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Sample records for lethal systemic gram-negative

  1. Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection

    PubMed Central

    Andonegui, Graciela; Zhou, Hong; Bullard, Daniel; Kelly, Margaret M.; Mullaly, Sarah C.; McDonald, Braedon; Long, Elizabeth M.; Robbins, Stephen M.; Kubes, Paul

    2009-01-01

    Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as EndotheliumTLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in EndotheliumTLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, EndotheliumTLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in EndotheliumTLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow–derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow–derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection. PMID:19603547

  2. Glucocorticoid and antibiotic effects on hepatic microcirculation and associated host responses in lethal gram-negative bacteremia.

    PubMed

    Balis, J U; Paterson, J F; Shelley, S A; Larson, C H; Fareed, J; Gerber, L I

    1979-01-01

    Liver changes and associated host responses were evaluated in four groups of male rats, weighing 300 +/- 20 gm., which received intravenous injection of 2.2 times 10(9) live Escherichia coli. This bolus was given either without additional treatment (group A) or prior to the following regimens: intramuscular injection of gentamicin sulfate, 5 mg. per kg. (group B); intravenous injection of methylprednisolone sodium succinate, 40 mg. per kg. (group C); and intramuscular injection of gentamicin immediately after methylprednisolone sodium succinate treatment (group D). Rats given injections of saline or methylprednisolone sodium succinate served as controls. Survival rates at 10 and 20 hours were 25 per cent and 4 per cent for group A; 44 per cent and 28 per cent for group B; 94 per cent and 70 per cent for group C; 98 per cent and 98 per cent for group D, respectively. In rats of groups A and B, killed at 1, 2, 4, and 6 hours, progressive liver changes included intravascular sequestration of rapidly degranulating leukocytes, fibrinous deposits, and platelet aggregates in sinusoids as well as in spaces of Disse adjacent to subendothelial collagen, and extensive Kupffer cell disruption in association with severe midzonal necrosis. These alterations were accompanied by progressive hypoglycemia and elevations of serum enzymes, glutamic pyruvic transaminase, lactate dehydrogenase, and glutamic oxaloacetic transaminase. Hematologic studies revealed that E. coli bacteremia results in rapid leukopenia and disseminated intravascular coagulation primarily due to activation of the intrinsic coagulation pathway. All above reactions were delayed and markedly reduced in rats treated with methylprednisolone sodium succinate. The results indicate that antibiotic treatment of lethal, Gram-negative bacteremia is effective only in conjunction with early steroid treatment. The protective effects of glucocorticoids on the liver microcirculation and polymorphonuclear leukocytes appear to

  3. Quorum sensing signal-response systems in Gram-negative bacteria.

    PubMed

    Papenfort, Kai; Bassler, Bonnie L

    2016-08-11

    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

  4. Meningitis - gram-negative

    MedlinePlus

    Gram-negative meningitis ... Acute bacterial meningitis can be caused by Gram-negative bacteria. Meningococcal and H. influenzae meningitis are caused by Gram-negative bacteria and are covered in detail in other articles. This article ...

  5. Surface Organelles Assembled by Secretion Systems of Gram-Negative Bacteria: Diversity in Structure and Function

    PubMed Central

    Thanassi, David G.; Bliska, James B.; Christie, Peter J.

    2012-01-01

    Gram-negative bacteria express a wide variety of organelles on their cell surface. These surface structures may be the end products of secretion systems, such as the hair-like fibers assembled by the chaperone/usher and type IV pilus pathways, which generally function in adhesion to surfaces and bacterial-bacterial and bacterial-host interactions. Alternatively, the surface organelles may be integral components of the secretion machinery itself, such as the needle complex and pilus extensions formed by the type III and type IV secretion systems, which function in the delivery of bacterial effectors inside host cells. Bacterial surface structures perform functions critical for pathogenesis and have evolved to withstand forces exerted by the external environment and cope with defenses mounted by the host immune system. Given their essential roles in pathogenesis and exposed nature, bacterial surface structures also make attractive targets for therapeutic intervention. This review will describe the structure and function of surface organelles assembled by four different Gram-negative bacterial secretion systems: the chaperone/usher pathway, the type IV pilus pathway, and the type III and type IV secretion systems. PMID:22545799

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

    PubMed

    Appelbaum, P C; Leathers, D J

    1984-10-01

    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

  7. Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation

    PubMed Central

    Fugier, Emilie; Dumont, Audrey; Malleron, Annie; Poquet, Enora; Mas Pons, Jordi; Baron, Aurélie; Vauzeilles, Boris; Dukan, Sam

    2015-01-01

    Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope. PMID:26061695

  8. Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens.

    PubMed

    Gerlach, Roman G; Hensel, Michael

    2007-10-01

    Protein secretion is a basic cellular function found in organisms of all kingdoms of life. Gram-negative bacteria have evolved a remarkable number of pathways for the transport of proteins across the cell envelope. The secretion systems fulfill general cellular functions but are also essential for pathogenic bacteria during the interaction with eukaryotic host cells. Secretion systems range from relatively simple structures such as type I secretion systems composed of three subunits that only secrete one substrate protein to complex machines such as type III and IV secretion systems composed of more than 20 subunits that can translocate large sets of effector proteins into eukaryotic target cells. In this review, the main structural and functional features of secretion systems are described. One subgroup of substrate proteins of secretion systems are protein adhesins. Despite the conserved function in binding to host cell ligands or to abiotic surfaces, the assembly of the various bacterial adhesins is highly divergent. Here we give an overview on the recent understanding of the assembly of fimbrial and non-fimbrial adhesins and the role of type I, III and V secretion systems and specialized branches of the general secretion pathway in their biogenesis. PMID:17482513

  9. Comparison of Rapid NFT system and conventional methods for identification of nonsaccharolytic gram-negative bacteria.

    PubMed

    Martin, R; Siavoshi, F; McDougal, D L

    1986-12-01

    This study examined the Rapid NFT system (Analytab Products, Plainview, N.Y.) to determine its ability to accurately identify 229 clinical isolates of mostly nonsaccharolytic gram-negative rods. Identifications were classified by the following scheme: correct (corresponding to excellent, very good, good, or acceptable identification as listed in the code book); low discrimination (correct identification among a range of listed possibilities, with additional tests necessary for accurate identification); incorrect. Correct identification was considered correct to species and subspecies for all organisms except Alcaligenes faecalis and "Alcaligenes odorans"; "A. faecalis/odorans" was considered a correct response. By using these criteria, 71.6% of the strains were correctly identified, 17.9% were identified with low discrimination, and 10.5% were incorrectly identified. When consideration was made for incorrect identification resulting from taxonomic problems (e.g., Alcaligenes and Moraxella spp.), incorrect identifications fell to 5.2%. The Rapid NFT system was truly rapid and was easy to use and interpret. Its use of carbon substrate assimilation enables it to provide more accurate identification of medically important nonsaccharolytic bacteria than do other commercially available systems. PMID:3536999

  10. The Type III secretion system of Gram-negative bacteria: a potential therapeutic target?

    PubMed

    Müller, Simone; Feldman, Mario F; Cornelis, Guy R

    2001-06-01

    Several pathogenic Gram-negative bacteria, including Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa and enteropathogenic Escherichia coli harbour a complex attack system called 'Type III secretion' which is, in every case, an essential virulence determinant. This system, activated by contact with an eukaryotic cell membrane, allows bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane, to reach the cell's cytosol and destroy or subvert the host cell. The Type III virulence mechanism consists of a secretion apparatus, made up of about 25 proteins, and a set of effector proteins released by this apparatus. The mechanism of protein secretion is highly conserved among the different bacteria, although they cause a variety of diseases with different symptoms and severities, from fatal septicaemia to mild diarrhoea or from fulgurant diarrhoea to chronic infection of the lung. This review focuses on the proteins that make up the secretion machinery and examine if it could be a potential target for novel antimicrobials. PMID:12540268

  11. Comparison of the Cathra Repliscan II, the AutoMicrobic system Gram-Negative General Susceptibility-Plus Card, and the Micro-Media System Fox Panel for dilution susceptibility testing of gram-negative bacilli.

    PubMed

    Reiber, N E; Kelly, M T; Latimer, J M; Tison, D L; Hysmith, R M

    1985-06-01

    A comparative evaluation was done to test the accuracy of the Cathra Repliscan II agar dilution system (Diagnostic Equipment, Inc., St. Paul, Minn.), the AutoMicrobic system with Gram-Negative General Susceptibility-Plus Card (Vitek Systems, Inc., Hazelwood, Mo.), and the Micro-Media Fox Panel micro broth dilution system (Micro-Media Systems, Inc., San Jose, Calif.) in determining MICs of 12 antibiotics for 200 gram-negative bacilli. Of the 200 strains tested, 12 isolates did not grow in one of the three systems. The 188 remaining organisms included 158 members of the family Enterobacteriaceae, 20 Pseudomonas spp., 5 Acinetobacter sp., 3 Aeromonas spp., and 2 Vibrio spp. A total of 2,256 organism-antibiotic combinations were analyzed for each system. An MIC was considered correct if two of the three systems were in agreement. When disagreements occurred, correct MICs were determined by the standard agar dilution method. With this criterion, overall agreements of the Cathra Repliscan II system, AutoMicrobic system, and Micro-Media Fox Panel system were 94.7, 94.9, and 95.5%, respectively. Tetracycline (20%), nitrofurantoin (20%), and ampicillin (16%) accounted for 56% of the discrepancies observed. These results indicate that all three systems perform with a high degree of accuracy for susceptibility testing of gram-negative bacilli. PMID:4008625

  12. 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.

  13. Systemic Activation of TLR3-Dependent TRIF Signaling Confers Host Defense against Gram-Negative Bacteria in the Intestine

    PubMed Central

    Ruiz, Jose; Kanagavelu, Saravana; Flores, Claudia; Romero, Laura; Riveron, Reldy; Shih, David Q.; Fukata, Masayuki

    2016-01-01

    Recognition of Gram-negative bacteria by toll-like receptor (TLR)4 induces MyD88 and TRIF mediated responses. We have shown that TRIF-dependent responses play an important role in intestinal defense against Gram-negative enteropathogens. In the current study, we examined underlying mechanisms of how systemic TRIF activation enhances intestinal immune defense against Gram-negative bacteria. First we confirmed that the protective effect of poly I:C against enteric infection of mice with Yersinia enterocolitica was dependent on TLR3-mediated TRIF signaling by using TLR3-deficient mice. This protection was unique in TRIF-dependent TLR signaling because systemic stimulation of mice with agonists for TLR2 (Pam3CSK4) or TLR5 (flagellin) did not reduce mortality on Y. enterocolitica infection. Systemic administration of poly I:C mobilized CD11c+, F4/80+, and Gr−1hi cells from lamina propria and activated NK cells in the mesenteric lymph nodes (MLN) within 24 h. This innate immune cell rearrangement was type I IFN dependent and mediated through upregulation of TLR4 followed by CCR7 expression in these innate immune cells found in the intestinal mucosa. Poly I:C induced IFN-γ expression by NK cells in the MLN, which was mediated through type I IFNs and IL-12p40 from antigen presenting cells and consequent activation of STAT1 and STAT4 in NK cells. This formation of innate immunity significantly contributed to the elimination of bacteria in the MLN. Our results demonstrated an innate immune network in the intestine that can be established by systemic stimulation of TRIF, which provides a strong host defense against Gram-negative pathogens. The mechanism underlying TRIF-mediated protective immunity may be useful to develop novel therapies for enteric bacterial infection. PMID:26793623

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

    PubMed Central

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

    2016-01-01

    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/PchnB 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]. PMID:26870759

  15. 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]. PMID:26870759

  16. 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

  17. Think (Gram) negative!

    PubMed Central

    2010-01-01

    The increasing prevalence of multiresistant Gram-negative bacteria of the Enterobacteriaceae family in Europe is a worrisome phenomenon. Extended spectrum betalactamase-producing Escherichia coli strains are widespread in the community and are frequently imported into the hospital. Of even more concern is the spread of carbapenem-resistant strains of Klebsiella spp. from regions where they are already endemic. Antibiotic use is a main driver of antibiotic resistance, which again increases broad spectrum antibiotic use, resulting in a vicious circle that is difficult to interrupt. The present commentary highlights important findings of a surveillance study of antimicrobial use and resistance in German ICUs over 8 years with a focus on Gram-negative resistance. PMID:20587087

  18. [Evaluation of the GNF computer-coding system for the identification of non-fermentative Gram-negative bacilli].

    PubMed

    Tarng, C M; Chen, M M; Tsai, W C

    1983-05-01

    In order to evaluate the effectiveness of the GNF computer-coding system for the identification of glucose non-fermenting gram-negative bacilli, we employed 406 strains of bacteria including 367 clinical isolates and 39 standard strains for testing. These strains were inoculated into the following eleven conventional biochemical test media: Triple Sugar Iron Agar, Simmon's Citrate Agar, Christensen's Urea Agar, Sulfide-Indole-Motility Medium, Semisolid Voges-Proskauer Test Medium, Moeller's Ornithine Decarboxylase Test Medium, Pyocyanin Test Medium, Oxidation/Fermentation (O/F) Glucose, O/F Fructose, Nitrate Broth, Moeller's Arginine Dihydrolase Test Medium. The results of these tests plus those from the hanging drop motility test and the oxidase test were converted into bacterial code number and then checked with the GNF computer-coding system. It was found that the first preference of agreement was 75.6%, second 15.3%, third 5.9%, and fourth or more 3.2%. In regard to the speed of bacterial identification by using the GNF system and information from hemolysis pattern and flagella stain, it was indicated that 84.7% would be correctly identified within 36-48 hours after isolation. If more confirmational tests were employed, the accurate identification rate would reach to 98.7% after 4 days of isolation. In addition, the use of the GNF computer-coding system can standardize identification procedures, shorten the identification period, and save cost in terms of materials supply, inoculation time, media preparation and media-storing space. Therefore, we conclude that the GNF computer-coding system is an effective tool in the identification of the glucose non-fermenting gram-negative bacilli. PMID:6617315

  19. Systemic toxoplasmosis and Gram-negative sepsis in a southern chamois (Rupicapra pyrenaica) from the Pyrenees in northeast Spain.

