin-fixed bacteria of the species used to induce infection. In the present study we investigated further the nature of the antigens responsible for the T cell proliferation and studied the ability of different bacterial strains and species to produce proliferative responses. We found that heat-killed bacteria were more stimulatory than formalin-fixed bacteria, and that soluble supernatants of heat-killed organism were also effective. The stimulatory effects of supernatants were destroyed by trypsin and the responses were MHC-restricted. Twelve different E. coli strains, with or without characteristics of uropathogenicity in humans, were all highly stimulatory to T cells derived from a kidney infected with a single E. coli strain. Strains of Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens--species of Enterobacteriaceae closely related to E. coli--were also stimulatory, whereas more distantly related bacteria--Proteus, Morganella, and P. aeruginosa--were not. T cells propagated from kidneys infected with P. aeruginosa responded to supernatants of this organism, but not to E. coli supernatants. We conclude that a protein antigen (or antigens) shared by strains of E. coli and related Enterobacteriaceae, but not by other gram-negative bacteria, produce MHC-restricted proliferative responses of CD4+ T cells that infiltrate rat kidneys infected with E. coli.

  16. Enhancing effect of serum ultrafiltrate on the activity of cephalosporins against gram-negative bacilli.

    PubMed Central

    Leggett, J E; Craig, W A

    1989-01-01

    A few studies have suggested that the inhibitory effect of serum on activity of broad-spectrum cephalosporins is less than that predicted by the degree of protein binding. Microdilution MICs of ceftriaxone, cefoperazone, moxalactam, and ceftizoxime were therefore determined against ATCC and clinical strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus in Mueller-Hinton broth containing either human albumin (as 0, 2.5, or 5% solution) or heat-inactivated human serum (as 0, 25, 50, or 95% solution). Arithmetic linear dilutions were used to improve accuracy. For standard bacterial strains, MICs in the presence of 5% albumin were higher than in broth alone by multiples of 10.9 to 21 for ceftriaxone, 5.5 to 16.4 for cefoperazone, 1.9 to 3.7 for moxalactam, and 1.1 to 1.4 for ceftizoxime, as expected by their protein binding. MICs in the presence of 95% serum were similar to those in 5% albumin for all four drugs against S. aureus and P. aeruginosa but were 2.2- to 4.8-fold lower (P less than 0.001) against E. coli and K. pneumoniae. Similar findings were observed at lower protein concentrations and with clinical isolates, except that for some strains of P. aeruginosa MICs were lower in serum than in albumin. Individual sera from five subjects gave comparable results. The addition of serum ultrafiltrate to albumin-containing solutions reduced MICs of ceftriaxone and cefoperazone 1.6- to 7.4-fold against E. coli and K. pneumoniae (P less than 0.01) but did not alter the MICs for S. aureus. Serum may contain an ultrafiltrable component(s) that enhances the activity of third-generation cephalosporins against many gram-negative bacilli. PMID:2496656

  17. Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

    PubMed Central

    Lauber, Frédéric; Cornelis, Guy Richard

    2016-01-01

    ABSTRACT Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes. Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2. We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. PMID:27795390

  18. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

    PubMed

    Alakomi, H L; Skyttä, E; Saarela, M; Mattila-Sandholm, T; Latva-Kala, K; Helander, I M

    2000-05-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

  19. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

    PubMed Central

    Alakomi, H.-L.; Skyttä, E.; Saarela, M.; Mattila-Sandholm, T.; Latva-Kala, K.; Helander, I. M.

    2000-01-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl2. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances. PMID:10788373

  20. The Structural Diversity of Carbohydrate Antigens of Selected Gram-Negative Marine Bacteria

    PubMed Central

    Nazarenko, Evgeny L.; Crawford, Russell J.; Ivanova, Elena P.

    2011-01-01

    Marine microorganisms have evolved for millions of years to survive in the environments characterized by one or more extreme physical or chemical parameters, e.g., high pressure, low temperature or high salinity. Marine bacteria have the ability to produce a range of biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents, and as a result, they have been a topic of research interest for many years. Among these biologically active molecules, the carbohydrate antigens, lipopolysaccharides (LPSs, O-antigens) found in cell walls of Gram-negative marine bacteria, show great potential as candidates in the development of drugs to prevent septic shock due to their low virulence. The structural diversity of LPSs is thought to be a reflection of the ability for these bacteria to adapt to an array of habitats, protecting the cell from being compromised by exposure to harsh environmental stress factors. Over the last few years, the variety of structures of core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been discovered. In this review, we discuss the most recently encountered structures that have been identified from bacteria belonging to the genera Aeromonas, Alteromonas, Idiomarina, Microbulbifer, Pseudoalteromonas, Plesiomonas and Shewanella of the Gammaproteobacteria phylum; Sulfitobacter and Loktanella of the Alphaproteobactera phylum and to the genera Arenibacter, Cellulophaga, Chryseobacterium, Flavobacterium, Flexibacter of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention is paid to the particular chemical features of the LPSs, such as the monosaccharide type, non-sugar substituents and phosphate groups, together with some of the typifying traits of LPSs obtained from marine bacteria. A possible correlation is then made between such features and the environmental adaptations undertaken by marine bacteria. PMID:22073003

  1. Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species.

    PubMed

    Potter, S; Yang, X; Boulanger, M J; Ishiguro, E E

    1998-04-01

    The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.

  2. Bioenergetics and the Role of Soluble Cytochromes c for Alkaline Adaptation in Gram-Negative Alkaliphilic Pseudomonas

    PubMed Central

    Matsuno, T.; Yumoto, I.

    2015-01-01

    Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles. The reversed difference of H+ concentration across the membrane will make energy production considerably difficult for Gram-negative as well as Gram-positive bacteria. Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21T grown at pH 10 under low-aeration intensity have a soluble cytochrome c content that is 3.6-fold higher than that of the cells grown at pH 7 under high-aeration intensity. Cytochrome c-552 content was higher (64% in all soluble cytochromes c) than those of cytochrome c-554 and cytochrome c-551. In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in μmax⁡ [h−1] (40%) and maximum cell turbidity (25%) relative to those of the wild type. Considering the high electron-retaining abilities of the three soluble cytochromes c, the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by the soluble cytochromes c acting as electron storages in the periplasmic space of the bacterium. These electron-retaining cytochromes c may play a role as electron and H+ condenser, which facilitate terminal oxidation at high pH under air-limited conditions, which is difficult to respire owing to less oxygen and less H+. PMID:25705691

  3. ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

    PubMed Central

    Paramasivam, Nagarajan; Linke, Dirk

    2011-01-01

    The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

  4. Gram-negative bacteria account for main differences between faecal microbiota from patients with ulcerative colitis and healthy controls.

    PubMed

    Vigsnæs, L K; Brynskov, J; Steenholdt, C; Wilcks, A; Licht, T R

    2012-12-01

    Detailed knowledge about the composition of the intestinal microbiota may be critical to unravel the pathogenesis of ulcerative colitis (UC), a human chronic inflammatory bowel disease, since the intestinal microbes are expected to influence some of the key mechanisms involved in the inflammatory process of the gut mucosa. The aim of this study was to investigate the faecal microbiota in patients either with UC in remission (n=6) or with active disease (n=6), and in healthy controls (n=6). The composition of Gram-negative bacteria and Gram-positive bacteria was examined. Antigenic structures of Gram-negative bacteria such as lipopolysaccharides have been related to the inflammatory responses and pathogenesis of inflammatory bowel disease. Dice cluster analysis and principal component analysis of faecal microbiota profiles obtained by denaturing gradient gel electrophoresis and quantitative PCR, respectively, revealed that the composition of faecal bacteria from UC patients with active disease differed from the healthy controls and that this difference should be ascribed to Gram-negative bacteria. The analysis did not show any clear grouping of UC patients in remission. Even with the relatively low number of subjects in each group, we were able to detect a statistically significant underrepresentation of Lactobacillus spp. and Akkermansia muciniphila in UC patients with clinically active disease compared to the healthy controls. In line with previous communications, we have shown that the microbiota in UC patients with active disease differ from that in healthy controls. Our findings indicate that alterations in the composition of the Gram-negative bacterial population, as well as reduced numbers of lactobacilli and A. muciniphila may play a role in UC.

  5. Effects of Photodynamic Therapy on Gram-Positive and Gram-Negative Bacterial Biofilms by Bioluminescence Imaging and Scanning Electron Microscopic Analysis

    PubMed Central

    Núñez, Silvia C.; Azambuja, Nilton; Fregnani, Eduardo R.; Rodriguez, Helena M.H.; Hamblin, Michael R.; Suzuki, Hideo; Ribeiro, Martha S.