    PubMed

    Marco, Ignasi; Velarde, Roser; López-Olvera, Jorge R; Cabezón, Oscar; Pumarola, Martí; Lavín, Santiago

    2009-03-01

    A 6-year-old, male southern chamois (Rupicapra pyrenaica) had an absence of flight response and was captured by hand in the Catalan Pyrenees in northeast Spain. On clinical examination, the animal was in good body condition, and only atrophy of the right eye was observed. Blood samples were collected and hematologic analysis performed, but no alterations were observed. The animal was sent to a Wildlife Rescue Centre, where it developed chronic wasting and died after 32 days in captivity. At necropsy, the animal was cachectic and had edematous, mottled lungs. Histopathologic examination revealed systemic toxoplasmosis and acute Gram-negative septicemia. The protozoan organisms were identified as Toxoplasma gondii based on immunohistochemistry. An indirect fluorescent antibody test was performed, and the animal was positive with an antibody titer of 150. PMID:19286506

  20. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    DOEpatents

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  1. Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

    PubMed Central

    Mazzola, Priscila G; Martins, Alzira MS; Penna, Thereza CV

    2006-01-01

    Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite

  2. Accuracy and reproducibility of the Oxi/Ferm system in identifying a select group of unusual gram-negative bacilli.

    PubMed Central

    Nadler, H; George, H; Barr, J

    1979-01-01

    The Oxi/Ferm (O/F) identification system was compared in a double-blind study to a conventional test battery for the characterization of 96 reference and clinical strains consisting of 83 nonfermentative and 13 oxidase-producing, fermentative gram-negative bacilli. The O/F tube and supplemental tests correctly identified 84% of the nonfermentative and 77% of the oxidase-producing, fermentative bacilli. However, when the supplemental tests were excluded and the biochemical profiles generated by all nine O/F tube reactions were examined, the profile accuracy reached 95% (79 of 83) for the nonfermentative and 93% (12 of 13) for oxidase-producing, fermentative bacilli. Seven of the nine O/F substrate reactions demonstrated less than or equal to 89% agreement with conventional reactions, whereas the urea and arginine reactions provided 82 and 85% agreement, respectively. Replicate O/F tests with six selected organisms demonstrated 97% identification reproducibility and 84% overall substrate reproducibility. The mean O/F identification time was 2.6 days as compared to 3.3 days for the conventional system. Although this study suggests that the O/F system is a convenient, rapid, and accurate alternative to conventional identification methods, several modifications are recommended. PMID:372222

  3. Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

    PubMed

    Branda, John A; 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 F; Westblade, Lars F; Ferraro, Mary Jane

    2014-02-01

    The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level. PMID:24321357

  4. Nissui Glucose Fermentative Gram-Negative Rod Identification System EB-20 Gives a Unique Profile for Typical Non-Sorbitol-Fermenting Escherichia coli O157:H7

    PubMed Central

    Kodaka, H.; Uesaka, Y.; Kashitani, F.

    2004-01-01

    The 98 non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 strains identified on a Nissui glucose fermentative gram-negative rod identification system (EB-20) gave a unique biochemical profile number that was not detected in 85 pathogenic and 13 nonpathogenic E. coli strains. Thus, EB-20 is useful for the identification of NSF E. coli O157:H7 and provides a simple, cost-effective, and reliable tool for clinical laboratories. PMID:14715777

  5. Meningitis - gram-negative

    MedlinePlus

    ... and children include: Infection Recent brain surgery Recent injury to the head Spinal abnormalities Spinal fluid shunt placement after brain surgery Urinary tract abnormalities Urinary tract infection Weakened immune system

  6. The Role of Bacterial Secretion Systems in the Virulence of Gram-Negative Airway Pathogens Associated with Cystic Fibrosis

    PubMed Central

    Depluverez, Sofie; Devos, Simon; Devreese, Bart

    2016-01-01

    Cystic fibrosis (CF) is the most common lethal inherited disorder in Caucasians. It is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene. A defect in the CFTR ion channel causes a dramatic change in the composition of the airway surface fluid, leading to a highly viscous mucus layer. In healthy individuals, the majority of bacteria trapped in the mucus layer are removed and destroyed by mucociliary clearance. However, in the lungs of patients with CF, the mucociliary clearance is impaired due to dehydration of the airway surface fluid. As a consequence, patients with CF are highly susceptible to chronic or intermittent pulmonary infections, often causing extensive lung inflammation and damage, accompanied by a decreased life expectancy. This mini review will focus on the different secretion mechanisms used by the major bacterial CF pathogens to release virulence factors, their role in resistance and discusses the potential for therapeutically targeting secretion systems. PMID:27625638

  7. The Role of Bacterial Secretion Systems in the Virulence of Gram-Negative Airway Pathogens Associated with Cystic Fibrosis.

    PubMed

    Depluverez, Sofie; Devos, Simon; Devreese, Bart

    2016-01-01

    Cystic fibrosis (CF) is the most common lethal inherited disorder in Caucasians. It is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene. A defect in the CFTR ion channel causes a dramatic change in the composition of the airway surface fluid, leading to a highly viscous mucus layer. In healthy individuals, the majority of bacteria trapped in the mucus layer are removed and destroyed by mucociliary clearance. However, in the lungs of patients with CF, the mucociliary clearance is impaired due to dehydration of the airway surface fluid. As a consequence, patients with CF are highly susceptible to chronic or intermittent pulmonary infections, often causing extensive lung inflammation and damage, accompanied by a decreased life expectancy. This mini review will focus on the different secretion mechanisms used by the major bacterial CF pathogens to release virulence factors, their role in resistance and discusses the potential for therapeutically targeting secretion systems. PMID:27625638

  8. 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

  9. 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

  10. Differentiation of gram-negative, nonfermentative bacteria isolated from biofilters on the basis of Fatty Acid composition, quinone system, and physiological reaction profiles.

    PubMed

    Lipski, A; Klatte, S; Bendinger, B; Altendorf, K

    1992-06-01

    Gram-negative, nonfermentative bacteria isolated from biofilters for off-gas treatment of animal-rendering-plant emissions were differentiated by whole-cell fatty acid analysis, quinone analysis, and numerical taxonomy based on their physiological reaction profiles. The last system consisted of 60 physiological tests and was arranged as a microtest system on microtitration plates. Based on fatty acid analyses, 31 isolates were separated into six clusters and five single-member clusters. The isolates of two clusters were identified as Alcaligenes faecalis and Pseudomonas diminuta. The remaining nine clusters were characterized by their fatty acid profiles, quinone systems, and physiological reaction profiles. Clusters resulting from fatty acid analyses were compared with those resulting from physiological reaction profiles. Six clusters could be confirmed this way. The efficiency of the physiological test system was increased by the prearrangement of the isolates according to their quinone type. PMID:16348724

  11. Carbapenem-resistant Gram-negative bacteria – analysis of the data obtained through a mandatory reporting system in the Rhine-Main region, Germany, 2012–2015

    PubMed Central

    Heudorf, Ursel; Büttner, Barbara; Hauri, Anja M.; Heinmüller, Petra; Hunfeld, Klaus-Peter; Kaase, Martin; Kleinkauf, Niels; Albert-Braun, Sabine; Tessmann, Rolf; Kempf, Volkhard A. J.

    2016-01-01

    Background: Multidrug-resistant Gram-negative bacteria (MRGN) and the infections they cause are a serious threat and a challenge to the healthcare system. This particularly applies to carbapenem-resistant Gram-negative bacteria (CRGN). Currently, the introduction of a nationwide mandatory notification system for CRGN in Germany is under consideration. Against this background, this paper presents an analysis of the mandatory reporting system for CRGN in effect since November 2011 in the federal state of Hesse (Germany). Materials and methods: All carbapenem-resistant Gram-negative bacteria and the detected carbapenemases reported to the public health department of the city of Frankfurt am Main, Hesse, Germany, on the basis of the mandatory notification system were analyzed. Results: 827 CRGN cases were reported to the public health department of Frankfurt/Main between April 2012 and December 2015. The following bacterial species were reported: Pseudomonas spp. (n=268), Acinetobacter spp. (n=183), Klebsiella spp. (n=195), Enterobacter spp. (n=77), Escherichia coli (n=75) and others (n=29). Between 2012 and 2015, a reduction of the CRGN reports was noticed, mainly due to changes in the reporting of Pseudomonas spp. Between 2012 and 2015, the total number of notifications decreased slightly, although the number of reported CRGN in screening samples increased, thus giving no indication of a decreased testing frequency. For 10.5% of the patients, the place of residence was not Germany, 18.0% of the patients had previously stayed in hospitals abroad, often in countries with a high CRGN prevalence. CRGN bacteria were reported from all of Frankfurt’s hospitals, and 3.9% were reported from out-patient care facilities. Carbapenemases were detected and reported in 251 CRGN bacteria, including 73 OXA-48, 76 OXA-23, 56 NDM subtypes, and 21 KPC subtypes. There have been no major epidemiological signs of outbreak scenarios. Discussion: CRGN bacteria are already widespread in

  12. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    PubMed

    Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

    2016-03-01

    The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. PMID:26833388

  13. Collaborative evaluation of the Abbott Avantage system for identification of frequently isolated nonfermentative or oxidase-positive gram-negative bacilli.

    PubMed Central

    Jorgensen, J H; Dyke, J W; Helgeson, N G; Cooper, B H; Redding, J S; Crawford, S A; Andruszewski, M T; Prowant, S A

    1984-01-01

    The capability of the Abbott Avantage system to identify 10 species of commonly isolated glucose nonfermentative or oxidase-positive gram-negative bacilli in a 5-h test period was evaluated in a collaborative study. The Avantage nonenteric data base uses 20 biochemical test reactions performed in an expanded Abbott bacterial identification cartridge plus the results of a manual oxidase test. The species included in the Avantage data base are Acinetobacter anitratus, Acinetobacter Iwoffi, Aeromonas hydrophila, Flavobacterium meningosepticum-IIb group, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens-putida group, Pseudomonas maltophilia, Pasteurella multocida, and Plesiomonas shigelloides. The collaborative study included the testing of 200 coded challenge strains in all three laboratories and the subsequent testing of an additional group of 100 to 200 clinical isolates recovered independently by each laboratory. Reference identifications for all isolates were determined by conventional biochemical test reactions. The overall accuracy of identification of the coded challenge strains for the three laboratories was 97%, whereas 95% of 437 clinical isolates and selected stock cultures of clinical derivation were identified correctly. PMID:6392323

  14. Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

    PubMed

    Cangelosi, Chris; Hannagan, Susan; Santiago, Clayton P; Wilson, James W

    2015-11-01

    Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria. PMID:26505312

  15. Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

    PubMed

    Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

    2010-06-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen. PMID:20363780

  16. Comparison of Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometer to BD Phoenix Automated Microbiology System for Identification of Gram-Negative Bacilli▿

    PubMed Central

    Saffert, Ryan T.; Cunningham, Scott A.; Ihde, Sherry M.; Monson Jobe, Kristine E.; Mandrekar, Jayawant; Patel, Robin

    2011-01-01

    We compared the BD Phoenix automated microbiology system to the Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) system for identification of Gram-negative bacilli, using biochemical testing and/or genetic sequencing to resolve discordant results. The BD Phoenix correctly identified 363 (83%) and 330 (75%) isolates to the genus and species level, respectively. The Bruker Biotyper correctly identified 408 (93%) and 360 (82%) isolates to the genus and species level, respectively. The 440 isolates were grouped into common (308) and infrequent (132) isolates in the clinical laboratory. For the 308 common isolates, the BD Phoenix and Bruker Biotyper correctly identified 294 (95%) and 296 (96%) of the isolates to the genus level, respectively. For species identification, the BD Phoenix and Bruker Biotyper correctly identified 93% of the common isolates (285 and 286, respectively). In contrast, for the 132 infrequent isolates, the Bruker Biotyper correctly identified 112 (85%) and 74 (56%) isolates to the genus and species level, respectively, compared to the BD Phoenix, which identified only 69 (52%) and 45 (34%) isolates to the genus and species level, respectively. Statistically, the Bruker Biotyper overall outperformed the BD Phoenix for identification of Gram-negative bacilli to the genus (P < 0.0001) and species (P = 0.0005) level in this sample set. When isolates were categorized as common or infrequent isolates, there was statistically no difference between the instruments for identification of common Gram-negative bacilli (P > 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated Gram-negative bacilli (P < 0.0001). PMID:21209160

  17. 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...

  18. V-antigen homologs in pathogenic gram-negative bacteria.

    PubMed

    Sawa, Teiji; Katoh, Hideya; Yasumoto, Hiroaki

    2014-05-01

    Gram-negative bacteria cause many types of infections in animals from fish and shrimps to humans. Bacteria use Type III secretion systems (TTSSs) to translocate their toxins directly into eukaryotic cells. The V-antigen is a multifunctional protein required for the TTSS in Yersinia and Pseudomonas aeruginosa. V-antigen vaccines and anti-V-antigen antisera confer protection against Yersinia or P. aeruginosa infections in animal models. The V-antigen forms a pentameric cap structure at the tip of the Type III secretory needle; this structure, which has evolved from the bacterial flagellar cap structure, is indispensable for toxin translocation. Various pathogenic gram-negative bacteria such as Photorhabdus luminescens, Vibrio spp., and Aeromonas spp. encode homologs of the V-antigen. Because the V-antigens of pathogenic gram-negative bacteria play a key role in toxin translocation, they are potential therapeutic targets for combatting bacterial virulence. In the USA and Europe, these vaccines and specific antibodies against V-antigens are in clinical trials investigating the treatment of Yersinia or P. aeruginosa infections. Pathogenic gram-negative bacteria are of great interest because of their ability to infect fish and shrimp farms, their potential for exploitation in biological terrorism attacks, and their ability to cause opportunistic infections in humans. Thus, elucidation of the roles of the V-antigen in the TTSS and mechanisms by which these functions can be blocked is critical to facilitating the development of improved anti-V-antigen strategies. PMID:24641673

  19. Relationship between immune system and gram negative bacteria. I. Spontaneous binding of smooth and rough Salmonella to human peripheral blood lymphocytes.

    PubMed Central

    Jirillo, E; Antonaci, S; Michalek, S M; Colwell, D E; McGhee, J R; Bonomo, L

    1984-01-01

    Over the past years many reports have emphasized that either Gram positive or Gram negative bacteria possess the ability to bind spontaneously to human peripheral blood lymphocytes (PBL). Here, bacterial binding to human PBL has been studied by using a smooth (S) Salmonella typhimurium LT-2 and two rough (R) mutants of Salmonella minnesota R 345 (Rb) and R 595 (Re), which possess specific deletions in their lipopolysaccharide (LPS) molecule. Our results provide evidence that all three bacterial strains spontaneously bind to PBL, even though Re and mostly Rb cells display the highest degree of adherence. The three major regions of LPS (O-polysaccharide chain, R core and lipid A) seem to be involved in the binding since adherence is specifically inhibited by pretreating PBL with S- or R-LPS extracted from homologous bacteria. Furthermore, using a panel of monoclonal antibodies to lymphocyte surface antigens, S- and R-Salmonella bacteria bind to T lymphocytes (preferentially T8+ cells), while few B cells are coated by bacteria. Additionally, bacterial binding is significantly reduced by trypsin pretreatment of PBL, this suggesting that proteins (or glycoproteins) of the PBL membrane are involved in the binding. PMID:6383666

  20. Veillonella, Firmicutes: Microbes disguised as Gram negatives

    PubMed Central

    Vesth, Tammi; Ozen, Aslı; Andersen, Sandra C.; Kaas, Rolf Sommer; Lukjancenko, Oksana; Bohlin, Jon; Nookaew, Intawat; Wassenaar, Trudy M.; Ussery, David W.