    2013-01-01

    Abstract Objective: The aim of this study was to test photodynamic therapy (PDT) as an alternative approach to biofilm disruption on dental hard tissue, We evaluated the effect of methylene blue and a 660 nm diode laser on the viability and architecture of Gram-positive and Gram-negative bacterial biofilms. Materials and methods: Ten human teeth were inoculated with bioluminescent Pseudomonas aeruginosa or Enterococcus faecalis to form 3 day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify and evaluate the bacterial viability, and scanning electron microscopic (SEM) imaging was used to assess architecture and morphology of bacterial biofilm before and after PDT employing methylene blue and 40 mW, 660 nm diode laser light delivered into the root canal via a 300 μm fiber for 240 sec, resulting in a total energy of 9.6 J. The data were statistically analyzed with analysis of variance (ANOVA) followed by Tukey test. Results: The bacterial reduction showed a dose dependence; as the light energy increased, the bioluminescence decreased in both planktonic suspension and in biofilms. The SEM analysis showed a significant reduction of biofilm on the surface. PDT promoted disruption of the biofilm and the number of adherent bacteria was reduced. Conclusions: The photodynamic effect seems to disrupt the biofilm by acting both on bacterial cells and on the extracellular matrix. PMID:23822168

  6. Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria.

    PubMed

    Townsend, D E; Esenwine, A J; George, J; Bross, D; Maguire, M E; Smith, R L

    1995-09-01

    The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation. The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S. typhimurium corA Mg2+ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.

  7. Effect of kojic acid-grafted-chitosan oligosaccharides as a novel antibacterial agent on cell membrane of gram-positive and gram-negative bacteria.

    PubMed

    Liu, Xiaoli; Xia, Wenshui; Jiang, Qixing; Xu, Yanshun; Yu, Peipei

    2015-09-01

    Our work here, for the first time, reported the antibacterial activity of kojic acid-grafted-chitosan oligosaccharides (COS/KA) against three gram-positive and three gram-negative bacteria. Integrity of cell membrane, outer membrane (OM) and inner membrane (IM) permeabilization assay, alkaline phosphatase (ALP) and glucose-6-phosphate dehydrogenase (G6PDH) assay, and SDS-PAGE assay techniques were used to investigate the interactions between COS/KA and bacterial membranes. The antibacterial activity of COS/KA was higher than those of unmodified COS. The electric conductivity of bacteria suspensions increased, followed by increasing of the units of average release for ALP and G6PDH. COS/KA can also rapidly increase the 1-N-phenylanphthylamine (NPN) uptake and the release of β-galactosidase via increasing the permeability of OM and IM in Escherichia coli. SDS-PAGE indicated the content of cellular soluble proteins decreased significantly in COS/KA-treated bacteria. Hence, COS/KA has potential in food industry and biomedical sciences.

  8. Anti-bacterial performance of azithromycin nanoparticles as colloidal drug delivery system against different gram-negative and gram-positive bacteria

    PubMed Central

    Azhdarzadeh, Morteza; Lotfipour, Farzaneh; Zakeri-Milani, Parvin; Mohammadi, Ghobad; Valizadeh, Hadi

    2012-01-01

    Purpose: Azithromycin (AZI) is a new macrolide antibiotic with a better activity against intracellular gram negative bacteria in comparison with Erythromycin. The purpose of this research was to prepare AZI nanoparticles (NPs) using PLGA polymer and to compare the effectiveness of prepared nanoparticles with untreated AZI solution. Methods: AZI NPs were prepared by Modified Quasi-Emulsion Solvent Diffusion method. The antibacterial activities of prepared NPs in comparison with AZI solution were assayed against indicator bacteria of Escherichia coli (PTCC 1330), Haemophilus influenzae (PTCC 1623) and Streptococcus pneumoniae (PTCC 1240) using agar well diffusion. Inhibition zone diameters (IZD) of nano-formulation were compared to the corresponding untreated AZI. Mean Inhibitory Concentration (MIC) values of AZI were also determined using serial dilution method in nutrient broth medium. Results: Mean IZD of nano-formulations for all indicator bacteria were significantly higher than that of untreated AZI (P<0.01). The enhanced antibacterial efficacy was more dominant in the gram positive species. The MIC values of NPs against the tested bacteria were reduced 8 times in comparison to those of untreated AZI. Conclusion: These results indicated an improved potency of AZI NPs which could be attributed to the modified surface characteristics as well as increased drug adsorption and uptake. PMID:24312766

  9. Sulfoxides, Analogues of L-Methionine and L-Cysteine As Pro-Drugs against Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    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

  10. ZL-2, a cathelicidin-derived antimicrobial peptide, has a broad antimicrobial activity against gram-positive bacteria and gram-negative bacteria in vitro and in vivo.

    PubMed

    Tu, Jiancheng; Wu, Geping; Zuo, Yun; Zhao, Lei; Wang, Shusheng

    2015-10-01

    Alloferons are a group of naturally occurring peptides primarily isolated from insects that are capable of stimulating mouse and human NK cell cytotoxicity toward cancer cells. In this study, we found that a modified antibacterial peptide had a broad range of action against both gram-positive and gram-negative bacteria. A time-course experiment showed that CFU counts rapidly decreased after ZL-2 treatment, with the bacteria nearly eliminated within 4 h. We also examined the synergy between the peptide and antibiotics. The peptide ZL-2 resulted in a significant synergistic improvement in the potencies of ampicillin, erythromycin and ceftazidime against methicillin-resistant bacteria. In addition, ZL-2 had no detectable cytotoxicity in mouse spleen cells or a mouse animal model. In the mouse model by i.p. inoculation with Escherichia coli, timely treatment of i.p. injection with ZL-2 resulted in 100-fold reduction in bacteria load in blood as well as 80% protection from death in the inoculated animals. In conclusion, we successfully identified a modified peptide with maximal bactericidal activity. This study also provides a potential therapeutic for the treatment of E. coli septicemia by increasing the activity of antimicrobials.

  11. First study on antimicriobial activity and synergy between isothiocyanates and antibiotics against selected Gram-negative and Gram-positive pathogenic bacteria from clinical and animal source.

    PubMed

    Dias, Carla; Aires, Alfredo; Bennett, Richard N; Rosa, Eduardo A S; Saavedra, Maria J

    2012-05-01

    The emergence of new diseases and the resurgence of several infections that were controlled in the past, associated with recent increase of bacterial resistance have created the necessity for more studies towards to the development of new antimicrobials and new treatment strategies. The aim of the present study was to evaluate the in vitro synergy between different classes of important glucosinolates hydrolysis products-isothiocyanates with antibiotics (gentamycin and vancomycin), against important pathogenic bacteria: Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus. A disc diffusion method was used to evaluate the antibacterial activity. The antimicrobial activity of phytochemicals and combinations between gentamycin, vancomycin and phytochemicals were quantitatively assessed by measuring the inhibitory halos. The results showed a selective antimicrobial effect of isothiocyanates, and this effect was strictly related with their chemical structure. In general the benzylisothiocyanate was the most effective compound against both Gram-positive and Gram-negative bacteria. The Listeria monocytogenes and Staphylococcus aureus were the bacteria most affected either by the phytochemicals alone or by the combination phytochemical-antibiotic. The bacteria Pseudomonas aeruginosa was the less affected pathogen. The most important synergism detected occurred between the commercial antibiotics with benzylisothiocyanate and 2-phenylethylisothiocyanate. In conclusion, some isothiocyanates are effective inhibitors of in vitro bacterial growth, and they can act synergistically with antibiotics.

  12. 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.

  13. A 980nm driven photothermal ablation of virulent and antibiotic resistant Gram-positive and Gram-negative bacteria strains using Prussian blue nanoparticles.

    PubMed

    Maaoui, Houcem; Jijie, Roxana; Pan, Guo-Hui; Drider, Djamel; Caly, Delphine; Bouckaert, Julie; Dumitrascu, Nicoleta; Chtourou, Radouane; Szunerits, Sabine; Boukherroub, Rabah

    2016-10-15

    A 980nm laser-driven antimicrobial photothermal therapy using poly(vinylpyrrolidone) -coated Prussian Blue nanoparticles (PVP/PB NPs) is demonstrated. This approach allows an efficient eradication of a virulent strain of Gram-negative Escherichia coli (E. coli) associated with urinary tract infection as well as for the ablation of antibiotic resistant pathogens such as methicillin resistant Staphylococcus aureus (MRSA) and extended spectrum β-lactamase (ESBL) E. coli. Interestingly the 980nm irradiation exhibits minimal effect on mammalian cells up to a PVP/PB NPs concentration of 50μgmL(-1), while at this concentration bacteria are completely eradicated. This feature is certainly very promising for the selective targeting of bacteria over mammalian cells.

  14. Permeability barrier of the gram-negative bacterial outer membrane with special reference to nisin.

    PubMed

    Helander, I M; Mattila-Sandholm, T

    2000-09-25

    The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to I mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure

  15. Intravenous Colistin Use for Multidrug-Resistant Gram-Negative Infections in Pediatric Patients

    PubMed Central

    Karaaslan, Ayşe; Çağan, Eren; Kadayifci, Eda Kepenekli; Atıcı, Serkan; Akkoç, Gülşen; Yakut, Nurhayat; Demir, Sevliya Öcal; Soysal, Ahmet; Bakır, Mustafa

    2016-01-01

    Background The emergence of infections due to multidrug-resistant Gram-negative bacilli (MDR-GNB) has led to the resurrection of colistin use. The data on colistin use and drug-related adverse effects in children are scarce. Aims In this study, we aimed to evaluate the clinical efficacy and safety of colistin use in critically ill pediatric patients. Study Design This study has a retrospective study design. Methods Sixty-one critically ill children were identified through the department’s patient files archive during the period from January 2011 to November 2014. Results Twenty-nine females and thirty-two males with a mean±standard deviation (SD) age of 61±9 months (range 0–216, median 12 months) received IV colistin due to MDR-GNB infections. Bacteremia (n=23, 37.7%) was the leading diagnosis, followed by pneumonia (n=19, 31%), clinical sepsis (n=7, 11.4%), wound infection (n=6, 9.8%), urinary tract infection (n=5, 8.1%) and meningitis (n=1, 1.6%). All of the isolates were resistant to carbapenems; however, all were susceptible to colistin. The isolated microorganisms in decreasing order of frequency were: Acinetobacter baumanni (n=27, 44.2%), Pseudomonas aeruginosa (n=17, 27.8%), Klebsiella pneumoniae (n=6, 9.8%), K. pneumoniae and Stenotrophomonas maltophilia (n=1, 1.6%), K. pneumoniae and A. baumanni (n=1, 1.6%), K. oxytoca (n=1, 1.6%) and Enterobacter cloacae (n=1, 1.6%). In seven patients, no microorganisms were detected; however, five of these patients were colonized by carbapenem-resistant K. pneumoniae. The mean duration of colistin therapy was 12 days (range 3–45). Colistin was administered concomitantly with one of the following antibiotics: carbapenem (n=50, %82), ampicillin-sulbactam (n=5, 8%), quinolones (n=5, 8%), rifampicin (n=1, 1.6%). Carbapenem was the most frequently used antibiotic. Nephrotoxicity was observed in only 1 patient, and we did not observe neurotoxicity in this study. All the patients received intravenous colistin

  16. Risk factors for tracheobronchial acquisition of resistant Gram-negative bacterial pathogens in mechanically ventilated ICU patients.

    PubMed

    Papakonstantinou, Ilias; Angelopoulos, Epameinondas; Baraboutis, Ioannis; Perivolioti, Efstathia; Parisi, Maria; Psaroudaki, Zoe; Kampisiouli, Efstathia; Argyropoulou, Athina; Nanas, Serafeim; Routsi, Christina

    2015-10-01

    The aim of this study was to identify risk factors for tracheobronchial acquisition with the most common resistant Gram-negative bacteria in the intensive care unit (ICU) during the first week after intubation and mechanical ventilation. Tracheobronchial and oropharyngeal cultures were obtained at admission, after 48 hours, and after 7 days of mechanical ventilation. Patient characteristics, interventions, and antibiotic usage were recorded. Among 71 eligible patients with two negative bronchial cultures for resistant Gram-negative bacteria (at admission and within 48 hours), 41 (58%) acquired bronchial resistant Gram-negative bacteria by day 7. Acquisition strongly correlated with presence of the same pathogens in the oropharynx: Acinetobacter baumannii [odds ratio (OR) = 20·2, 95% confidence interval (CI): 5·5-73·6], Klebsiella pneumoniae (OR = 8·0, 95% CI: 1·9-33·6), and Pseudomonas aeruginosa (OR = 27, 95%: CI 2·7-273). Bronchial acquisition with resistant K. pneumoniae also was associated with chronic liver disease (OR = 3·9, 95% CI: 1·0-15·3), treatment with aminoglycosides (OR = 4·9, 95% CI: 1·4-18·2), tigecycline (OR = 4·9, 95% CI: 1·4-18·2), and linezolid (OR = 3·9, 95% CI: 1·1-15·0). In multivariate analysis, treatment with tigecycline and chronic liver disease were independently associated with bronchial resistant K. pneumoniae acquisition. Our results show a high incidence of tracheobronchial acquisition with resistant Gram-negative microorganisms in the bronchial tree of newly intubated patients. Oropharynx colonization with the same pathogens and specific antibiotics use were independent risk factors.

  17. Epidemiology of bacteremia episodes in a single center: increase in Gram-negative isolates, antibiotics resistance, and patient age.

    PubMed

    Marchaim, D; Zaidenstein, R; Lazarovitch, T; Karpuch, Y; Ziv, T; Weinberger, M

    2008-11-01

    Increased resistance among isolates causing bacteremia constitutes a major challenge to medical practitioners and institutions. Variability between institutes is substantial, and requires the individual analysis of local trends. An eight-year (1997-2004) surveillance study of episodes of bacteremia was conducted in an 850-bed university hospital in central Israel. Trends of incidence, resistance, age, and mortality were analyzed. We studied 6,096 patient-unique episodes of bacteremia, of which, 2,722 (45.3%) were nosocomial and 523 (9.2%) involved children less than 18 years of age. The overall incidence of bacteremia episodes has increased over the study years by 39% and the patient mean age by 7.5 years. Gram-negative organisms accounted for 72% of hospital-acquired cases and 69% of community-acquired cases. There was a substantial increase in the incidence of nosocomial episodes, predominantly due to Gram-negative isolates, mainly Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. Increased resistance to broad-spectrum antibiotics was noted among Gram-negative organisms, including quinolones (in K. pneumoniae), imipenem (A. baumannii and P. aeruginosa), piperacillin-tazobactam (K. pneumoniae), and amikacin (A. baumannii and P. aeruginosa). Increased resistance to oxacillin among coagulase-negative staphylococci was also noted. The all-cause mortality rates showed a significant rise. The patient age, intensive care unit (ICU) stay, and hospital acquisition were independently associated with mortality. We describe an increase in the incidence and resistance of Gram-negative organisms causing bacteremia and concomitant ageing of the patients with bacteremia. Similar patterns have been reported from other localities, and are of real concern.

  18. Comparative evaluation of the Vitek-2 Compact and Phoenix systems for rapid identification and antibiotic susceptibility testing directly from blood cultures of Gram-negative and Gram-positive isolates.

    PubMed

    Gherardi, Giovanni; Angeletti, Silvia; Panitti, Miriam; Pompilio, Arianna; Di Bonaventura, Giovanni; Crea, Francesca; Avola, Alessandra; Fico, Laura; Palazzo, Carlo; Sapia, Genoveffa Francesca; Visaggio, Daniela; Dicuonzo, Giordano

    2012-01-01

    We performed a comparative evaluation of the Vitek-2 Compact and Phoenix systems for direct identification and antimicrobial susceptibility testing (AST) from positive blood culture bottles in comparison to the standard methods. Overall, 139 monomicrobial blood cultures, comprising 91 Gram-negative and 48 Gram-positive isolates, were studied. Altogether, 100% and 92.3% of the Gram-negative isolates and 75% and 43.75% of the Gram-positive isolates showed concordant identification between the direct and the standard methods with Vitek and Phoenix, respectively. AST categorical agreements of 98.7% and 99% in Gram-negative and of 96.2% and 99.5% in Gram-positive isolates with Vitek and Phoenix, respectively, were observed. In conclusion, direct inoculation procedures for Gram-negative isolates showed an excellent performance with both automated systems, while for identification of Gram-positive isolates they proved to be less reliable, although Vitek provided acceptable results. This approach contributes to reducing the turnaround time to result of blood cultures, with a positive impact on patient care.

  19. 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.

  20. 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

  1. 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

    Rychert, Jenna; 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

    2013-07-01

    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.

  2. Detection of gram-negative bacteremia by limulus amebocyte lysate assay: evaluation in a rat model of peritonitis.

    PubMed

    du Moulin, G C; Lynch, S E; Hedley-Whyte, J; Broitman, S A

    1985-01-01

    A spectrophotometric Limulus amebocyte lysate assay using lysis filtration and centrifugation has been developed for the detection of gram-negative bacteria in blood. The assay is directed at detection of endotoxin in viable and nonviable bacteria present in the blood-stream and not detection of free endotoxin in plasma. The assay was evaluated in a model of peritonitis in which rats were challenged with an inoculum consisting of sterilized human feces, barium sulfate, and one of eight species of bacteria. This assay was able to detect gram-negative bacteremia due to Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, and Klebsiella pneumoniae in the rat model when compared with sham-inoculated uninfected rats. The assay failed to detect bacteremia due to Bacteroides fragilis or Staphylococcus aureus, nor was there a significant rise in absorbance when a pellet containing sterilized feces was implanted in the rat.