    2013-01-01

    The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the

  1. 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. PMID:27062655

  2. Recombinant α1-Antitrypsin Pittsburgh Attenuates Experimental Gram-Negative Septicemia

    PubMed Central

    Colman, Robert W.; Flores, Daniel N.; De La Cadena, Raul A.; Scott, Cheryl F.; Cousens, Laurence; Barr, Philip J.; Hoffman, Ian B.; Kueppers, Friedrich; Fisher, Donald; Idell, Steven; Pisarello, Jorge

    1988-01-01

    Alpha1-antitrypsin-Pittsburgh (AT-P), a naturally occurring lethal mutation (358Met → Arg), has been genetically engineered (rAT-P). The protein has been shown to be a potent active site-directed inhibitor of thrombin and the contact enzymes Factor XIIf, Factor XIa, and kallikrein. Because activation of the contact system is known to occur in gram-negative septicemia, the authors have hypothesized that the administration of rAT-P might modulate the course of this syndrome. Yorkshire piglets anesthetized with pentobarbital and infused with viable Pseudomonas aeruginosa (2 X 108 CFU) were untreated (Group I) or treated with rAT-P (Group II) and studied in a 6-hour protocol. Coagulation studies revealed that rAT-P significantly inhibited the rapid decrease in the functional concentrations of Antithrombin III, Factor XI, and fibrinogen. In addition, rAT-P markedly reduced the serum levels of fibrinogen degradation products. Survival in Group II was significantly increased during 2-5 hours but not at 6 hours when the functional levels of rAT-P in plasma were the lowest. These results indicate that this recombinant inhibitor, even at low concentrations, affords protection in experimental gram-negative septicemia. PMID:3257651

  3. The Lethality Test System

    NASA Astrophysics Data System (ADS)

    Parsons, W. M.; Sims, J. R.; Parker, J. V.

    1986-11-01

    The Lethality Test System (LTS) under construction at Los Alamos is an electromagnetic launcher facility designed to perform impact experiments at velocities up to 15 km/sec. The launcher is a 25 mm round bore, plasma armature railgun 22 m in length. Preinjection is accomplished with a two-stage light gas gun capable of 7 km/sec. The railgun power supply utilizes traction motors, vacuum interrupters, and pulse transformers. An assembly of 28 traction motors, equipped with flywheels, stores approximately 80 MJ at 92 percent of full speed and energizes the primary windings of three pulse transformers at a current of 50 kA. At peak current an array of vacuum interrupters disconnects the transformer primary windings and forces the current to flow in the secondary windings. The secondary windings are connected to the railgun, and by staging the vacuum interrupter openings, a 1-1.3 MA ramped current waveform will be delivered to the railgun.

  4. Lethality test system

    SciTech Connect

    Parsons, W.M.; Sims, J.R.; Parker, J.V.

    1986-01-01

    The Lethality Test System (LTS), presently under construction at Los Alamos, is an electromagnetic launcher facility designed to perform impact experiments at velocities up to 15 km/s. The launcher is a 25 mm round bore, plasma armature railgun extending 22 m in length. Preinjection is accomplished with a two-stage gas gun capable of 7 km/s. The railgun power supply utilizes traction motors, vacuum interrupters, and pulse transformers. An assembly of 28 traction motors, equipped with flywheels, stores approximately 80 MJ at 92% of full speed and energizes the primary windings of three pulse transformers at a current of 50 kA. At peak current an array of vacuum interrupters disconnects the transformer primary windings and forces the current to flow in the secondary windings. The secondary windings are connected to the railgun, and by staging the vacuum interrupter openings, a 1 MA to 1.3 MA ramped current waveform will be delivered to the railgun.

  5. Direct comparison of the BD phoenix system with the MicroScan WalkAway system for identification and antimicrobial susceptibility testing of Enterobacteriaceae and nonfermentative gram-negative organisms.

    PubMed

    Snyder, J W; Munier, G K; Johnson, C L

    2008-07-01

    The Phoenix automated microbiology system (BD Diagnostics, Sparks, MD) is designed for the rapid identification (ID) and antimicrobial susceptibility testing (AST) of clinically significant human bacterial pathogens. We evaluated the performance of the Phoenix instrument in comparison with that of the MicroScan WalkAway system (Dade Behring, West Sacramento, CA) in the ID and AST of gram-negative clinical strains and challenge isolates of Enterobacteriaceae (n = 150) and nonfermentative gram-negative bacilli (NFGNB; 45 clinical isolates and 8 challenge isolates). ID discrepancies were resolved with the API 20E and API 20NE conventional biochemical ID systems (bioMerieux, Durham, NC). The standard disk diffusion method was used to resolve discordant AST results. The overall percentages of agreement between the Phoenix ID results and the MicroScan results at the genus and species levels for clinical isolates of Enterobacteriaceae were 98.7 and 97.7%, respectively; following resolution with conventional biochemical testing, the accuracy of the Phoenix system was determined to be 100%. For NFGNB, the levels of agreement were 100 and 97.7%, respectively. Both systems incorrectly identified the majority of the uncommon nonfermentative nonpseudomonal challenge isolates recovered from cystic fibrosis patients; these isolates are not included in the databases of the respective systems. For AST of Enterobacteriaceae, the rate of complete agreement between the Phoenix results and the MicroScan results was 97%; the rates of very major, major, and minor errors were 0.3, 0.2, and 2.7%, respectively. For NFGNB, the rate of complete agreement between the Phoenix results and the MicroScan results was 89.1%; the rates of very major, major, and minor errors were 0, 0.5, and 7.7%, respectively. Following the confirmatory testing of nine clinical isolates initially screened by the MicroScan system as possible extended-spectrum-beta-lactamase (ESBL)-producing organisms (seven Klebsiella

  6. 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. PMID:27226088

  7. 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

  8. The genetics of glycosylation in Gram-negative bacteria.

    PubMed

    Power, P M; Jennings, M P

    2003-01-28

    In recent years there has been a dramatic increase in reports of glycosylation of proteins in various Gram-negative systems including Neisseria meningitidis, Neisseria gonorrhoeae, Campylobacter jejuni, Pseudomonas aeruginosa, Escherichia coli, Caulobacter crescentus, Aeromonas caviae and Helicobacter pylori. Although this growing list contains many important pathogens (reviewed by Benz and Schmidt [Mol. Microbiol. 45 (2002) 267-276]) and the glycosylations are found on proteins important in pathogenesis such as pili, adhesins and flagella the precise role(s) of the glycosylation of these proteins remains to be determined. Furthermore, the details of the glycosylation biosynthetic process have not been determined in any of these systems. The definition of the precise role of glycosylation and the mechanism of biosynthesis will be facilitated by a detailed understanding of the genes involved. PMID:12586395

  9. Lipopolysaccharide modification in Gram-negative bacteria during chronic infection.

    PubMed

    Maldonado, Rita F; Sá-Correia, Isabel; Valvano, Miguel A

    2016-07-01

    The Gram-negative bacterial lipopolysaccharide (LPS) is a major component of the outer membrane that plays a key role in host-pathogen interactions with the innate immune system. During infection, bacteria are exposed to a host environment that is typically dominated by inflammatory cells and soluble factors, including antibiotics, which provide cues about regulation of gene expression. Bacterial adaptive changes including modulation of LPS synthesis and structure are a conserved theme in infections, irrespective of the type or bacteria or the site of infection. In general, these changes result in immune system evasion, persisting inflammation and increased antimicrobial resistance. Here, we review the modifications of LPS structure and biosynthetic pathways that occur upon adaptation of model opportunistic pathogens (Pseudomonas aeruginosa, Burkholderia cepacia complex bacteria, Helicobacter pylori and Salmonella enterica) to chronic infection in respiratory and gastrointestinal sites. We also discuss the molecular mechanisms of these variations and their role in the host-pathogen interaction. PMID:27075488

  10. Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies.

    PubMed Central

    Teng, N N; Kaplan, H S; Hebert, J M; Moore, C; Douglas, H; Wunderlich, A; Braude, A I

    1985-01-01

    Hybridomas producing human monoclonal IgM antibodies (mAbs) against bacterial lipopolysaccharide (LPS) were generated by fusion of B lymphocytes from sensitized human spleen with heteromyeloma cells. The splenocytes were from patients undergoing splenectomy during staging for Hodgkin disease after vaccination with the J5 mutant of Escherichia coli, which is deficient in O antigenic side chains. This deficiency exposes the core oligosaccharide, common to LPS of all Gram-negative bacteria. The mAbs cross-reacted strongly with endotoxins from a wide range of unrelated species of Gram-negative bacteria. The mAbs also gave strong protection against LPS in the dermal Shwartzman reaction and against lethal Gram-negative bacteremia in mice. These findings indicate that monoclonal IgM against LPS endotoxin can neutralize its toxicity in vivo and might be valuable for treatment of patients with Gram-negative bacteremia. Analysis of one of the hybridoma clones, A6(H4C5), showed that the IgM mAb is directed against the covalently bound lipid A, which represents the most conservative and least variable structural element of LPS. Images PMID:3856860

  11. Vaccine Approaches for Multidrug Resistant Gram negative infections

    PubMed Central

    Campfield, Brian; Chen, Kong; Kolls, Jay K.

    2014-01-01

    Multidrug resistant (MDR) Gram negative bacterial infections are increasing in frequency and are associated with significant financial costs, morbidity and mortality. Current antibiotic therapies are associated with unacceptably poor clinical outcomes and toxicity. Unfortunately, the development of novel antimicrobials is stagnant leaving a significant clinical need for alternative treatments of MDR Gram negative rod infections. Recent preclinical studies have identified Th17 cells as critical mediators of broadly protective adaptive immunity, including protection against MDR infections. Studies of Th17 eliciting antigens, adjuvants and routes of immunization have identified potential vaccine strategies that may confer long-lived adaptive immunity against MDR Gram negative bacterial infections. PMID:24637162

  12. 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

  13. Procalcitonin Levels in Gram-Positive, Gram-Negative, and Fungal Bloodstream Infections

    PubMed Central

    Ferranti, Marta; Moretti, Amedeo; Al Dhahab, Zainab Salim; Cenci, Elio; Mencacci, Antonella

    2015-01-01

    Procalcitonin (PCT) can discriminate bacterial from viral systemic infections and true bacteremia from contaminated blood cultures. The aim of this study was to evaluate PCT diagnostic accuracy in discriminating Gram-positive, Gram-negative, and fungal bloodstream infections. A total of 1,949 samples from patients with suspected bloodstream infections were included in the study. Median PCT value in Gram-negative (13.8 ng/mL, interquartile range (IQR) 3.4–44.1) bacteremias was significantly higher than in Gram-positive (2.1 ng/mL, IQR 0.6–7.6) or fungal (0.5 ng/mL, IQR 0.4–1) infections (P < 0.0001). Receiver operating characteristic analysis showed an area under the curve (AUC) for PCT of 0.765 (95% CI 0.725–0.805, P < 0.0001) in discriminating Gram-negatives from Gram-positives at the best cut-off value of 10.8 ng/mL and an AUC of 0.944 (95% CI 0.919–0.969, P < 0.0001) in discriminating Gram-negatives from fungi at the best cut-off of 1.6 ng/mL. Additional results showed a significant difference in median PCT values between Enterobacteriaceae and nonfermentative Gram-negative bacteria (17.1 ng/mL, IQR 5.9–48.5 versus 3.5 ng/mL, IQR 0.8–21.5; P < 0.0001). This study suggests that PCT may be of value to distinguish Gram-negative from Gram-positive and fungal bloodstream infections. Nevertheless, its utility to predict different microorganisms needs to be assessed in further studies. PMID:25852221

  14. 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

  15. Biogenesis of outer membranes in Gram-negative bacteria.

    PubMed

    Tokuda, Hajime

    2009-03-23

    The outer membrane, an essential organelle of Gram-negative bacteria, is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins. The mechanisms underlying the transport of these components to outer membranes are currently under extensive examination. Among them, the sorting of lipoproteins to the outer membrane of Escherichia coli has been clarified in detail. The Lol system, composed of five proteins, catalyzes outer membrane sorting of lipoproteins. Various Lpt proteins have recently been identified as factors involved in the transport of lipopolysaccharide to the outer membrane, although the mechanism remains largely unknown. Proteins with alpha-helical membrane spanning segments are found in the inner membrane, whereas amphipathic beta-barrel proteins span the outer membrane. These beta-barrel proteins are inserted into the outer membranes through a central core protein BamA (YaeT) with the help of four outer membrane lipoproteins. In contrast, little is known about how phospholipids are transported to the outer membrane. PMID:19270402

  16. Forming crosslinked peptidoglycan from synthetic Gram-negative Lipid II

    PubMed Central

    Lebar, Matthew D.; Lupoli, Tania J.; Tsukamoto, Hirokazu; May, Janine M.; Walker, Suzanne; Kahne, Daniel

    2013-01-01

    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 crosslinking 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. PMID:23480167

  17. In vitro activity of imipenem towards gram-negative bacilli.

    PubMed

    Speciale, A; Caccamo, F; Pellegrino, M B; Blandino, G; Nicoletti, G

    1986-10-01

    The authors studied the in vitro antimicrobial activity of imipenem towards 355 gram-negative bacterial strains, taking into particular consideration unusual or dangerous species. The study was carried out on a comparative basis with piperacillin, cefotaxime, ceftazidime and gentamicin. Ninety percent of the fermentative gram-negative strains were inhibited at concentrations less than or equal to 2 mg/l. Imipenem inhibited 100% of strains of Alcaligenes faecalis, Alcaligenes denitrificans, Flavobacterium odoratum, Acinetobacter lwoffii, Acinetobacter anitratum, Pseudomonas fluorescens, Pseudomonas stutzeri and 90% of strains of Pseudomonas aeruginosa and Pseudomonas putida. The excellent bactericidal activity of imipenem was indicated by its minimum bactericidal concentrations equal to or slightly greater than its minimum inhibitory concentrations (MIC). As far as the other parameters were concerned (influence of the dimensions of inoculum and culture medium on MICs), imipenem confirmed its excellent in vitro microbiologic characteristics. PMID:3466723

  18. 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.