  3. Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan.

    PubMed

    Sato, Maiko; Ahmed, Ashraf M; Noda, Ayako; Watanabe, Hitoshi; Fukumoto, Yukio; Shimamoto, Tadashi

    2009-11-24

    Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, beta-lactamase-encoding genes, blaTEM-1 and blaCTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.

  4. A brief report of gram-negative bacterial endotoxin levels in airborne and settled dusts in animal confinement buildings

    SciTech Connect

    Thedell, T.D.; Mull, J.C.; Olenchock, S.A.

    1980-01-01

    Gram-negative bacterial endotoxins, implicated in adverse worker health responses, were found in settled and airborne dust samples obtained from poultry and swine confinement units. Results of the Limulus amebocyte lysate gel test found endotoxin levels in dust samples ranged from 4.5 to 47.7 micrograms of FDA Klebsiella endotoxin equivalents/gm. Differences in endotoxin levels between dust samples may have been due to variables in time, geographic locations, confined animals, confinement buildings and equipment, and methods of sample collection. Animal confinement workers are potentially exposed to large amounts of gram-negative bacterial endotoxins; however, the respiratory health effects of such exposures to animal confinement workers have yet to be determined.

  5. Prior colonization is associated with increased risk of antibiotic-resistant Gram-negative bacteremia in cancer patients☆,☆☆

    PubMed Central

    Kleinberg, Michael; Sorkin, John D.; Netzer, Giora; Johnson, Jennifer K.; Shardell, Michelle; Thom, Kerri A.; Harris, Anthony D.; Roghmann, Mary-Claire

    2015-01-01

    We hypothesized that prior colonization with antibiotic-resistant Gram-negative bacteria is associated with increased risk of subsequent antibiotic-resistant Gram-negative bacteremia among cancer patients. We performed a matched case-control study. Cases were cancer patients with a blood culture positive for antibiotic-resistant Gram-negative bacteria. Controls were cancer patients with a blood culture not positive for antibiotic-resistant Gram-negative bacteria. Prior colonization was defined as any antibiotic-resistant Gram-negative bacteria in surveillance or non-sterile-site cultures obtained 2–365 days before the bacteremia. Thirty-two (37%) of 86 cases and 27 (8%) of 323 matched controls were previously colonized by any antibiotic-resistant Gram-negative bacteria. Prior colonization was strongly associated with antibiotic-resistant Gram-negative bacteremia (odds ratio [OR] 7.2, 95% confidence interval [CI] 3.5–14.7) after controlling for recent treatment with piperacillin-tazobactam (OR 2.5, 95% CI 1.3–4.8). In these patients with suspected bacteremia, prior cultures may predict increased risk of antibiotic-resistant Gram-negative bacteremia. PMID:24582582

  6. Frequency of Resistance to Kanamycin, Tobramycin, Netilmicin, and Amikacin in Gentamicin-Resistant Gram-Negative Bacteria

    PubMed Central

    Seligman, Stephen J.

    1978-01-01

    In vitro evaluation of 66 epidemiologically distinct, gentamicin-resistant, gram-negative isolates from four hospitals revealed that 92% were kanamycin resistant, 44% were netilmicin resistant, 41% were tobramycin resistant, and 6% were amikacin resistant. Combined resistance to gentamicin, tobramycin, and netilmicin occurred in 30% of the strains. Although the resistance percentage to amikacin was the lowest of the three newer agents, two strains were resistant to all of the aminoglycosides tested. PMID:626492

  7. Gram-negative bacteraemia; a multi-centre prospective evaluation of empiric antibiotic therapy and outcome in English acute hospitals.

    PubMed

    Fitzpatrick, J M; Biswas, J S; Edgeworth, J D; Islam, J; Jenkins, N; Judge, R; Lavery, A J; Melzer, M; Morris-Jones, S; Nsutebu, E F; Peters, J; Pillay, D G; Pink, F; Price, J R; Scarborough, M; Thwaites, G E; Tilley, R; Walker, A S; Llewelyn, M J

    2016-03-01

    Increasing antibiotic resistance makes choosing antibiotics for suspected Gram-negative infection challenging. This study set out to identify key determinants of mortality among patients with Gram-negative bacteraemia, focusing particularly on the importance of appropriate empiric antibiotic treatment. We conducted a prospective observational study of 679 unselected adults with Gram-negative bacteraemia at ten acute english hospitals between October 2013 and March 2014. Appropriate empiric antibiotic treatment was defined as intravenous treatment on the day of blood culture collection with an antibiotic to which the cultured organism was sensitive in vitro. Mortality analyses were adjusted for patient demographics, co-morbidities and illness severity. The majority of bacteraemias were community-onset (70%); most were caused by Escherichia coli (65%), Klebsiella spp. (15%) or Pseudomonas spp. (7%). Main foci of infection were urinary tract (51%), abdomen/biliary tract (20%) and lower respiratory tract (14%). The main antibiotics used were co-amoxiclav (32%) and piperacillin-tazobactam (30%) with 34% receiving combination therapy (predominantly aminoglycosides). Empiric treatment was inappropriate in 34%. All-cause mortality was 8% at 7 days and 15% at 30 days. Independent predictors of mortality (p <0.05) included older age, greater burden of co-morbid disease, severity of illness at presentation and inflammatory response. Inappropriate empiric antibiotic therapy was not associated with mortality at either time-point (adjusted OR 0.82; 95% CI 0.35-1.94 and adjusted OR 0.92; 95% CI 0.50-1.66, respectively). Although our study does not exclude an impact of empiric antibiotic choice on survival in Gram-negative bacteraemia, outcome is determined primarily by patient and disease factors.

  8. Rapid Differentiation of Fermentative from Nonfermentative Gram-Negative Bacilli in Positive Blood Cultures by an Impedance Method

    PubMed Central

    Chang, Tsung Chain; Huang, Ay Huey

    2000-01-01

    Rapid differentiation of fermentative gram-negative bacilli (fermenters) from nonfermentative gram-negative bacilli (nonfermenters) in positive blood cultures may help physicians to narrow the choice of appropriate antibiotics for empiric treatment. An impedance method for direct differentiation of fermenters from nonfermenters was investigated. The bacterial suspensions (or positive culture broths containing gram-negative bacteria) were inoculated into the module wells of a Bactometer (bioMérieux, Inc., Hazelwood, Mo.) containing 1 ml of Muller-Hinton broth. The inoculated modules were incubated at 35°C, and the change in impedance in each well was continuously monitored. The amount of time required to cause a series of significant deviations from baseline impedance values was defined as the detection time (DT). The percent change of impedance was defined as the change of impedance at the time interval from DT to DT plus 1 h. After testing 857 strains of pure cultures (586 strains of fermenters and 271 strains of nonfermenters), a breakpoint (2.98%) of impedance change was obtained by discriminant analysis. Strains displaying impedance changes of greater than 2.98% were classified as fermenters; the others were classified as nonfermenters. By using this breakpoint, 98.6% (340 of 345) of positive blood cultures containing fermenters and 98% (98 of 100) of positive blood cultures containing nonfermenters were correctly classified. The impedance method was simple, and the results were normally available within 2 to 4 h after direct inoculation of positive blood culture broths. PMID:11015369

  9. Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes

    PubMed Central

    Amos, Sarah-Beth T. A.; Vermeer, Louic S.; Ferguson, Philip M.; Kozlowska, Justyna; Davy, Matthew; Bui, Tam T.; Drake, Alex F.; Lorenz, Christian D.; Mason, A. James

    2016-01-01

    The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency. PMID:27874065

  10. Antibacterial Activity of Thymus Syriacus Boiss Essential Oil and Its Components against Some Syrian Gram-Negative Bacteria Isolates

    PubMed Central

    Al-Mariri, Ayman; Swied, Ghayath; Oda, Adnan; Al Hallab, Laila

    2013-01-01

    Background: Despite the medical discoveries of different medicines and advanced ways of treatment, statistics have shown that the number of patients is increasing. This may be due to chemical drugs used in healthcare, agriculture, and diets. This soaring demand in medicines urges us to look for natural sources such as aromatic plants and essential oils, which are rich in efficient compounds. Methods: Extraction of essential oils was performed using a Clevenger-type apparatus. Identification was achieved using the GC-FID technique. Confirmation was made using the GC-MS technique, and isolation was done using a preparative HPLC, equipped with an aliquots collector. The microdilution broth susceptibility assay was utilized to determine minimum inhibitory concentrations (MICs). Results: Our in vitro study demonstrated the antibacterial activity of the Thymus syriacus Boiss essential oil and its components against the tested isolates at levels between 0.375 and 50 µl/ml. The main components of the T. syriacus essential oil were carvacrol, γ-terpinene, and ß–caryophyllene. MIC90 values for the T. syriacus essential oil against the gram-negative organisms varied between 3.125 and 12.5 µl/ml. The most effective components against the gram-negative bacteria were thymol, carvacrol, dihydro-carvon, and linalool respectively. Conclusions: The T. syriacus essential oil and some of its components exhibited very good inhibitory effects against Syrian gram-negative isolates. PMID:24031109

  11. Structural and Enzymatic Characterization of ABgp46, a Novel Phage Endolysin with Broad Anti-Gram-Negative Bacterial Activity

    PubMed Central

    Oliveira, Hugo; Vilas Boas, Diana; Mesnage, Stéphane; Kluskens, Leon D.; Lavigne, Rob; Sillankorva, Sanna; Secundo, Francesco; Azeredo, Joana

    2016-01-01

    The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0–10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 h. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (>5 log) and more than 4 logs Pseudomonas aeruginosa and Salmonella typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine. PMID:26955368

  12. Evaluation of Gram Negative Bacterial Contamination in Dental Unit Water Supplies in a University Clinic in Tabriz, Iran

    PubMed Central

    Pouralibaba, Firoz; Balaei, Esrafil; Kashefimehr, Atabak

    2011-01-01

    Background and aims Bacterial contamination of dental unit water supplies (DUWS) has attracted a lot of attention in recent years due to the emergence of serious infectionsin susceptible dental patients. The aim of the present study was to evaluate the presence of gram-negative bacterial contamination in DUWS at Tabriz University of Medical Sciences Faculty of Dentistry. Materials and methods This descriptive study was carried out on 51 active dental units in different departments. Con-tamination was determined by taking samples from the unit's water supply before dental procedures and the use of specific culture media. The cultures were evaluated after 48 hours. Results Gram-negative bacterial contamination was identical in all the departments. In the departments on the ground floor, namely Departments of Periodontics and Oral and Maxillofacial Surgery, Pseudomonas contamination was observed in 71% of units; in the departments on the first floor, namely Departments of Prosthodontics, Orthodontics and Pedodon-tics, 46.8% of the units had Pseudomonas contamination; and in the departments on the second floor, namely Departments of Operative Dentistry and Endodontics, 37.7% of the units demonstrated Pseudomonas contamination. Conclusion Gram-negative bacterial contamination was evident in the evaluated DUWS. The contamination type was identical but the number of contaminated units decreased with the increase in the height of the floors. PMID:22991613

  13. Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes

    NASA Astrophysics Data System (ADS)

    Amos, Sarah-Beth T. A.; Vermeer, Louic S.; Ferguson, Philip M.; Kozlowska, Justyna; Davy, Matthew; Bui, Tam T.; Drake, Alex F.; Lorenz, Christian D.; Mason, A. James

    2016-11-01

    The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.

  14. Multiple antimicrobial resistance of gram-negative bacteria from natural oligotrophic lakes under distinct anthropogenic influence in a tropical region.

    PubMed

    Pontes, D S; Pinheiro, F A; Lima-Bittencourt, C I; Guedes, R L M; Cursino, L; Barbosa, F; Santos, F R; Chartone-Souza, E; Nascimento, A M A

    2009-11-01

    The aim of this study was to evaluate the resistance to ten antimicrobial agents and the presence of bla ( TEM1 ) gene of Gram-negative bacteria isolated from three natural oligotrophic lakes with varying degrees of anthropogenic influence. A total of 272 indigenous bacteria were recovered on eosin methylene blue medium; they were characterized for antimicrobial resistance and identified taxonomically by homology search and phylogenetic comparisons. Based on 16S ribosomal RNA sequences analysis, 97% of the isolates were found to be Gram-negative bacteria; they belonged to 11 different genera. Members of the genera Acinetobacter, Enterobacter, and Pseudomonas predominated. Most of the bacteria were resistant to at least one antimicrobial. The incidence of resistance to beta-lactams, chloramphenicol, and mercury was high, whereas resistance to tetracycline, aminoglycosides, and nalidixic acid was low. There was a great frequency of multiple resistances among the isolates from the three lakes, although no significant differences were found among the disturbed and reference lakes. The ampicillin resistance mechanism of 71% of the isolates was due to the gene bla ( TEM1 ). Our study suggests that multiresistant Gram-negative bacteria and the bla ( TEM1 ) gene are common in freshwater oligotrophic lakes, which are subject to different levels of anthropogenic inputs.

  15. Antimicrobial resistance pattern of gram-negative bacteria of nosocomial origin at a teaching hospital in the Islamic Republic of Iran.

    PubMed

    Khalili, H; Soltani, R; Safhami, S; Dashti-Khavidaki, S; Alijani, B

    2012-02-01

    The emergence of antimicrobial resistance is a global problem in the community and in hospitals. Antibiotic resistance of Gram-negative bacteria from nosocomial infections were evaluated during a 6-month period at Shariati teaching hospital, Tehran, Islamic Republic of Iran. Susceptibility tests were performed on 570 Gram-negative isolates obtained from clinical samples of patients infected after at least 72 hours stay in the hospital. Escherichia coli was the most frequently isolated Gram-negative organism (42.6%). The highest rate of resistance in Gram-negative isolates was seen in the intensive care unit, with Acinetobacter spp. as the most resistant organisms. Gentamicin was the most effective antibiotic against E. coli and all other isolates, while ciprofloxacin was also effective against a wide range of other species. Antibiotic resistant Gram-negative nosocomial infection is prevalent in this teaching hospital in Tehran.

  16. Treatment of Amaranthus cruentus with chemical and biological inducers of resistance has contrasting effects on fitness and protection against compatible Gram positive and Gram negative bacterial pathogens.

    PubMed

    Casarrubias-Castillo, Kena; Martínez-Gallardo, Norma A; Délano-Frier, John P

    2014-07-01

    Amaranthus cruentus (Ac) plants were treated with the synthetic systemic acquired resistance (SAR) inducer benzothiadiazole (BTH), methyl jasmonate (MeJA) and the incompatible pathogen, Pseudomonas syringae pv. syringae (Pss), under greenhouse conditions. The treatments induced a set of marker genes in the absence of pathogen infection: BTH and Pss similarly induced genes coding for pathogenesis-related and antioxidant proteins, whereas MeJA induced the arginase, LOX2 and amarandin 1 genes. BTH and Pss were effective when tested against the Gram negative pathogen Ps pv. tabaci (Pst), which was found to have a compatible interaction with grain amaranth. The resistance response appeared to be salicylic acid-independent. However, resistance against Clavibacter michiganensis subsp. michiganensis (Cmm), a Gram positive tomato pathogen also found to infect Ac, was only conferred by Pss, while BTH increased susceptibility. Conversely, MeJA was ineffective against both pathogens. Induced resistance against Pst correlated with the rapid and sustained stimulation of the above genes, including the AhPAL2 gene, which were expressed both locally and distally. The lack of protection against Cmm provided by BTH, coincided with a generalized down-regulation of defense gene expression and chitinase activity. On the other hand, Pss-treated Ac plants showed augmented expression levels of an anti-microbial peptide gene and, surprisingly, of AhACCO, an ethylene biosynthetic gene associated with susceptibility to Cmm in tomato, its main host. Pss treatment had no effect on productivity, but compromised growth, whereas MeJA reduced yield and harvest index. Conversely, BTH treatments led to smaller plants, but produced significantly increased yields. These results suggest essential differences in the mechanisms employed by biological and chemical agents to induce SAR in Ac against bacterial pathogens having different infection strategies. This may determine the outcome of a particular

  17. 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.