  19. 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

  20. A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of all Gram-negative bacteria and is largely responsible for evoking the de...

  1. 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.

  2. 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

  3. Alteco endotoxin hemoadsorption in Gram-negative septic shock patients

    PubMed Central

    Shum, Hoi Ping; Leung, Yuk Wah; Lam, Sin Man; Chan, King Chung; Yan, Wing Wa

    2014-01-01

    Background and Aims: Severe sepsis and septic shock are common causes of mortality and morbidity in an intensive care unit setting. Endotoxin, derived from the outer membranes of Gram-negative bacteria, is considered a major factor in the pathogenesis of sepsis. This study investigated the effect of Alteco endotoxin hemoadsorption device on Gram-negative septic shock patients. Materials and Methods: An open, controlled, prospective, randomized, single-center trial was conducted between February 2010 and June 2012. Patients with septic shock due to intra-abdominal sepsis were randomized to either conventional therapy (n = 8) or conventional therapy plus two 2-hourly sessions of Alteco endotoxin hemoadsorption (n = 7). Primary endpoint was the Sequential Organ Failure Assessment (SOFA) score changes from 0 to 72 h. Secondary end points included vasopressor requirement, PaO2/FiO2 ratio (PFR), length of stay (LOS), and 28-day mortality. Results: This study was terminated early as interim analysis showed a low probability of significant findings. No significant difference was noted between the two groups with respect to change in SOFA score, vasopressor score, PFR, LOS, and 28-day mortality. Side-effect was minimal. Conclusions: We could not identify any clinical benefit on the addition of Alteco endotoxin hemoadsorption to conventional therapy in patients who suffered from intra-abdominal sepsis with shock. The side effect profile of this novel device was acceptable. PMID:25538412

  4. Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria.

    PubMed

    Lee, Kyung-Jo; Lee, Mi-Ae; Hwang, Won; Park, Hana; Lee, Kyu-Ho

    2016-08-01

    The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria. PMID:27294580

  5. Current Epidemiology and Growing Resistance of Gram-Negative Pathogens

    PubMed Central

    2012-01-01

    In the 1980s, Gram-negative pathogens appeared to have been beaten by oxyimino-cephalosporins, carbapenems, and fluoroquinolones. Yet these pathogens have fought back, aided by their membrane organization, which promotes the exclusion and efflux of antibiotics, and by a remarkable propensity to recruit, transfer, and modify the expression of resistance genes, including those for extended-spectrum β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and even a quinolone-modifying variant of an aminoglycoside-modifying enzyme. Gram-negative isolates -both fermenters and non-fermenters-susceptible only to colistin and, more variably, fosfomycin and tigecycline, are encountered with increasing frequency, including in Korea. Some ESBLs and carbapenemases have become associated with strains that have great epidemic potential, spreading across countries and continents; examples include Escherichia coli sequence type (ST)131 with CTX-M-15 ESBL and Klebsiella pneumoniae ST258 with KPC carbapenemases. Both of these high-risk lineages have reached Korea. In other cases, notably New Delhi Metallo carbapenemase, the relevant gene is carried by promiscuous plasmids that readily transfer among strains and species. Unless antibiotic stewardship is reinforced, microbiological diagnosis accelerated, and antibiotic development reinvigorated, there is a real prospect that the antibiotic revolution of the 20th century will crumble. PMID:22707882

  6. Current epidemiology and growing resistance of gram-negative pathogens.

    PubMed

    Livermore, David M

    2012-06-01

    In the 1980s, gram-negative pathogens appeared to have been beaten by oxyimino-cephalosporins, carbapenems, and fluoroquinolones. Yet these pathogens have fought back, aided by their membrane organization, which promotes the exclusion and efflux of antibiotics, and by a remarkable propensity to recruit, transfer, and modify the expression of resistance genes, including those for extended-spectrum β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and even a quinolone-modifying variant of an aminoglycoside-modifying enzyme. Gram-negative isolates--both fermenters and non-fermenters--susceptible only to colistin and, more variably, fosfomycin and tigecycline, are encountered with increasing frequency, including in Korea. Some ESBLs and carbapenemases have become associated with strains that have great epidemic potential, spreading across countries and continents; examples include Escherichia coli sequence type (ST)131 with CTX-M-15 ESBL and Klebsiella pneumoniae ST258 with KPC carbapenemases. Both of these high-risk lineages have reached Korea. In other cases, notably New Delhi Metallo carbapenemase, the relevant gene is carried by promiscuous plasmids that readily transfer among strains and species. Unless antibiotic stewardship is reinforced, microbiological diagnosis accelerated, and antibiotic development reinvigorated, there is a real prospect that the antibiotic revolution of the 20th century will crumble. PMID:22707882

  7. 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

  8. Synergistic interaction of eugenol with antibiotics against Gram negative bacteria.

    PubMed

    Hemaiswarya, S; Doble, M

    2009-11-01

    Eugenol, the principal chemical component of clove oil from Eugenia aromatica has been long known for its analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. The interaction of the eugenol with ten different hydrophobic and hydrophilic antibiotics was studied against five different Gram negative bacteria. The MIC of the combination was found to decrease by a factor of 5-1000 with respect to their individual MIC. This synergy is because of the membrane damaging nature of eugenol, where 1mM of its concentration is able to damage nearly 50% of the bacterial membrane. Eugenol was also able to enhance the activities of lysozyme, Triton X-100 and SDS in damaging the bacterial cell membrane. The hydrophilic antibiotics such as vancomycin and beta-lactam antibiotics which have a marginal activity on these gram negative bacteria exhibit an enhanced antibacterial activity when pretreated with eugenol. Reduced usage of antibiotics could be employed as a treatment strategy to slow down the onset of antibiotic resistance as well as decrease its toxicity. Experiments performed with human blood cells indicated that the concentration of eugenol used for the combination studies were below its cytotoxic values. Pharmacodynamic studies of the combinations need to be performed to decide on the effective dosage. PMID:19540744

  9. Killing of gram-negative bacteria by lactoferrin and lysozyme.

    PubMed Central

    Ellison, R T; Giehl, T J

    1991-01-01

    Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense. Images PMID:1918365

  10. Three-Dimensional Distribution of Phospholipids in Gram Negative Bacteria.

    PubMed

    Furse, Samuel; Scott, David J

    2016-08-30

    Exploration of the molecular structure of the bacterial cell envelope informs our understanding of its role in bacterial growth. This is crucial for research into both inhibiting and promoting bacterial growth as well as fundamental studies of cell cycle control. The spatial arrangement of the lipids in the cell envelope of Gram negative bacteria in particular has attracted considerable research attention in recent years. In this mini-review, we explore advances in understanding the spatial distribution of lipids in the model Gram negative prokaryote Escherichia coli. This includes the distribution of lipids in three dimensions, (a) lateral distribution within a monolayer, (b) asymmetry between bilayers and monolayers, and (c) distribution as a function of progress through membrane division (temporal shifts). We conclude that lipid distribution in E. coli and probably all bacteria is dynamic despite a narrow lipid profile and that the biophysical properties of the membrane are inhomogeneous as a result. Finally, we suggest that further work in this field may indicate how lipid distribution is controlled and what this means for bacterial growth and metabolism and even cell cycle control. PMID:27509296

  11. Marine Compounds with Therapeutic Potential in Gram-Negative Sepsis

    PubMed Central

    Solov’eva, Tamara; Davydova, Viktoria; Krasikova, Inna; Yermak, Irina

    2013-01-01

    This paper concerns the potential use of compounds, including lipid A, chitosan, and carrageenan, from marine sources as agents for treating endotoxemic complications from Gram-negative infections, such as sepsis and endotoxic shock. Lipid A, which can be isolated from various species of marine bacteria, is a potential antagonist of bacterial endotoxins (lipopolysaccharide (LPSs)). Chitosan is a widespread marine polysaccharide that is derived from chitin, the major component of crustacean shells. The potential of chitosan as an LPS-binding and endotoxin-neutralizing agent is also examined in this paper, including a discussion on the generation of hydrophobic chitosan derivatives to increase the binding affinity of chitosan to LPS. In addition, the ability of carrageenan, which is the polysaccharide of red alga, to decrease the toxicity of LPS is discussed. We also review data obtained using animal models that demonstrate the potency of carrageenan and chitosan as antiendotoxin agents. PMID:23783404

  12. Poplar lignin decomposition by gram-negative aerobic bacteria

    SciTech Connect

    Odier, E.; Janin, G.; Monties, B.

    1981-02-01

    Eleven gram-negative aerobic bacteria (Pseudomonadaceae and Neisseriaceae) out of 122 soil isolates were selected for their ability to assimilate poplar dioxane lignin without a cosubstrate. Dioxane lignin and milled wood lignin degradation rates ranged between 20 and 40% of initial content after 7 days in mineral medium, as determined by a loss of absorbance at 280 nm; 10 strains could degrade in situ lignin, as evidenced by the decrease of the acetyl bromide lignin content of microtome wood sections. No degradation of wood polysaccharides was detected. Lignin biodegradation by Pseudomonas 106 was confirmed by 14CO2 release from labeled poplar wood, although in lower yields compared with results obtained through chemical analysis based on acetyl bromide residual lignin determination. (Refs. 31).

  13. 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.

  14. [Identification of Gram negative rods and evaluation of their antibiotic sensitivity: comparison of the BIOTEST MHK/ID-system with the API 20E-system and the agar diffusion test].

    PubMed

    Stanek, G; Hirschl, A; Rotter, M

    1982-10-01

    BIOTEST MHK/ID is a new system for the identification of gramnegative fermentative and non fermentative rods and for the simultaneous determination of the minimal inhibitory concentrations (MICs) against 15 antimicrobial drugs. Its identification system was compared with the API 20E kit. When the computer generated identification manuals for each of the systems were used 99.5% of the 190 strains tested were identified. Of these identifications 98.4% were identical. Comparing the results of altogether 16 biochemical reactions 92.3% (table 4) were conformable. Considering each single test low agreement was found with the results of the Citrate-, Urea-, Voges-Proskauer- and Arginindehydrolase-tests (76.8, 79.5, 85.3 and 86.8% agreement, respectively). The BIOTEST MHK/ID system yielded more positive reactions. But despite of these differences there was no influence on the accuracy in identifying the species. Thus, both systems must be considered equally suited to identify gramnegative rods. The results of both BIOTEST MHK/ID system (MIC-values) and the agardiffusion test (inhibition zone sizes) were transferred into the interpretative criteria "sensitive", "intermediate" and "resistant" (table 1) and compared for each strain. The overall-agreement amounted to 93.0% (table 7). With the MIC-test, however, more (7.2%) strains were found "intermediate" than with the agardiffusion test (3.1%, table 8). PMID:6817546

  15. New unified nomenclature for genes involved in the oxidation of methanol in gram-negative bacteria.

    PubMed

    Lidstrom, M E; Anthony, C; Biville, F; Gasser, F; Goodwin, P; Hanson, R S; Harms, N

    1994-03-15

    The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox, for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups. PMID:8181704

  16. The Changing Role of the Clinical Microbiology Laboratory in Defining Resistance in Gram-negatives.

    PubMed

    Endimiani, Andrea; Jacobs, Michael R

    2016-06-01

    The evolution of resistance in Gram-negatives has challenged the clinical microbiology laboratory to implement new methods for their detection. Multidrug-resistant strains present major challenges to conventional and new detection methods. More rapid pathogen identification and antimicrobial susceptibility testing have been developed for use directly on specimens, including fluorescence in situ hybridization tests, automated polymerase chain reaction systems, microarrays, mass spectroscopy, next-generation sequencing, and microfluidics. Review of these methods shows the advances that have been made in rapid detection of resistance in cultures, but limited progress in direct detection from specimens. PMID:27208762

  17. [Antibiotic sensitivity of nonfermenting gram-negative bacteria].

    PubMed

    Iskhakova, Kh I

    1988-11-01

    Antibiotic resistance of 132 strains of nonfermenting gram-negative bacteria (NGNB) was studied. 43, 20, 17, 14 and 12 of them belonged to Acinetobacter calcoaceticus (anitratus and lwoffi), Pseudomonas cepacia, Alcaligenes faecalis, P. stutzeri and P. maltophilia, respectively. With rare exceptions all the strains were resistant to benzylpenicillin, oxacillin, lincomycin, ampicillin and cephaloridine. Sensitivity to the other antibiotics varied within wide ranges. Amikacin (94.3 per cent) and tobramycin (90.8 per cent), as well as polymyxin, rifampicin and gentamicin (71.7-66.9 per cent) had the highest effect. The majority of the antibiotics had higher activity (p less than 0.01) against the tested NGNB as compared to their activity against P. aeruginosa. Antibioticograms of every of the tested species of NGNB revealed that P. cepacia and P. stutzeri were the most resistant species. The biovars of Acinetobacter varied in their antibiotic resistance: A. subsp. lwoffi was more sensitive to the majority of the antibiotics though some of them, i.e. doxycycline, carbenicillin, and polymyxin were more active against A. subsp. anitratus. PMID:3228322

  18. 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

  19. [Gram-negative bacteria resistant to antibiotics in foods].

    PubMed

    Dias, J C; Hofer, E

    1985-01-01

    From 154 food samples, including vegetables (lettuce), milk and meals served at school it was possible to isolate and identify 400 Gram negative bacilli distributed among 339 enteric bacteria (Escherichia, Shigella, Citrobacter, Klebsiella, Enterobacter, Serratia and Proteus) and other 61 non enteric bacilli (Acinetobacter, Flavobacterium, Aeromonas and Pseudomonas). Submitting this cultures to the drugs sulfadiazine (Su), streptomycin (Sm), tetracycline (Tc), chloramphenicol (Cm), kanamycin (Km), ampicillin (Ap), nalidixic acid (Nal) and gentamycin (Gm) it was observed only six stocks susceptible to all drugs and total sensibility to Gm. Among enteric bacteria the profiles Su (27,6%) and Su-Ap (39,6%) predominated, while for the non enteric bacilli percentages of 18.0 for Ap and 9.8 for Su-Ap were detected. Aiming to better characterization of resistance, experiments of conjugation were made with standard strains of Escherichia coli K 12. Great concern was raised by the recognition of these cultures due to the elevated R+ taxes for the enteric bacilli that were close to 90% (milk and food at school) and about 70% in relation to lettuce. PMID:3837834

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

    PubMed

    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

  1. 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

  2. Occurrence of gram-negative bacteria in hens' eggs depending on their source and storage conditions.

    PubMed

    Stepień-Pyśniak, D

    2010-01-01

    The aim of this study was to analyse the qualitative composition of Gram-negative microbes, mainly of the family Enterobacteriaceae, including pathogenic bacteria such as Salmonella, in the albumens and yolks and on the shells of hens' eggs, depending on their source and on the temperature and duration of their storage. A total of 375 table eggs were studied, from a large-scale poultry farm, a small-scale poultry farm and a supermarket. Each group was divided into 5 subgroups according to the temperature and duration of their storage during the study. Two serotypes of bacteria of the genus Salmonella were identified: S. Enteritidis and S. Arizonae. Strains of Salmonella spp. were also isolated. Apart from Salmonella and Escherichia coli, among the most frequently isolated bacteria of the family Enterobacteriaceae were Enterobacter spp., Klebsiella spp. and Citrobacter freundii. Qualitative analysis of the bacterial microflora of the eggs also showed the presence of other Gram negative bacteria, including Acinetobacter spp., Pseudomonas spp., Tatumella ptyseos, Providencia stuartii, Serratia liquefaciens, Flavimonas oryzihabitans, Vibrio metschnikovii, Leclercia adecarboxylata, Kluyvera spp., Rahnella aquatilis, Proteus mirabilis, and Achromobacter spp. The study demonstrated that the conditions applied, i.e., the temperature and duration of storage, did not significantly influence the prevalence of particular species of Gram-negative bacteria in the eggs. However, based on the analysis of contamination of eggs with Salmonella depending on their source, it can be concluded that the system in which the hens are housed affects the risk of contamination of eggs with these pathogens. PMID:21033566

  3. Mechanisms of antimicrobial resistance in Gram-negative bacilli.

    PubMed

    Ruppé, Étienne; Woerther, Paul-Louis; Barbier, François

    2015-12-01

    The burden of multidrug resistance in Gram-negative bacilli (GNB) now represents a daily issue for the management of antimicrobial therapy in intensive care unit (ICU) patients. In Enterobacteriaceae, the dramatic increase in the rates of resistance to third-generation cephalosporins mainly results from the spread of plasmid-borne extended-spectrum beta-lactamase (ESBL), especially those belonging to the CTX-M family. The efficacy of beta-lactam/beta-lactamase inhibitor associations for severe infections due to ESBL-producing Enterobacteriaceae has not been adequately evaluated in critically ill patients, and carbapenems still stands as the first-line choice in this situation. However, carbapenemase-producing strains have emerged worldwide over the past decade. VIM- and NDM-type metallo-beta-lactamases, OXA-48 and KPC appear as the most successful enzymes and may threaten the efficacy of carbapenems in the near future. ESBL- and carbapenemase-encoding plasmids frequently bear resistance determinants for other antimicrobial classes, including aminoglycosides (aminoglycoside-modifying enzymes or 16S rRNA methylases) and fluoroquinolones (Qnr, AAC(6')-Ib-cr or efflux pumps), a key feature that fosters the spread of multidrug resistance in Enterobacteriaceae. In non-fermenting GNB such as Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia, multidrug resistance may emerge following the sole occurrence of sequential chromosomal mutations, which may lead to the overproduction of intrinsic beta-lactamases, hyper-expression of efflux pumps, target modifications and permeability alterations. P. aeruginosa and A. baumannii also have the ability to acquire mobile genetic elements encoding resistance determinants, including carbapenemases. Available options for the treatment of ICU-acquired infections due to carbapenem-resistant GNB are currently scarce, and recent reports emphasizing the spread of colistin resistance in environments with high

  4. 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

  5. ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria.

    PubMed

    Narita, Shin-ichiro

    2011-01-01

    The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed. PMID:21670534

  6. 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

  7. 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. PMID:25388855

  8. 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

  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. [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. PMID:26084076

  11. Role of the platelet-activating factor (PAF) receptor during pulmonary infection with gram negative bacteria

    PubMed Central

    Soares, A C; Pinho, V S; Souza, D G; Shimizu, T; Ishii, S; Nicoli, J R; Teixeira, M M

    2002-01-01

    The lipid mediator PAF plays an important role in the phagocytosis of particles, including bacteria, and consequent production of pro-inflammatory cytokines, such as TNF-α and IL-8. Using a PAF receptor antagonist (UK-74,505) and PAF receptor knock-out mice, we have investigated the relevance of PAF for the inflammatory changes and lethality after pulmonary infection with the gram-negative bacteria Klebsiella pneumoniae in mice. At an inoculum of 3×106 bacteria, there was marked pulmonary (bronchoalveolar lavage and lung) neutrophilia that started early (2.5 h after infection) and peaked at 48 h. All animals were dead by day 4 of infection. The chemokine KC and the pro-inflammatory cytokine TNF-α increased rapidly and persisted for 48 h in the lungs. Pretreatment with UK-74,505 (30 mg kg−1 per day, p.o.) had no significant effects on the number of infiltrating neutrophils in BAL fluid or lung tissue, as assessed by histology and measuring myeloperoxidase, or on the concentrations of KC. In contrast, concentrations of TNF-α and the number of bacteria inside neutrophils were significantly diminished. In order to support a role for the PAF during K. pneumoniae infection, experiments were also carried out in PAFR-deficient mice. In the latter animals, lethality occurred earlier than in wild-type controls. This was associated with greater number of bacteria in lung tissue and diminished percentage of neutrophils containing bacteria in their cytoplasm. Our results suggest that PAF, acting on its receptor, plays a protective role during infection with K. pneumoniae in mice. PMID:12381675

  12. 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

  13. Gram Negative Wound Infection in Hospitalised Adult Burn Patients-Systematic Review and Metanalysis-

    PubMed Central

    Azzopardi, Ernest A.; Azzopardi, Elayne; Camilleri, Liberato; Villapalos, Jorge; Boyce, Dean E.; Dziewulski, Peter; Dickson, William A.; Whitaker, Iain S.

    2014-01-01

    Background Gram negative infection is a major determinant of morbidity and survival. Traditional teaching suggests that burn wound infections in different centres are caused by differing sets of causative organisms. This study established whether Gram-negative burn wound isolates associated to clinical wound infection differ between burn centres. Methods Studies investigating adult hospitalised patients (2000–2010) were critically appraised and qualified to a levels of evidence hierarchy. The contribution of bacterial pathogen type, and burn centre to the variance in standardised incidence of Gram-negative burn wound infection was analysed using two-way analysis of variance. Primary Findings Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanni, Enterobacter spp., Proteus spp. and Escherichia coli emerged as the commonest Gram-negative burn wound pathogens. Individual pathogens’ incidence did not differ significantly between burn centres (F (4, 20) = 1.1, p = 0.3797; r2 = 9.84). Interpretation Gram-negative infections predominate in burn surgery. This study is the first to establish that burn wound infections do not differ significantly between burn centres. It is the first study to report the pathogens responsible for the majority of Gram-negative infections in these patients. Whilst burn wound infection is not exclusive to these bacteria, it is hoped that reporting the presence of this group of common Gram-negative “target organisms” facilitate clinical practice and target research towards a defined clinical demand. PMID:24751699

  14. Resistance trends in gram-negative bacteria: surveillance results from two Mexican hospitals, 2005–2010

    PubMed Central

    2012-01-01

    Background Hospital-acquired infections caused by multiresistant gram-negative bacteria are difficult to treat and cause high rates of morbidity and mortality. The analysis of antimicrobial resistance trends of gram-negative pathogens isolated from hospital-acquired infections is important for the development of antimicrobial stewardship programs. The information obtained from antimicrobial resistant programs from two hospitals from Mexico will be helpful in the selection of empiric therapy for hospital-acquired gram-negative infections. Findings Two thousand one hundred thirty two gram-negative bacteria collected between January 2005 and December 2010 from hospital-acquired infections occurring in two teaching hospitals in Mexico were evaluated. Escherichia coli was the most frequently isolated gram-negative bacteria, with >50% of strains resistant to ciprofloxacin and levofloxacin. Klebsiella spp. showed resistance rates similar to Escherichia coli for ceftazidime (33.1% vs 33.2%), but exhibited lower rates for levofloxacin (18.2% vs 56%). Of the samples collected for the third most common gram-negative bacteria, Pseudomonas aeruginosa, >12.8% were resistant to the carbapenems, imipenem and meropenem. The highest overall resistance was found in Acinetobacter spp. Enterobacter spp. showed high susceptibility to carbapenems. Conclusions E. coli was the most common nosocomial gram-negative bacilli isolated in this study and was found to have the second-highest resistance to fluoroquinolones (>57.9%, after Acinetobacter spp. 81.2%). This finding represents a disturbing development in a common nosocomial and community pathogen. PMID:22676813

  15. The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology

    PubMed Central

    Santander, Javier; Xin, Wei; Yang, Zhao; Curtiss, Roy

    2010-01-01

    asdA mutants of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric Gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry. PMID:21209920

  16. A comparison of media used in vitro to isolate non-sporing Gram-negative anaerobes from blood.

    PubMed

    Forgan-Smith, W R; Darrell, J H

    1974-04-01

    Five anaerobic media were compared in a model blood culture system for their ability to recover small inocula of Gram-negative non-sporing anaerobes. Dehydrated cooked meat medium was the least effective; USP thioglycollate medium was the most effective isolation medium and is recommended for routine use. Freshly prepared cooked meat medium has the advantage of allowing prolonged survival of strains. PMID:4850178

  17. Gram-negative diabetic foot osteomyelitis: risk factors and clinical presentation.

    PubMed

    Aragón-Sánchez, Javier; Lipsky, Benjamin A; Lázaro-Martínez, Jose L

    2013-03-01

    Osteomyelitis frequently complicates infections in the feet of patients with diabetes. Gram-positive cocci, especially Staphylococcus aureus, are the most commonly isolated pathogens, but gram-negative bacteria also cause some cases of diabetic foot osteomyelitis (DFO). These gram-negatives require different antibiotic regimens than those commonly directed at gram-positives. There are, however, few data on factors related to their presence and how they influence the clinical picture. We conducted a retrospective study to determine the variables associated with the isolation of gram-negative bacteria from bone samples in cases of DFO and the clinical presentation of these infections. Among 341 cases of DFO, 150 had a gram-negative isolate (alone or combined with a gram-positive isolate) comprising 44.0% of all patients and 50.8% of those with a positive bone culture. Compared with gram-positive infections, wounds with gram-negative organisms more often had a fetid odor, necrotic tissue, signs of soft tissue infection accompanying osteomyelitis, and clinically severe infection. By multivariate analysis, the predictive variables related to an increased likelihood of isolating gram-negatives from bone samples were glycated hemoglobin <7% (odds ratio [OR] = 2.0, 95% confidence interval [CI] = 1.1-3.5) and a wound caused by traumatic injury (OR = 2.0, 95% CI = 1.0-3.9). Overall, patients whose bone samples contained gram-negatives had a statistically significantly higher prevalence of leukocytosis and higher white blood cell counts than those without gram-negatives. In conclusion, gram-negative organisms were isolated in nearly half of our cases of DFO and were associated with more severe infections, higher white blood cell counts, lower glycated hemoglobin levels, and wounds of traumatic etiology. PMID:23446368

  18. 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

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

    DOE PAGESBeta

    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.

  20. Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood.

    PubMed Central

    Parker, T S; Levine, D M; Chang, J C; Laxer, J; Coffin, C C; Rubin, A L

    1995-01-01

    We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood. PMID:7528733

  1. 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

  2. Levels of gram-negative bacteria, Aspergillus fumigatus, dust, and endotoxin at compost plants.

    PubMed Central

    Clark, C S; Rylander, R; Larsson, L

    1983-01-01

    Airborne gram-negative bacteria, endotoxins, dust, and Aspergillus fumigatus were measured in four compost plants in Sweden. At sites where material was processed, the number of airborne A. fumigatus exceeded 10(6)/m3, whereas the number of gram-negative bacteria was usually lower. Dust levels were moderate, and endotoxin levels were well below 0.5 micrograms/m3. Medical studies to evaluate the effects of this type of microbial exposure are recommended. PMID:6347061

  3. Experimental gram-negative bacterial sepsis: prevention of mortality not preventable by antibiotics alone.

    PubMed Central

    Greisman, S E; DuBuy, J B; Woodward, C L

    1979-01-01

    Outbred Swiss mice were inoculated intraperitoneally or intravenously with one 90 to 100% lethal dose of Escherichia coli O:18, Proteus mirabilis, or Klebsiella pneumoniae. After carefully timed intervals, aminoglycoside antibiotics were begun at dosages nnd intervals predetermined to constitute optimal therapy. With progressive increases in delay of antibiotic therapy, mortality rates increased progressively from 0% to 90 to 100%. Standardized models of infection were developed by selecting delay periods before initiating antibiotic therapy such that 50 to 70% mortalities resulted. Utilizing these models, agents with reputed anti-endotoxin activity were administered concomitantly with the delayed antibiotic therapy to determine if any could prevent gram-negative septic mortality no longer preventable by the antibiotics alone. The following were observed: (i) adrenal corticosteroids prevented mortality that was no longer preventable by optimal aminoglycoside antibiotics alone. The following were preventable by optimal aminoglycoside antibiotic therapy alone; (ii) specific antisera also did so, provided anaphylaxis was circumvented; (iii) in one model (P. mirabilis), such protection by adrenal corticosteroids and specific antiserum could be additive; (iv) adrenal corticosteroids and specific antiserum acted synergistically with the aminoglycoside antibiotics--no protection was achieved by delayed administration of the steroids or antiserum alone; (v) timing was crucial--the synergistic protective activity of adrenal corticosteroids and of specific antiserum with aminoglycosides declined rapidly as infection progressed; (vi) cyclophosphamide pretreatment markedly impaired the synergistic protective activity of specific antiserum and of adrenal corticosteroids with aminoglycosides; (vii) no reputed anti-endotoxin agents other than adrenal corticosteroids and specific antiserum proved capable of preventing mortality not preventable by aminoglycoside antibiotics alone

  4. Appraising contemporary strategies to combat multidrug resistant gram-negative bacterial infections--proceedings and data from the Gram-Negative Resistance Summit.