  18. Silver nanocrystallites: Facile biofabrication using Shewanella oneidensis, and an evaluation of their comparative toxicity on Gram-negative and Gram-positive bacteria

    SciTech Connect

    Suresh, Anil K; Wang, Wei; Pelletier, Dale A; Moon, Ji Won; Gu, Baohua; Mortensen, Ninell P; Allison, David P; Joy, David Charles; Phelps, Tommy Joe; Doktycz, Mitchel John

    2010-01-01

    Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the -proteobacterium, Shewanella oneidensis strain MR-1, upon incubation with an aqueous solution of silver nitrate. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the size range 2 11 nm (with an average of 4 1.5 nm). The bactericidal effect of these biologically synthesized silver nanoparticles (biogenic-Ag) are compared to similar chemically synthesized nanoparticles (colloidal silver [colloidal-Ag] and oleate capped silver [oleate-Ag]). The determination of the bactericidal effect of these different silver nanoparticles was assessed using both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria and based on the diameter of the inhibition zone in disc diffusion tests, minimum inhibitory concentrations, Live/Dead staining assays, and atomic force microscopy. From a toxicity perspective, a clear synthesis procedure, and a surface coat- and strain-dependent inhibition were observed for silver nanoparticles. Biogenic-Ag was found to be of higher toxicity when compared to colloidal-Ag for both E. coli and B. subtilis. E. coli was found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, Oleate-Ag was not toxic to either of the bacteria. These findings have important implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

  19. Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor

    PubMed Central

    Card, Roderick M.; Mafura, Muriel; Hunt, Theresa; Kirchner, Miranda; Weile, Jan; Rashid, Mamun-Ur; Weintraub, Andrej; Nord, Carl Erik

    2015-01-01

    The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum β-lactamase blaCTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein. PMID:25987611

  20. Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor.

    PubMed

    Card, Roderick M; Mafura, Muriel; Hunt, Theresa; Kirchner, Miranda; Weile, Jan; Rashid, Mamun-Ur; Weintraub, Andrej; Nord, Carl Erik; Anjum, Muna F

    2015-08-01

    The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum β-lactamase blaCTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein.

  1. Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against Gram-negative bacteria.

    PubMed

    Sofrata, Abier; Santangelo, Ellen M; Azeem, Muhammad; Borg-Karlson, Anna-Karin; Gustafsson, Anders; Pütsep, Katrin

    2011-01-01

    Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate.

  2. Benzyl Isothiocyanate, a Major Component from the Roots of Salvadora Persica Is Highly Active against Gram-Negative Bacteria

    PubMed Central

    Sofrata, Abier; Santangelo, Ellen M.; Azeem, Muhammad; Borg-Karlson, Anna-Karin; Gustafsson, Anders; Pütsep, Katrin

    2011-01-01

    Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate. PMID:21829688

  3. Significance of postgrowth processing of ZnO nanostructures on antibacterial activity against gram-positive and gram-negative bacteria

    PubMed Central

    Mehmood, Shahid; Rehman, Malik A; Ismail, Hammad; Mirza, Bushra; Bhatti, Arshad S

    2015-01-01

    In this work, we highlighted the effect of surface modifications of one-dimensional (1D) ZnO nanostructures (NSs) grown by the vapor–solid mechanism on their antibacterial activity. Two sets of ZnO NSs were modified separately – one set was modified by annealing in an Ar environment, and the second set was modified in O2 plasma. Annealing in Ar below 800°C resulted in a compressed lattice, which was due to removal of Zn interstitials and increased O vacancies. Annealing above 1,000°C caused the formation of a new prominent phase, Zn2SiO4. Plasma oxidation of the ZnO NSs caused an expansion in the lattice due to the removal of O vacancies and incorporation of excess O. Photoluminescence (PL) spectroscopy was employed for the quantification of defects associated with Zn and O in the as-grown and processed ZnO NS. Two distinct bands were observed, one in the ultraviolet (UV) region, due to interband transitions, and other in the visible region, due to defects associated with Zn and O. PL confirmed the surface modification of ZnO NS, as substantial decrease in intensities of visible band was observed. Antibacterial activity of the modified ZnO NSs demonstrated that the surface modifications by Ar annealing limited the antibacterial characteristics of ZnO NS against Staphylococcus aureus. However, ZnO NSs annealed at 1,000°C or higher showed a remarkable antibacterial activity against Escherichia coli. O2 plasma–treated NS showed appreciable antibacterial activity against both E. coli and S. aureus. The minimum inhibition concentration was determined to be 0.5 mg/mL and 1 mg/mL for Ar-annealed and plasma-oxidized ZnO NS, respectively. It was thus proved that the O content at the surface of the ZnO NS was crucial to tune the antibacterial activity against both selected gram-negative (E. coli) and gram-positive (S. aureus) bacterial species. PMID:26213466

  4. A resurgence of β-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens.

    PubMed

    Bush, Karen

    2015-11-01

    β-Lactamase inhibitors (BLIs) have played an important role in combatting β-lactam resistance in Gram-negative bacteria, but their effectiveness has diminished with the evolution of diverse and deleterious varieties of β-lactamases. In this review, a new generation of BLIs and inhibitor combinations is presented, describing epidemiological information, pharmacodynamic studies, resistance identification and current clinical status. Novel serine BLIs of major interest include the non-β-lactams of the diazabicyclo[3.2.1]octanone (DBO) series. The DBOs avibactam, relebactam and RG6080 inhibit most class A and class C β-lactamases, with selected inhibition of class D enzymes by avibactam. The novel boronic acid inhibitor RPX7009 has a similar inhibitory profile. All of these inhibitors are being developed in combinations that are targeting primarily carbapenemase-producing Gram-negative pathogens. Two BLI combinations (ceftolozane/tazobactam and ceftazidime/avibactam) were recently approved by the US Food and Drug Administration (FDA) under the designation of a Qualified Infectious Disease Product (QIDP). Other inhibitor combinations that have at least completed phase 1 clinical trials are ceftaroline fosamil/avibactam, aztreonam/avibactam, imipenem/relebactam, meropenem/RPX7009 and cefepime/AAI101. Although effective inhibitor combinations are in development for the treatment of infections caused by Gram-negative bacteria with serine carbapenemases, better options are still necessary for pathogens that produce metallo-β-lactamases (MBLs). The aztreonam/avibactam combination demonstrates inhibitory activity against MBL-producing enteric bacteria owing to the stability of the monobactam to these enzymes, but resistance is still an issue for MBL-producing non-fermentative bacteria. Because all of the inhibitor combinations are being developed as parenteral drugs, an orally bioavailable combination would also be of interest.

  5. Antimicrobial activity of doripenem against Gram-negative pathogens: results from INVITA-A-DORI Brazilian study.

    PubMed

    Gales, Ana Cristina; Azevedo, Heber D; Cereda, Rosângela Ferraz; Girardello, Raquel; Xavier, Danilo Elias

    2011-01-01

    In vitro activity of doripenem and comparator antimicrobial agents was evaluated against Gram-negative bacilli recently isolated from Brazilian private hospitals that were enrolled in the INVITA-A-DORI Brazilian Study. A total of 805 unique Gram-negative bacilli were collected from patients hospitalized at 18 medical centers between May/08 and March/09. Each hospital was asked to submit 50 single Gram-negative bacilli isolated from blood, lower respiratory tract or intraabdominal secretions. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using Clinical Laboratory Standards Institute (CLSI) microdilution method at a central laboratory. CLSI M100-S21 (2011) or US-FDA package insert criteria (tigecycline) was used for interpretation of the antimicrobial susceptibility results. Doripenem was as active as meropenem and more active than imipenem against E. coli and K. pneumoniae isolates. A total of 50.0% of Enterobacter spp. isolates were resistant to ceftazidime but 85.7% of them were inhibited at doripenem MICs < 1 µg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, < 0.5/1 µg/mL) and P. aeruginosa (MIC50/90, 1/2 µg/mL). Although high rates of imipenem (53.1%) and meropenem (44.5%) resistance were detected among P. aeruginosa, doripenem showed MIC50 of 16 µg/mL against imipenem-resistant P. aeruginosa and inhibited a greater number of imipenem-resistant P. aeruginosa (10.5%) at MIC values of < 4 µg/mL than did meropenem (0.0%). In this study, doripenem showed similar in vitro activity to that of meropenem and retained some activity against imipenem-resistant P. aeruginosa isolated from Brazilian medical centers.

  6. Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of gram-negative bacterial pathogens.

    PubMed

    Card, Roderick; Zhang, Jiancheng; Das, Priya; Cook, Charlotte; Woodford, Neil; Anjum, Muna F

    2013-01-01

    A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.

  7. Prevalence and Characteristics of Surgical Site Infections Caused by Gram-negative Rod-shaped Bacteria from the Family Enterobacteriacae and Gram-positive Cocci from the Genus Staphylococcus in Patients who Underwent Surgical Procedures on Selected Surgical Wards.

    PubMed

    Tomaszewska-Kowalska, Małgorzata; Kołomecki, Krzysztof; Wieloch-Torzecka, Maria

    2016-10-01

    Surgical site infections on surgical wards are the most common cause of postoperative complications. Prevalence of surgical site infections depends on the surgical specialization. Analysis of the causes of surgical site infections allows to conclude that microorganisms from the patient's own microbiota - Gram-negative rod-shaped bacteria from the family Enterobacteriacae and from the patient's skin microbiota - Gram-positive cocci - Staphylococcus are the most common agents inducing surgical site infections. The aim of the study was to assess prevalence and characteristics of surgical site infections caused by Gram-negative rod-shaped bacteria from the family Eneterobacteriacae and Gram-positive cocci from the genus Staphylococcus in patients who underwent surgical procedures at the Regional Specialist Hospital named after M. Copernika in Łódź on selected surgical wards.