    PubMed

    Kollef, Marin H; Golan, Yoav; Micek, Scott T; Shorr, Andrew F; Restrepo, Marcos I

    2011-09-01

    The emerging problem of antibiotic resistance, especially among Gram-negative bacteria (GNB), has become a serious threat to global public health. Very few new antibacterial classes with activity against antibiotic-resistant GNB have been brought to market. Renewed and growing attention to the development of novel compounds targeting antibiotic-resistant GNB, as well as a better understanding of strategies aimed at preventing the spread of resistant bacterial strains and preserving the efficacy of existing antibiotic agents, has occurred. The Gram-Negative Resistance Summit convened national opinion leaders for the purpose of analyzing current literature, epidemiologic trends, clinical trial data, therapeutic options, and treatment guidelines related to the management of antibiotic-resistant GNB infections. After an in-depth analysis, the Summit investigators were surveyed with regard to 4 clinical practice statements. The results then were compared with the same survey completed by 138 infectious disease and critical care physicians and are the basis of this article. PMID:21868447

  5. 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

    2016-03-01

    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. PMID:26827160

  6. Multidrug-resistant Gram-negative bacterial infections: the emerging threat and potential novel treatment options.

    PubMed

    Vergidis, Paschalis I; Falagas, Matthew E

    2008-02-01

    Gram-negative bacterial infections constitute an emerging threat because of the development of multidrug-resistant organisms. There is a relative shortage of new drugs in the antimicrobial development pipeline that have been tested in vitro and evaluated in clinical studies. Antibiotics that are in the pipeline for the treatment of serious Gram-negative bacterial infections include the cephalosporins, ceftobiprole, ceftarolin and FR-264205. Tigecycline is the first drug approved from a new class of antibiotics called glycylcyclines, and there has been renewed interest in this drug for the treatment of some multidrug-resistant Gram-negative organisms. Carbapenems in the pipeline include tomopenem, with the approved drugs doripenem and faropenem, an oral agent, under evaluation for activity against multidrug-resistant Gram-negative bacterial infections. Polymyxins are old antibiotics traditionally considered to be toxic, but which are being used because of their activity against resistant Gram-negative organisms. New pharmacokinetic and pharmacodynamic data are available regarding the use of these agents. Finally, antimicrobial peptides and efflux pump inhibitors are two new classes of agents under development. This review of investigational antibiotics shows that several new agents will become available in the coming years, even though the pace of antimicrobial research is far from ideal. PMID:18246520

  7. Epidemiology and Outcome of Gram-Negative Bloodstream Infection in Children: A Population-Based Study

    PubMed Central

    Al-Hasan, M. N.; Huskins, W. C.; Lahr, B. D.; Eckel-Passow, J. E.; Baddour, L. M.

    2010-01-01

    SUMMARY Population-based studies of gram-negative bloodstream infection (BSI) in children are lacking. Therefore, we performed this population-based investigation in Olmsted County, Minnesota, to determine the incidence rate, site of acquisition, and outcome of gram-negative BSI in children under 18 years old. We used Kaplan-Meier method and Cox proportional hazard regression for mortality analysis. We identified 56 unique children with gram-negative BSI during the past decade. The gender-adjusted incidence rate of gram-negative BSI per 100,000 person-years was 129.7 (95% confidence interval [CI]: 77.8-181.6]) in infants, with a sharp decline to 14.6 (95% CI: 6.0-23.2) and 7.6 (95% CI: 4.3-10.9) in children 1-4 and 5-18 years old, respectively. The urinary tract was the most common identified source of infection (34%) and Escherichia coli was the most common pathogen isolated (38%). Over two-thirds (68%) of children had underlying medical conditions that predisposed to gram-negative BSI. The overall 28-day and 1-year all-cause mortality rates were 11% (95% CI: 3-18%) and 18% (95% CI: 8-28%), respectively. Younger age and number of underlying medical conditions were associated with 28-day and 1-year mortality, respectively. Nosocomial or healthcare-associated acquisition was associated with both 28-day and 1-year mortality. PMID:20598212

  8. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers.

    PubMed

    Lam, Shu J; O'Brien-Simpson, Neil M; Pantarat, Namfon; Sulistio, Adrian; Wong, Edgar H H; Chen, Yu-Yen; Lenzo, Jason C; Holden, James A; Blencowe, Anton; Reynolds, Eric C; Qiao, Greg G

    2016-01-01

    With the recent emergence of reports on resistant Gram-negative 'superbugs', infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed 'structurally nanoengineered antimicrobial peptide polymers' (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria. PMID:27617798

  9. 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

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

    PubMed

    Zhang, Weiwei; Li, Chenghua

    2015-01-01

    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

  11. [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. PMID:21413423

  12. Fluoroquinolone Resistance Among Gram-Negative Urinary Tract Pathogens: Global Smart Program Results, 2009-2010

    PubMed Central

    Bouchillon, Sam; Hoban, Daryl J; Badal, Robert; Hawser, Stephen

    2012-01-01

    OBJECTIVES: To determine the rates of fluoroquinolone resistant (FQR) in gram-negative bacilli urinary tract infections (UTIs) in a global population. METHODS: The Study for Monitoring Antimicrobial Resistance Trends (SMART) collected 1,116 FQR gram-negative urinary pathogens from hospitalized patients in 33 countries during 2009-2010. Amikacin, ertapenem, and imipenem were the most active agents tested against FQR UTI pathogens, including extended-spectrum beta-lactamase producers. RESULTS: FQR rates vary widely country to country with a range of 6% to 75%. Regional FQR rates were 23.5% in North America, 29.4% in Europe, 33.2% in Asia, 38.7% in Latin America, and 25.5% in the South Pacific. CONCLUSIONS: These observations suggest that fluoroquinolones may no longer be effective as first-line therapy for gram-negative UTI in hospitalized patients. PMID:23002406

  13. Continual Gram-negative bacterial challenge accelerates stroke onset in stroke-prone spontaneously hypertensive rats.

    PubMed

    Kawato, Takayuki; Tanaka, Hideki; Tabuchi, Masaki; Ooshima, Kana; Nakai, Kumiko; Yamashita, Yoshihisa; Maeno, Masao

    2013-01-01

    This study examined the effects of continual Gram-negative bacterial challenge on stroke onset. Stroke onset occurred significantly earlier in stroke-prone spontaneously hypertensive rats (SHRSP) injected with a bacterial cell suspension of Gram-negative rods or lipopolysaccharides (LPSs) than in uninjected controls. Paralysis of the hindlimb, piloerection, hypokinesis, and hyperkinesis were observed in LPS-injected SHRSP but not in uninjected controls during stroke onset. The serum levels of NOx, thiobarbituric acid reactive substance, and 8-hydroxydeoxyguanosine increased in LPS-injected SHRSP. These results suggest that continual Gram-negative bacterial challenge induces accelerated stroke onset in SHRSP, probably caused by oxidative stress responses derived from LPSs. PMID:22630606

  14. Combination Therapy for Treatment of Infections with Gram-Negative Bacteria

    PubMed Central

    Cosgrove, Sara E.; Maragakis, Lisa L.

    2012-01-01

    Summary: Combination antibiotic therapy for invasive infections with Gram-negative bacteria is employed in many health care facilities, especially for certain subgroups of patients, including those with neutropenia, those with infections caused by Pseudomonas aeruginosa, those with ventilator-associated pneumonia, and the severely ill. An argument can be made for empiric combination therapy, as we are witnessing a rise in infections caused by multidrug-resistant Gram-negative organisms. The wisdom of continued combination therapy after an organism is isolated and antimicrobial susceptibility data are known, however, is more controversial. The available evidence suggests that the greatest benefit of combination antibiotic therapy stems from the increased likelihood of choosing an effective agent during empiric therapy, rather than exploitation of in vitro synergy or the prevention of resistance during definitive treatment. In this review, we summarize the available data comparing monotherapy versus combination antimicrobial therapy for the treatment of infections with Gram-negative bacteria. PMID:22763634

  15. The "other" gram-negative bacteria in mastitis: Klebsiella, serratia, and more.

    PubMed

    Schukken, Ynte; Chuff, Matt; Moroni, Paolo; Gurjar, Abhijit; Santisteban, Carlos; Welcome, Frank; Zadoks, Ruth

    2012-07-01

    Mastitis caused by gram-negative infections is of increasing importance on modern and well-managed dairy farms. Without a doubt, E coli tends to be the most important cause of these gram-negative infections when the data are tallied across farms.1 However, more precise investigation of individual farms often reveals a farm-specific infection pattern where a single gram-negative bacterial species predominates. Several farms with a predominance of “other” gram-negative IMIs may be observed. We have shown the presence of outbreaks on individual dairy farms with K pneumoniae, S marcescens, and Enterobacter cloacae. On farms with a predominance of these “other” gram-negative infections, a detailed epidemiologic investigation may reveal the source of these infections. It is quite surprising to identify the difference in host immune response pattern and the associated clinical and subclinical presentations of IMIs due to the different gram-negative organisms. Experimental and field observations would suggest that among the gram-negative bacterial causes of mastitis, Klebsiella spp are causing the most severe cases, closely followed by E coli and then much less clinical severity is observed in Serratia spp and Enterobacter spp cases. The precise mechanisms that would explain the difference in clinical severity are not known, but the most likely explanation appears to be the structure of the lipid A fraction of the LPS of the bacterial species. Important differences in the lipid A fraction of LPS between and within bacterial species are observed. The prevention of IMIs with gram-negative bacteria has components that are generic across species and components that are species specific. Generic prevention may be obtained by improving hygiene and reducing exposure of teat ends to environmental contamination. Also the use of a J5 bacterin is expected to provide some reduction in severity of gram-negative IMIs across bacterial species. Specific prevention programs will

  16. 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

  17. Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

    PubMed

    McLeod, Sarah M; Fleming, Paul R; MacCormack, Kathleen; McLaughlin, Robert E; Whiteaker, James D; Narita, Shin-Ichiro; Mori, Makiko; Tokuda, Hajime; Miller, Alita A

    2015-03-01

    In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy. PMID:25583975

  18. Carbapenemase Genes among Multidrug Resistant Gram Negative Clinical Isolates from a Tertiary Hospital in Mwanza, Tanzania

    PubMed Central

    Mushi, Martha F.; Mshana, Stephen E.; Imirzalioglu, Can; Bwanga, Freddie

    2014-01-01

    The burden of antimicrobial resistance (AMR) is rapidly growing across antibiotic classes, with increased detection of isolates resistant to carbapenems. Data on the prevalence of carbapenem resistance in developing countries is limited; therefore, in this study, we determined the prevalence of carbapenemase genes among multidrug resistant gram negative bacteria (MDR-GNB) isolated from clinical specimens in a tertiary hospital in Mwanza, Tanzania. A total of 227 MDR-GNB isolates were analyzed for carbapenem resistance genes. For each isolate, five different PCR assays were performed, allowing for the detection of the major carbapenemase genes, including those encoding the VIM-, IMP-, and NDM-type metallo-beta-lactamases, the class A KPC-type carbapenemases, and the class D OXA-48 enzyme. Of 227 isolates, 80 (35%) were positive for one or more carbapenemase gene. IMP-types were the most predominant gene followed by VIM, in 49 (21.59%) and 28 (12%) isolates, respectively. Carbapenemase genes were most detected in K. pneumoniae 24 (11%), followed by P. aeruginosa 23 (10%), and E. coli with 19 isolates (8%). We have demonstrated for the first time a high prevalence of MDR-GNB clinical isolates having carbapenem resistance genes in Tanzania. We recommend routine testing for carbapenem resistance among the MDR-GNB particularly in systemic infections. PMID:24707481

  19. 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

  20. 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.

  1. The TFPI-2 Derived Peptide EDC34 Improves Outcome of Gram-Negative Sepsis

    PubMed Central

    Papareddy, Praveen; Kalle, Martina; Sørensen, Ole E.; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-01-01

    Sepsis is characterized by a dysregulated host-pathogen response, leading to high cytokine levels, excessive coagulation and failure to eradicate invasive bacteria. Novel therapeutic strategies that address crucial pathogenetic steps during infection are urgently needed. Here, we describe novel bioactive roles and therapeutic anti-infective potential of the peptide EDC34, derived from the C-terminus of tissue factor pathway inhibitor-2 (TFPI-2). This peptide exerted direct bactericidal effects and boosted activation of the classical complement pathway including formation of antimicrobial C3a, but inhibited bacteria-induced activation of the contact system. Correspondingly, in mouse models of severe Escherichia coli and Pseudomonas aeruginosa infection, treatment with EDC34 reduced bacterial levels and lung damage. In combination with the antibiotic ceftazidime, the peptide significantly prolonged survival and reduced mortality in mice. The peptide's boosting effect on bacterial clearance paired with its inhibiting effect on excessive coagulation makes it a promising therapeutic candidate for invasive Gram-negative infections. PMID:24339780

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

    PubMed

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

    2016-05-01

    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

  3. How Porin Heterogeneity and Trade-Offs Affect the Antibiotic Susceptibility of Gram-Negative Bacteria

    PubMed Central

    Ferenci, Thomas; Phan, Katherine

    2015-01-01

    Variations in porin proteins are common in Gram-negative pathogens. Altered or absent porins reduce access of polar antibiotics across the outer membrane and can thus contribute to antibiotic resistance. Reduced permeability has a cost however, in lowering access to nutrients. This trade-off between permeability and nutritional competence is the source of considerable natural variation in porin gate-keeping. Mutational changes in this trade-off are frequently selected, so susceptibility to detergents and antibiotics is polymorphic in environmental isolates as well as pathogens. Understanding the mechanism, costs and heterogeneity of antibiotic exclusion by porins will be crucial in combating Gram negative infections. PMID:26506392

  4. Antibiotic-Resistant Gram-Negative Bacterial Infections in Patients With Cancer

    PubMed Central

    Perez, Federico; Adachi, Javier; Bonomo, Robert A.

    2014-01-01

    Patients with cancer are at high risk for infections caused by antibiotic resistant gram-negative bacteria. In this review, we summarize trends among the major pathogens and clinical syndromes associated with antibiotic resistant gram-negative bacterial infection in patients with malignancy, with special attention to carbapenem and expanded-spectrum β-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia—all major threats to our cancer patients. Optimal therapy for these antibiotic-resistant pathogens still remains to be determined. PMID:25352627

  5. Antibiotic-resistant gram-negative bacterial infections in patients with cancer.

    PubMed

    Perez, Federico; Adachi, Javier; Bonomo, Robert A

    2014-11-15

    Patients with cancer are at high risk for infections caused by antibiotic resistant gram-negative bacteria. In this review, we summarize trends among the major pathogens and clinical syndromes associated with antibiotic resistant gram-negative bacterial infection in patients with malignancy, with special attention to carbapenem and expanded-spectrum β-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia--all major threats to our cancer patients. Optimal therapy for these antibiotic-resistant pathogens still remains to be determined. PMID:25352627

  6. Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria.

    PubMed

    Ni, Wentao; Li, Yanjun; Guan, Jie; Zhao, Jin; Cui, Junchang; Wang, Rui; Liu, Youning

    2016-05-01

    We tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially in Acinetobacter baumannii and Stenotrophomonas maltophilia These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP. PMID:26953203

  7. 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

    2015-05-01

    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). PMID:25857842

  8. 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

  9. A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria.

    PubMed

    Jeeves, Mark; Knowles, Timothy J

    2015-08-01

    The understanding of the biogenesis of the outer membrane of Gram-negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β-barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram-negative outer membrane that is independent of the BAM complex and TAM. PMID:26059329

  10. 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. PMID:25744116

  11. 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.

  12. Evaluation of the 4-hour RapID NF Plus method for identification of 345 gram-negative nonfermentative rods.

    PubMed

    Kitch, T T; Jacobs, M R; Appelbaum, P C

    1992-05-01

    The ability of the RapID NF Plus system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to identify 345 nonfermentative gram-negative rods was evaluated. Kits were inoculated with no. 1 McFarland suspensions, and reactions were interpreted after a 4-h incubation at 35 degrees C. Overall, the method correctly identified 311 strains (90.1%) without additional tests and 21 strains (6.1%) with additional tests, and 13 strains (3.8%) were misidentified. Five of 13 misidentified strains were Alcaligenes faecalis-Alcaligenes odorans misidentified as Alcaligenes xylosoxidans; however, all strains were xylose negative but nitrate positive and could have been A. faecalis group I-Alcaligenes piechaudii. The system does not differentiate between Pseudomonas fluorescens and Pseudomonas putida, and all Acinetobacter species are identified as Acetinobacter calcoaceticus. Additionally, no subspecies differentiation is made between A. xylosoxidans subsp. xylosoxidans and A. xylosoxidans subsp. denitrificans. All strains of the former Flavobacterium group IIb are identified as Flavobacterium indologenes-Flavobacterium gleum, and no species identification of the genus Methylobacterium is attempted. The system is easy to set up and interpret and provides an accurate commercial nonautomated method for same-day identification of gram-negative nonfermenters. PMID:1583129

  13. Procalcitonin Is a Marker of Gram-Negative Bacteremia in Patients With Sepsis

    PubMed Central

    Guo, Shun Yuan; Zhou, Yin; Hu, Qing Feng; Yao, Jiong

    2015-01-01

    Abstract: Background: Prediction of the species of pathogen among patients with sepsis within hours would be helpful in accelerating proper treatment. As a potential method of shortening the time to identification, this study considered the usefulness of measuring procalcitonin (PCT) to predict blood culture (BC) results. Methods: The authors retrospectively analyzed the data of patients with a diagnosis of sepsis in their hospital from December 2012 to December 2013. The authors analyzed all diagnostic episodes consisting of BC and PCT concentration. The diagnostic performance of PCT to predict gram-negative bacteremia was tested using a receiver operative characteristic curve. Logistic regression was constructed using the presence of gram-negative bacteria as the dependent variable. Results: A total of 262 diagnostic episodes met the inclusion criteria. According to BC classifications, a significantly higher value of PCT was observed in bloodstream infections caused by gram-negative bacteria (26.7 ng/mL, 0.09–188.3) than that in bloodstream infections caused by gram-positive bacteria (0.84 ng/mL, 0.05–18.79) or Candida spp. (1.12 ng/mL, 0.07–49.68). A cutoff value of ≥3.39 ng/mL for PCT showed a sensitivity of 80%, a specificity of 71%, a positive predictive value of 35%, a negative predictive value of 91% and an area under the curve of 0.73 for gram-negative bacteremia identification by BC. Among the 122 diagnostic episodes with positive BC results, a cutoff value of ≥6.47 ng/mL for PCT yielded a sensitivity of 74%, a specificity of 81%, a positive predictive value of 82%, a negative predictive value of 75% and an area under the curve of 0.81 for gram-negative bacteremia identification. Conclusions: PCT may represent a useful tool for differentiating gram-positive from gram-negative bloodstream infection with a significantly higher PCT level indicating gram-negative bacteremia. PMID:25992537

  14. Clinical efficacy of ciprofloxacin therapy for gram-negative bacillary osteomyelitis.

    PubMed

    Hessen, M T; Ingerman, M J; Kaufman, D H; Weiner, P; Santoro, J; Korzeniowski, O M; Boscia, J; Topiel, M; Bush, L M; Kaye, D

    1987-04-27

    The efficacy and toxicity of ciprofloxacin, an orally administered fluoroquinolone, were evaluated in 24 infections in 23 patients with osteomyelitis caused by aerobic gram-negative bacilli. The diagnosis was confirmed by surgical findings and the results of bone biopsy and culture of bone or deep soft tissue. The aerobic gram-negative bacilli were Pseudomonas aeruginosa (15 isolates), Serratia marcescens (five isolates), Escherichia coli (three isolates), Enterobacter species (three isolates), Proteus mirabilis (one isolate), Pseudomonas fluorescens (one isolate), and Klebsiella pneumoniae (one isolate). Minimal bactericidal concentrations (MBCs) were 1.56 micrograms/ml or less for all but one isolate. Nine infections were polymicrobial, involving aerobic gram-positive cocci or anaerobes in addition to aerobic gram-negative bacilli. Additional antibiotics to which the aerobic gram-negative bacilli were resistant were given when the additional organisms were resistant to ciprofloxacin. Patients received 750 mg of ciprofloxacin twice daily for a mean of 62 days. Peak serum levels of ciprofloxacin were at least threefold higher than the MBCs in 20 of 24 patients. Twenty of 22 infections in which a full course of therapy was completed were without evidence of active disease at one to 17 months posttreatment. A sternotomy wound infection relapsed after eight weeks of therapy with a newly resistant S. marcescens strain, and an infection of a compound fracture relapsed two months posttreatment with a still sensitive P. aeruginosa strain. Toxicity was minimal in most patients: eosinophilia (six patients), nausea (eight patients), mild elevation in transaminase levels (three patients), pruritus (one patient), diarrhea (two patients), thrush (two patients), rash (two patients), and mild leukopenia (one patient). Two additional patients had severe side effects (vertigo in one and acute renal failure in another) that required discontinuation of ciprofloxacin therapy. Overall

  15. Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts

    PubMed Central

    Misra, Rajeev

    2012-01-01

    In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts.

  16. The Resveratrol Tetramer (-)-Hopeaphenol Inhibits Type III Secretion in the Gram-Negative Pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa

    PubMed Central

    Zetterström, Caroline E.; Hasselgren, Jenny; Salin, Olli; Davis, Rohan A.; Quinn, Ronald J.; Sundin, Charlotta; Elofsson, Mikael

    2013-01-01

    Society faces huge challenges, as a large number of bacteria have developed resistance towards many or all of the antibiotics currently available. Novel strategies that can help solve this problem are urgently needed. One such strategy is to target bacterial virulence, the ability to cause disease e.g., by inhibition of type III secretion systems (T3SSs) utilized by many clinically relevant gram-negative pathogens. Many of the antibiotics used today originate from natural sources. In contrast, most virulence-blocking compounds towards the T3SS identified so far are small organic molecules. A recent high-throughput screening of a prefractionated natural product library identified the resveratrol tetramer (-)-hopeaphenol as an inhibitor of the T3SS in Yersinia pseudotuberculosis. In this study we have investigated the virulence blocking properties of (-)-hopeaphenol in three different gram-negative bacteria. (-)-Hopeaphenol was found to have micromolar activity towards the T3SSs in Yersinia pseudotuberculosis and Pseudomonas aeruginosa in cell-based infection models. In addition (-)-hopeaphenol reduced cell entry and subsequent intracellular growth of Chlamydia trachomatis. PMID:24324737

  17. Legionella and other gram-negative bacteria in potable water from various rural and urban sources.

    PubMed

    Stojek, Nimfa; Dutkiewicz, Jacek

    2006-01-01

    A total of 107 potable water samples were collected from various rural and urban sources located in the Lublin region (eastern Poland). 54 samples from rural sources comprised 32 samples of untreated well water and 22 samples of treated (chlorinated) tap water from rural dwellings distributed by the municipal water supply system (MWSS). 53 samples of treated water from urban sources were supplied by the city of Lublin MWSS. They comprised: 11 samples of tap water from offices and shops, 8 samples of tap water from dwellings, 19 samples from showerheads in health care units, and 15 samples from the outlets of medical appliances used for hydrotherapy in a rehabilitation centre. Water samples were examined for the presence and species composition of Legionella, Yersinia, Gram-negative bacteria belonging to family Enterobacteriaceae (GNB-E) and Gram-negative bacteria not belonging to family Enterobacteriaceae (GNB-NE), by filtering through cellulose filters and culture on respectively GVPC, CIN, EMB and tryptic soya agar media. Legionella was recovered from samples of well water, tap water from rural dwellings, tap water from urban dwellings, and water from medical appliances - with the isolation frequency of 27.8-50.0 %, and the low concentrations ranging from 0.7-13.3 x 10 (1) cfu/l. No Legionella strains were detected in tap water from offices and shops, and in water from showerheads in health care units. Strains of the Legionella pneumophila types 2-14 predominated, forming 89.9 % of total Legionella isolates, while other species of Legionella formed 10.1 %. Neither Legionella pneumophila type 1 strains nor Yersinia strains were isolated from the examined water samples. The isolation frequency and mean concentration of GNB-E in water samples from rural sources was significantly greater than in water samples from urban sources (respectively 61.1 % vs. 20.8 %, 17.1 vs. 3.4 x 10(1) cfu/l, p < 0.001). Isolation frequency of GNB-NE in water samples from rural sources

  18. Systems biology-guided identification of synthetic lethal gene pairs and its potential use to discover antibiotic combinations

    PubMed Central

    Aziz, Ramy K.; Monk, Jonathan M.; Lewis, Robert M.; In Loh, Suh; Mishra, Arti; Abhay Nagle, Amrita; Satyanarayana, Chitkala; Dhakshinamoorthy, Saravanakumar; Luche, Michele; Kitchen, Douglas B.; Andrews, Kathleen A.; Fong, Nicole L.; Li, Howard J.; Palsson, Bernhard O.; Charusanti, Pep

    2015-01-01

    Mathematical models of metabolism from bacterial systems biology have proven their utility across multiple fields, for example metabolic engineering, growth phenotype simulation, and biological discovery. The usefulness of the models stems from their ability to compute a link between genotype and phenotype, but their ability to accurately simulate gene-gene interactions has not been investigated extensively. Here we assess how accurately a metabolic model for Escherichia coli computes one particular type of gene-gene interaction, synthetic lethality, and find that the accuracy rate is between 25% and 43%. The most common failure modes were incorrect computation of single gene essentiality and biological information that was missing from the model. Moreover, we performed virtual and biological screening against several synthetic lethal pairs to explore whether two-compound formulations could be found that inhibit the growth of Gram-negative bacteria. One set of molecules was identified that, depending on the concentrations, inhibits E. coli and S. enterica serovar Typhimurium in an additive or antagonistic manner. These findings pinpoint specific ways in which to improve the predictive ability of metabolic models, and highlight one potential application of systems biology to drug discovery and translational medicine. PMID:26531810

  19. Investigational Agents for the Treatment of Gram-Negative Bacterial Infections: A Reality Check.

    PubMed

    Bush, Karen

    2015-11-13

    Antibiotic-resistant Gram-negative bacteria are, arguably, the most difficult organisms to treat, with a limited number of new antibiotics in the development pipeline. Currently 24 new agents in phase 1, phase 2, or phase 3 clinical development were identified for the potential treatment of infections caused by Gram-negative bacteria. Of these agents, most are improved iterations of known antibiotic classes, including new aminoglycosides, β-lactams, β-lactamase inhibitors, quinolones, and tetracyclines with greater potency or a broader spectrum of activity. However, novel structures also appear, with host defense peptide mimetics, boronic acid, and bridged diazabicyclooctane β-lactamase inhibitors and unique bacterial topoisomerase inhibitors. Most of the new agents have received a Qualified Infectious Disease Product (QIDP) designation that may help to accelerate FDA drug approvals. Because resistance will inevitably arise to any antibacterial agent, it will be necessary to continue to identify additional new agents in the future. PMID:27623407

  20. Lipopolysaccharide deacylation by an endogenous lipase controls innate antibody responses to Gram-negative bacteria.

    PubMed

    Lu, Mingfang; Zhang, Mei; Takashima, Akira; Weiss, Jerrold; Apicella, Michael A; Li, Xiang-Hong; Yuan, Dorothy; Munford, Robert S

    2005-10-01

    T cell-independent type 1 agonists such as Gram-negative bacterial lipopolysaccharides can stimulate B lymphocytes to proliferate and produce antibodies by signaling through Toll-like receptors. This phenomenon is well established in vitro, yet polyclonal B cell responses after bacterial infection in vivo are often weak and short-lived. We show here that B cell proliferation and polyclonal antibody production in response to Gram-negative bacterial infection are modulated by acyloxyacyl hydrolase, a host enzyme that deacylates bacterial lipopolysaccharides. Deacylation of lipopolysaccharide occurred over several days, allowing lipopolysaccharide to act as an innate immune stimulant yet limiting the eventual amount of B cell proliferation and polyclonal antibody production. Control of lipopolysaccharide activation by acyloxyacyl hydrolase indicates that mammals can regulate immune responses to bacterial infection by chemical modification of a Toll-like receptor agonist. PMID:16155573

  1. Resistance of gram-negative bacilli as related to hospital use of antimicrobial agents.

    PubMed Central

    Ma, M Y; Goldstein, E J; Friedman, M H; Anderson, M S; Mulligan, M E

    1983-01-01

    The development of resistance of gram-negative bacilli, which are common nosocomial pathogens, is an increasing problem. It is generally accepted that this resistance may directly reflect the frequency of use of various antimicrobial agents. Because our institution experienced in 1976 a dramatic change in the pattern of antimicrobial use, primarily a marked decrease in prescribing cephalosporins, we attempted to evaluate retrospectively the effects of this change upon the resistance of gram-negative bacilli that are common nosocomial pathogens. Susceptibilities of Klebsiella and Providencia spp., Pseudomonas aeruginosa, and Serratia marcescens were determined for the years 1975 to 1979. Not unexpectedly, we observed a substantial decrease in cephalosporin resistance. An unexpected finding was a decrease in aminoglycoside resistance, despite increased use of these agents. The possibility that decreased cephalosporin use may lead to decreased aminoglycoside resistance is an intriguing and provocative thesis which can only be speculative at this time but which would seem worthy of additional formal investigation. PMID:6638994

  2. Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry.