  8. RND efflux pump mediated antibiotic resistance in Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a major issue worldwide.

    PubMed

    Puzari, Minakshi; Chetia, Pankaj

    2017-02-01

    Therapeutic failures against diseases due to resistant Gram-negative bacteria have become a major threat nowadays as confirmed by surveillance reports across the world. One of the methods of development of multidrug resistance in Escherichia coli and Pseudomonas aeruginosa is by means of RND efflux pumps. Inhibition of these pumps might help to combat the antibiotic resistance problem, for which the structure and regulation of the pumps have to be known. Moreover, judicious antibiotic use is needed to control the situation. This paper focuses on the issue of antibiotic resistance as well as the structure, regulation and inhibition of the efflux pumps present in Escherichia coli and Pseudomonas aeruginosa.

  9. [Postmortem forensic medical diagnostics of fulminant sepsis caused by Gram-negative bacterium (Capnocitophaga canimorsus) following a dog bite].

    PubMed

    Kovalev, A V; Putintsev, V A; Bogomolov, D V; Gribunov, Iu P; Bogomolov, B P; Deviatkin, A V

    2015-01-01

    This article provides the example of postmortem forensic medical diagnostics of fulminant sepsis caused by Gram-negative bacterium (Capnocitophaga canimorsus) following a dog bite. In order to identify the etiological factor of fulminant sepsis, the expert carried out the study of the autopsy materials with the use of polymerase chain reaction (PCR). This method has only recently been introduced into postmortem diagnostics of fulminant sepsis in this country; it has no analogs abroad and can be employed for the purpose of forensic medical expertise and pathological anatomic studies.

  10. Emergence of Carbapenem Resistant Non-Fermenting Gram-Negative Bacilli Isolated in an ICU of a Tertiary Care Hospital

    PubMed Central

    Agarwal, Sonika; Khanduri, Sushant; Gupta, Shalini

    2017-01-01

    Introduction The emergence and spread of Multi-Drug Resistant (MDR) Non-Fermenting Gram-Negative Bacilli (NFGNB) in Intensive Care Units (ICU) and their genetic potential to transmit diverse antibiotic resistance regardless of their ability to ferment glucose poses a major threat in hospitals. The complex interplay of clonal spread, persistence, transmission of resistance elements and cell-cell interaction leads to the difficulty in controlling infections caused by these multi drug-resistant strains. Among non-fermenting Gram-negative rods, the most clinically significant species Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia are increasingly acquiring resistant to carbapenems. Carbapenems once considered as a backbone of treatment of life threatening infections appears to be broken as the resistance to carbapenems is on rise. Aim To document the prevalence of carbapenem resistance in non-fermenting Gram-negative bacilli isolated from patients with respiratory tract infections in the ICU of Himalayan Institute of Medical Sciences, Dehradun. Materials and Methods This is a cross-sectional study conducted in ICU patients between October 2015 to March 2016. A total of 366 lower respiratory tract samples were collected from 356 patients with clinical evidence of lower respiratory tract infections in form of Endotracheal (ET) aspirate, Tracheal Tube (TT) aspirate and Bronchoalveolar Lavage (BAL) specimen. Organism identification and the susceptibility testing was done by using an automated system VITEK 2. Results Out of 366 samples received 99 NFGNB were isolated and most common sample was ET aspirate sample 256 (64.5%). Acinetobacter baumannii was the most common NFGNB isolated 63 (63.63%) followed by Pseudomonas aeruginosa 25 (25.25%), Elizabethkingia meningoseptica seven (7.07%) and Strenotrophomonas maltophilia four (4.04%). We observed that 90.5% Acinetobacter baumannii were resistant to imipenem and 95.2% resistant to meropenem

  11. Prevalence of antibiotic-resistant Gram-negative bacteria associated with the red-eared slider (Trachemys scripta elegans).

    PubMed

    Liu, Dandan; Wilson, Cailin; Hearlson, Jodie; Singleton, Jennifer; Thomas, R Brent; Crupper, Scott S

    2013-09-01

    Free-ranging Red-eared Sliders (Trachemys scripta elegans) were captured from farm ponds located in the Flint Hills of Kansas and a zoo pond in Emporia, Kansas, USA, to evaluate their enteric bacterial flora and associated antibiotic resistance. Bacteria obtained from cloacal swabs were composed of six different Gram-negative genera. Although antibiotic resistance was present in turtles captured from both locations, 40 and 49% of bacteria demonstrated multiple antibiotic resistance to four of the antibiotics tested from the zoo captured and Flint Hills ponds turtles, respectively. These data illustrate environmental antibiotic resistance is widespread in the bacterial flora obtained from Red-eared Sliders in east central Kansas.

  12. The reaction mechanism for dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica

    NASA Astrophysics Data System (ADS)

    Yao, Yuan; Li, Ze-Sheng

    2012-01-01

    The fundamental reaction mechanism for the dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica has been studied by density functional theory calculations. The results indicate that the dehydration process undergoes a two-step cis-elimination mechanism, which is different from the previously proposed one. The catalytic roles of both the highly conserved residue His143 and the Schiff base formed between the substrate and Lys170 have also been elucidated. The structural and mechanistic insight presented here may direct the design of type I dehydroquinate dehydratase enzyme inhibitors as non-toxic antimicrobials, anti-fungals, and herbicides.

  13. Establishment of Experimental Murine Peritonitis Model with Hog Gastric Mucin for Carbapenem-Resistant Gram-Negative Bacteria

    PubMed Central

    Park, Chulmin; Chun, Hye-Sun; Byun, Ji-Hyun; Cho, Sung-Yeon

    2017-01-01

    Animal models are essential to studies of infectious diseases. The use of mice to test bacterial infection has been extensively reported. However, methods applied to clinical isolates, particularly for carbapenem-resistant bacteria, must be tailored according to the infection models and bacteria used. In this study, we infected 6-week-old female BALB/c mice intraperitoneally with different strains of resistant bacteria plus 3% hog gastric mucin. This method was found to be efficient and readily applicable for investigation of carbapenem-resisant Gram-negative pathogens (e.g., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii) detected in Korea. PMID:28271653

  14. Localization of membrane-derived oligosaccharides in the outer envelope of Escherichia coli and their occurrence in other Gram-negative bacteria.

    PubMed Central

    Schulman, H; Kennedy, E P

    1979-01-01

    The glucose-containing, membrane-derived oligosaccharides of Escherichia coli are localized in the external envelope of that organism, most probably in the periplasmic space. The membrane-derived oligosaccharides appear to be generally occurring cell constituents of gram-negative (but not gram-positive) bacteria. PMID:104978

  15. Hemodynamic and hormonal responses to lower body negative pressure in men with varying profiles of strength and aerobic power

    NASA Technical Reports Server (NTRS)

    Convertino, V. A.; Mathes, K. L.; Lasley, M. L.; Tomaselli, C. M.; Frey, M. A.; Hoffler, G. W.

    1993-01-01

    Hemodynamic, cardiac, and hormonal responses to lower-body negative pressure (LBNP) were examined in 24 healthy men to test the hypothesis that responsiveness of reflex control of blood pressure during orthostatic challenge is associated with interactions between strength and aerobic power. Subjects underwent treadmill tests to determine peak oxygen uptake (VO2max) and isokinetic dynamometer tests to determine knee extensor strength. Based on predetermined criteria, subjects were classified into one of four fitness profiles of six subjects each, matched for age, height, and body mass: (a) low strength/average aerobic fitness, (b) low strength/high aerobic fitness, (c) high strength/average aerobic fitness, and (d) high strength/high aerobic fitness. Following 90 min of 0.11 rad (6 degrees) head-down tilt (HDT), each subject underwent graded LBNP to -6.7 kPa or presyncope, with maximal duration 15 min, while hemodynamic, cardiac, and hormonal responses were measured. All groups exhibited typical hemodynamic, hormonal, and fluid shift responses during LBNP, with no intergroup differences between high and low strength characteristics. Subjects with high aerobic power exhibited greater (P < 0.05) stroke volume and lower (P < 0.05) heart rate, vascular peripheral resistance, and mean arterial pressure during rest, HDT, and LBNP. Seven subjects, distributed among the four fitness profiles, became presyncopal. These subjects showed greatest reduction in mean arterial pressure during LBNP, had greater elevations in vasopressin, and lesser increases in heart rate and peripheral resistance. Neither VO2max nor leg strength were associated with fall in arterial pressure or with syncopal episodes. We conclude that interactions between aerobic and strength fitness characteristics do not influence responses to LBNP challenge.

  16. Phototherapeutic spectrum expansion through synergistic effect of mesoporous silica trio-nanohybrids against antibiotic-resistant gram-negative bacterium.

    PubMed

    Kuthati, Yaswanth; Kankala, Ranjith Kumar; Busa, Prabhakar; Lin, Shi-Xiang; Deng, Jin-Pei; Mou, Chung-Yuan; Lee, Chia-Hung

    2017-04-01

    The extensive impact of antibiotic resistance has led to the exploration of new anti-bacterial modalities. We designed copper impregnated mesoporous silica nanoparticles (Cu-MSN) with immobilizing silver nanoparticles (SNPs) to apply photodynamic inactivation (PDI) of antibiotic-resistant E. coli. SNPs were decorated over the Cu-MSN surfaces by coordination of silver ions on diamine-functionalized Cu-MSN and further reduced to silver nanoparticles with formalin. We demonstrate that silver is capable of sensitizing the gram-negative bacteria E. coli to a gram-positive specific phototherapeutic agent in vitro; thereby expanding curcumin's phototherapeutic spectrum. The mesoporous structure of Cu-MSN remains intact after the exterior decoration with silver nanoparticles and subsequent curcumin loading through an enhanced effect from copper metal-curcumin affinity interaction. The synthesis, as well as successful assembly of the functional nanomaterials, was confirmed by various physical characterization techniques. Curcumin is capable of producing high amounts of reactive oxygen species (ROS) under light irradiation, which can further improve the silver ion release kinetics for antibacterial activity. In addition, the positive charged modified surfaces of Cu-MSN facilitate antimicrobial response through electrostatic attractions towards negatively charged bacterial cell membranes. The antibacterial action of the synthesized nanocomposites can be activated through a synergistic mechanism of energy transfer of the absorbed light from SNP to curcumin.

  17. High-level and novel mechanisms of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria.

    PubMed

    Ogbolu, D O; Webber, M A

    2014-05-01

    To determine the occurrence and molecular basis of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria, 182 non-duplicate Gram-negative bacterial isolates were investigated for antimicrobial susceptibility, presence of carbapenemases (tested phenotypically and genotypically), random amplified polymorphic DNA (RAPD) typing, plasmid sizing and replicon typing. Minimum inhibitory concentrations of carbapenems showed a high degree of resistance, with 67 isolates (36.8%) being resistant to all carbapenems, of which 40 (59.7%) produced enzymes able to hydrolyse imipenem. PCR and sequencing identified only 10 isolates (5.5%) carrying known carbapenemase genes, including bla(NDM), bla(VIM) and bla(GES). The majority of phenotypically carbapenem-resistant and carbapenemase-producing isolates did not carry a known carbapenemase gene. Transconjugant or transformant plasmid sizes were estimated to be 115 kb for bla(NDM)- and 93 kb for bla(VIM)-carrying plasmids. These plasmids were untypeable for replicon/incompatibility and transferred various other genes including plasmid-mediated quinolone resistance (PMQR) genes and bla(CTX-M-15). Typing showed that the isolates in this study were not clonally related. There is a high level of carbapenem resistance in Nigeria. As well as the globally relevant carbapenemases (bla(NDM), bla(VIM) and bla(GES)), there are other unknown gene(s) or variant(s) in circulation able to hydrolyse carbapenems and confer high-level resistance.

  18. Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation.

    PubMed

    de Haas, C J; van Leeuwen, E M; van Bommel, T; Verhoef, J; van Kessel, K P; van Strijp, J A

    2000-04-01

    Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.

  19. Complete genome sequence and cell structure of Limnochorda pilosa, a Gram-negative spore former within the phylum Firmicutes.

    PubMed

    Watanabe, Miho; Kojima, Hisaya; Fukui, Manabu

    2016-01-06

    Limnochorda pilosa is a pleomorphic facultative anaerobe and the sole species in the class Limnochordia, which has tentatively been placed in the phylum Firmicutes. In the present study, the complete genome sequence of L. pilosa HC45T was obtained and analyzed. The genome size was 3.82 Mbp and the G+C content was 69.73%. Phylogenetic analyses based on the 30S-50S ribosomal proteins and 23S rRNA gene consistently indicated that L. pilosa is phylogenetically isolated from the other members of the phylum Firmicutes. Ultrastructural observation revealed that L. pilosa possesses a Gram-negative-type cell wall and the capacity to form endospores. Accordingly, the L. pilosa genome has characteristics that are specific to Gram-negative bacteria and contains many genes that are involved in sporulation. On the other hand, several sporulation genes were absent in L. pilosa genome although they have been regarded as essential for endospore-forming system of the phylum Firmicutes. The gyrB gene of L. pilosa possesses an intein sequence. The genome has a high percentage of GTG start codons and lacks several conserved genes related to cell division.

  20. Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway.

    PubMed

    Sutterlin, Holly A; Shi, Handuo; May, Kerrie L; Miguel, Amanda; Khare, Somya; Huang, Kerwyn Casey; Silhavy, Thomas J

    2016-03-15

    Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.

  1. Polyclonal B-cell activation induced by extracts of Gram-negative bacteria isolated from periodontally diseased sites.

    PubMed Central

    Bick, P H; Carpenter, A B; Holdeman, L V; Miller, G A; Ranney, R R; Palcanis, K G; Tew, J G

    1981-01-01

    The objective of this research was to determine whether gram-negative bacteria frequently isolated from periodontally diseased sites contained polyclonal B-cell activators. Polyclonal B-cell activation, which results in nonspecific activation of multiple B-cell clones was analyzed by a hemolysis-in-gel assay designed to detect a broad range of antibody specificities. Extracts from numerous bacterial strains, including Bacteroides gingivalis, Bacteroides melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. intermedius, Fusobacterium nucleatum, Selenomonas sputigena, Capnocytophaga ochracea, and Actinobacillus actinomycetemcomitans, were tested. Extracts of the above organisms were found to stimulate polyclonal antibody responses in cultures of normal human peripheral blood lymphocytes, although the magnitude of stimulation varied among the extracts. Optimal antibody-forming cell responses were found at stimulator doses between 5 and 1,000 micrograms/ml. We conclude that the resident gram-negative subgingival flora associated with periodontal lesions possesses potent polyclonal B-cell activators. These activators may contribute to disease pathogenesis by inducing B lymphocytes to produce antibody, osteolytic factors, or both and possibly other mediators of inflammation. PMID:6975240

  2. Antibiotic Resistance and Regulation of the Gram-Negative Bacterial Outer Membrane Barrier by Host Innate Immune Molecules

    PubMed Central

    2016-01-01

    ABSTRACT The Gram-negative outer membrane is an important barrier that provides protection against toxic compounds, which include antibiotics and host innate immune molecules such as cationic antimicrobial peptides. Recently, significant research progress has been made in understanding the biogenesis, regulation, and functioning of the outer membrane, including a recent paper from the laboratory of Dr. Brett Finlay at the University of British Columbia (J. van der Heijden et al., mBio 7:e01238-16, 2016, http://dx.doi.org/10.1128/mBio.01541-16). These investigators demonstrate that toxic oxygen radicals, such as those found in host tissues, regulate outer membrane permeability by altering the outer membrane porin protein channels to regulate the influx of oxygen radicals as well as β-lactam antibiotics. This commentary provides context about this interesting paper and discusses the prospects of utilizing increased knowledge of outer membrane biology to develop new antibiotics for antibiotic-resistant Gram-negative bacteria. PMID:27677793

  3. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings

    PubMed Central

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J.

    2016-01-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests – ELISA and BIAcore – the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  4. Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

    PubMed

    Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita

    2015-04-07

    Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

  5. Morphological analysis of the antimicrobial action of nitric oxide on Gram-negative pathogens using atomic force microscopy

    PubMed Central

    Deupree, Susan M.; Schoenfisch, Mark H.

    2013-01-01

    Atomic force microscopy (AFM) was used to study the morphological changes of two Gram-negative pathogens, Pseudomonas aeruginosa and Escherichia coli, after exposure to nitric oxide (NO). The time-dependent effects of NO released from a xerogel coating and the concentration-dependent effects rendered by a small-molecule that releases NO in a bolus were examined and compared. Bacteria exhibited irregular and degraded exteriors. With NO-releasing surfaces, an increase in surface debris and disorganized adhesion patterns were observed compared to controls. Analysis of cell surface topography revealed that increasing membrane roughness correlated with higher doses of NO. At a lower total dose, NO delivered via a bolus resulted in greater membrane roughness than NO released from a surface via a sustained flux. At sub-inhibitory levels, treatment with amoxicillin, an antibiotic known to compromise the integrity of the cell wall, led to morphologies resembling those resulting from NO tr