    PubMed

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S; Schellenberger, Wolfgang; Rodloff, Arne C; Eschrich, Klaus

    2013-01-01

    Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli. PMID:23919091

  3. Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry

    PubMed Central

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S; Schellenberger, Wolfgang; Rodloff, Arne C; Eschrich, Klaus

    2013-01-01

    Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli. PMID:23919091

  4. Natural transfer of conjugative transposon Tn916 between gram-positive and gram-negative bacteria.

    PubMed

    Bertram, J; Strätz, M; Dürre, P

    1991-01-01

    The conjugative streptococcal transposon Tn916 was found to transfer naturally between a variety of gram-positive and gram-negative eubacteria. Enterococcus faecalis hosting the transposon could serve as a donor for Alcaligenes eutrophus, Citrobacter freundii, and Escherichia coli at frequencies of 10(-6) to 10(-8). No transfer was observed with several phototrophic species. Mating of an E. coli strain carrying Tn916 yielded transconjugants with Bacillus subtilis, Clostridium acetobutylicum, Enterococcus faecalis, and Streptococcus lactis subsp. diacetylactis at frequencies of 10(-4) to 10(-6). Acetobacterium woodii was the only gram-positive organism tested that did not accept the transposon from a gram-negative donor. The results prove the ability of conjugative transposable elements such as Tn916 for natural cross-species gene transfer, thus potentially contributing to bacterial evolution. PMID:1846142

  5. Community-acquired multidrug-resistant Gram-negative bacterial infective endocarditis

    PubMed Central

    Naha, Sowjanya; Naha, Kushal; Acharya, Vasudev; Hande, H Manjunath; Vivek, G

    2014-01-01

    We describe two cases of bacterial endocarditis secondary to multidrug-resistant Gram-negative organisms. In both cases, the diagnosis was made in accordance with the modified Duke's criteria and confirmed by histopathological analysis. Furthermore, in both instances there were no identifiable sources of bacteraemia and no history of contact with hospital or other medical services prior to the onset of symptoms. The patients were managed in similar fashion with prolonged broad-spectrum antibiotic therapy and surgical intervention and made complete recoveries. These cases highlight Gram-negative organisms as potential agents for endocarditis, as well as expose the dissemination of such multidrug-resistant bacteria into the community. The application of an integrated medical and surgical approach and therapeutic dilemmas encountered in managing these cases are described. PMID:25096655

  6. 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

  7. NDM-1 (New Delhi metallo beta lactamase-1) producing Gram-negative bacilli: Emergence & clinical implications

    PubMed Central

    Fomda, Bashir Ahmad; Khan, Asiya; Zahoor, Danish

    2014-01-01

    Backgound & objectives: Resistance to carbapenems in Gram-negative bacteria conferred by NDM-1 is a global health problem. We investigated the occurrence of NDM-1 in clinical isolates of Gram-negative bacilli in a tertiary care hospital in Kashmir valley, India. Methods: Gram-negative bacilli from different clinical isolates were included in the study. Antimicrobial susceptibility was performed by Kirby Bauer disk diffusion method and interpreted using Clinical Laboratory Standards Institute (CLSI) guidelines. Isolates resistant to carbapenems were subjected to different phenotypic test such as modified Hodge test (MHT), boronic acid and oxacillin based MHT (BA-MHT and OXA-MHT), combined disk test and minimum inhibitory concentration (MIC) with imipenem and imipenem -EDTA for determination of class B metallo enzymes. Presence of blaNDM-1 gene was established by PCR and confirmed by sequencing. Results: Of the total 1625 Gram-negative isolates received, 100 were resistant to imipenem. Of the 100 isolates, 55 (55%) were positive by modified Hodge test indicating carbapenemase production. Of the 100 isolates tested by MHT, BA-MHT and OXA-MHT, 29 (29%) isolates belonged to Class A and 15 (15%) to Class B, while 56 (56%) isolates were negative. Of the 15 class B metallo beta lactamase producers, nine carried the blaNDM-1 gene. NDM-1 was found among Escherichia coli (2 isolates), Klebsiella pneumoniae (2 isolates), Citrobacter freundii (3 isolates), Acinetobacter spp (1 isolate), and one isolate of Pseudomonas aeruginosa. Isolates were resistant to all antibiotic tested except polymyxin B and tigecycline. Interpretation & conclusions: Our study showed the presence of clinical isolates expressing NDM-1 in Srinagar, Jammu & Kashmir, India. These isolates harbour plasmid mediated multiple drug resistant determinants and can disseminate easily across several unrelated genera. To halt their spread, early identification of these isolates is mandatory. PMID:25579151

  8. Molecular Studies Neglect Apparently Gram-Negative Populations in the Human Gut Microbiota

    PubMed Central

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

    2013-01-01

    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 1010 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. PMID:23885002

  9. [The effect of netilmicin on gram negative rods isolated in the Konya region].

    PubMed

    Sengil, A Z; Ozenci, H; Altindiş, M; Erdoğan, E

    1988-01-01

    Netilmicin, was tested for the effect against 276 isolates of gram negative bacteria, before had used in Konya region. 15.9% of the isolates were resistant and 84.1% were sensitive to netilmicin. The effects of Netilmicin and gentamicin for 50 Pseudomonas isolates were compared. The other aminoglycosides also were tested against isolates of the bacterium, amikacin was the most active one. PMID:3252118

  10. Hand carriage of aerobic Gram-negative rods by health care personnel

    PubMed Central

    Adams, Brian G.; Marrie, Thomas J.

    1982-01-01

    A quantitative culture technique (hand washed in a glove containing broth for 30 s) was used to determine the frequency of hand carriage of aerobic Gram-negative rods by various groups of health care workers and 104 control subjects. Overall, 31% of health care workers carried aerobic Gram-negative rods on their hands compared to 59% of control subjects (P < 0·001). Enterobacter agglomerans accounted for 40% of the isolates, and other Enterobacter spp. 7%. Other organisms included Acinetobacter calcoaceticus 21%, Serratia spp. 11%, Klebsiella spp. 10%, Moraxella spp. 3%, Pseudomonas spp. 3%, Proteus spp. 1·5%, Escherichia coli 1%; Morganella morganii, Citrobacter freundii, Aeromonas sp. and an isolate that was not speciated accounted for 0·5% each. We conclude that endemic hand carriage of aerobic Gram-negative rods by health care personnel is common, but significantly less than that of control subjects. Enterobacter agglomerans is found so frequently on the hands of control subjects that it must be considered part of the normal hand flora. PMID:7097001

  11. 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. PMID:26344957

  12. Toll-like receptor genetic variants are associated with Gram-negative infections in VLBW infants

    PubMed Central

    Sampath, Venkatesh; Mulrooney, Neil P.; Garland, Jeffery S.; He, Jie; Patel, Aloka L.; Cohen, Jonathan D.; Simpson, Pippa M.; Hines, Ronald N.

    2015-01-01

    OBJECTIVE To test the hypothesis that single nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes alter susceptibility to bacterial infections and modulate WBC counts during infections in very low birth-weight infants (birth weight <1500g, VLBW). STUDY DESIGN VLBW infants recruited in a multi-center study were genotyped for 9 functional TLR SNPs and associations between SNPs and infection rates examined. WBC counts obtained during infections were compared among infants with and without SNPs. RESULTS In our cohort (n=408), 90 infants developed bacterial infections. Presence of TLR4 (rs4986790 & 4986791) variants were associated with Gram-negative infections. Female infants heterozygous for the X-linked IRAK1 (rs1059703) SNP had less Gram-negative infections. In regression models controlling for confounders, the TLR4 (rs4986790) SNP was associated with increased Gram-negative infections. The TLR5 (rs5744105) variant was associated with elevated WBC counts during infections. CONCLUSION TLR genetic variants can contribute to increased risk of bacterial infections and altered immune responses in VLBW infants. PMID:23867959

  13. The growing threat of multidrug-resistant Gram-negative infections in patients with hematologic malignancies.

    PubMed

    Baker, Thomas M; Satlin, Michael J

    2016-10-01

    Prolonged neutropenia and chemotherapy-induced mucositis render patients with hematologic malignancies highly vulnerable to Gram-negative bacteremia. Unfortunately, multidrug-resistant (MDR) Gram-negative bacteria are increasingly encountered globally, and current guidelines for empirical antibiotic coverage in these patients may not adequately treat these bacteria. This expansion of resistance, coupled with traditional culturing techniques requiring 2-4 days for bacterial identification and antimicrobial susceptibility results, have grave implications for these immunocompromised hosts. This review characterizes the epidemiology, risk factors, resistance mechanisms, recommended treatments, and outcomes of the MDR Gram-negative bacteria that commonly cause infections in patients with hematologic malignancies. We also examine the infection prevention strategies in hematology patients, such as infection control practices, antimicrobial stewardship, and targeted decolonization. Finally, we assess the strategies to improve outcomes of the infected patients, including gastrointestinal screening to guide empirical antibiotic therapy, new rapid diagnostic tools for expeditious identification of MDR pathogens, and use of two new antimicrobial agents, ceftolozane/tazobactam and ceftazidime/avibactam. PMID:27339405

  14. 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

  15. Prognostic factors and monomicrobial necrotizing fasciitis: gram-positive versus gram-negative pathogens

    PubMed Central

    2011-01-01

    Background Monomicrobial necrotizing fasciitis is rapidly progressive and life-threatening. This study was undertaken to ascertain whether the clinical presentation and outcome for patients with this disease differ for those infected with a gram-positive as compared to gram-negative pathogen. Methods Forty-six patients with monomicrobial necrotizing fasciitis were examined retrospectively from November 2002 to January 2008. All patients received adequate broad-spectrum antibiotic therapy, aggressive resuscitation, prompt radical debridement and adjuvant hyperbaric oxygen therapy. Eleven patients were infected with a gram-positive pathogen (Group 1) and 35 patients with a gram-negative pathogen (Group 2). Results Group 2 was characterized by a higher incidence of hemorrhagic bullae and septic shock, higher APACHE II scores at 24 h post-admission, a higher rate of thrombocytopenia, and a higher prevalence of chronic liver dysfunction. Gouty arthritis was more prevalent in Group 1. For non-survivors, the incidences of chronic liver dysfunction, chronic renal failure and thrombocytopenia were higher in comparison with those for survivors. Lower level of serum albumin was also demonstrated in the non-survivors as compared to those in survivors. Conclusions Pre-existing chronic liver dysfunction, chronic renal failure, thrombocytopenia and hypoalbuminemia, and post-operative dependence on mechanical ventilation represent poor prognostic factors in monomicrobial necrotizing fasciitis. Patients with gram-negative monobacterial necrotizing fasciitis present with more fulminant sepsis. PMID:21208438

  16. Tetracycline tethered to titanium inhibits colonization by Gram-negative bacteria.

    PubMed

    Davidson, Helen; Poon, Martin; Saunders, Ray; Shapiro, Irving M; Hickok, Noreen J; Adams, Christopher S

    2015-10-01

    As peri-prosthetic infection is one of the most devastating complications associated with implant placement, we have reasoned that such infection can be largely subverted by development of antibacterial implants. Our previous work demonstrated that covalent coupling of vancomycin to titanium alloy prevented colonization by the Gram-positive pathogens, Staphylococcus aureus and Staphylococcus epidermidis. Some orthopedic devices, including permanent prosthesis anchors, and most dental implants are transcutaneous or transmucosal and can be prone to colonization by Gram-negative pathogens. We report here the successful covalent coupling of the broad-spectrum antibiotic, tetracycline (TET), to titanium surfaces (Ti-TET) to retard Gram-negative colonization. Synthetic progress was followed by changes in water contact angle, while the presence of TET was confirmed by immunofluorescence. Ti-TET actively prevented colonization in the presence of bathing Escherichia coli, both by fluorescence microscopy and direct counting. Finally, the Ti-TET surface supported osteoblastic cell adhesion and proliferation over a 72-h period. Thus, this new surface offers a powerful means to protect transcutaneous implants from adhesion of Gram-negative pathogens, decreasing the need for replacement of this hardware. PMID:25389082

  17. 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

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

    PubMed

    Duarte, Silvana; e Silva, Flávia Cristina de Paula; 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. Platelet Mass Predicts Intracranial Hemorrhage in Neonates With Gram-negative Sepsis.

    PubMed

    Mitsiakos, Georgios; Pana, Zoe-Dorothea; Chatziioannidis, Ilias; Piltsouli, Dimitra; Lazaridou, Eleni; Koulourida, Vasiliki; Papadimitriou, Aikaterini; Nikolaidis, Nikolaos; Roilides, Emmanuel

    2015-10-01

    Neonatal sepsis due to gram-negative bacteria is associated with severe hemorrhagic conditions, such as intracranial hemorrhage (ICH). The aim of the study was to investigate the significance of platelet (PLT) count and platelet mass (PM) in predicting promptly neonatal ICH. Demographics, species, PLT, PM, ICH, and outcome for neonates with gram-negative sepsis for the period 2005 to 2012 were retrospectively recorded. Eighty-four infants were enrolled with median gestational age 30 weeks, median birthweight 1481.5 g, and median age at sepsis diagnosis 23 days. The most frequently isolated bacteria were Enterobacter spp. (38.1%). ICH occurred in 16 neonates (19%), whereas the mortality rate was 25% (21 neonates). The median PLT count and PM at days 1, 2, and 3 after diagnosis of gram-negative sepsis was significantly associated with the presence of ICH. Regression analysis revealed the cutoff predictive value of 355 fL/nL for the PM at day 3 (area under the curve: 75, sensitivity 90%, P=0.002). PM levels could play an important role in predicting the occurrence of ICH in high-risk neonates. PMID:26376234

  20. Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin.

    PubMed Central

    Ellison, R T; Giehl, T J; LaForce, F M

    1988-01-01

    Many studies have shown that lactoferrin and transferrin have antimicrobial activity against gram-negative bacteria, but a mechanism of action has not been defined. We hypothesized that the iron-binding proteins could affect the gram-negative outer membrane in a manner similar to that of the chelator EDTA. The ability of lactoferrin and transferrin to release radiolabeled lipopolysaccharide (LPS) from a UDP-galactose epimerase-deficient Escherichia coli mutant and from wild-type Salmonella typhimurium strains was tested. Initial studies in barbital-acetate buffer showed that EDTA and lactoferrin cause significant release of LPS from all three strains. Further studies found that LPS release was blocked by iron saturation of lactoferrin, occurred between pH 6 and 7.5, was comparable for bacterial concentrations from 10(4) to 10(7) CFU/ml, and increased with increasing lactoferrin concentrations. Studies using Hanks balanced salt solution lacking calcium and magnesium showed that transferrin also could cause LPS release. Additionally, both lactoferrin and transferrin increased the antibacterial effect of a subinhibitory concentration of rifampin, a drug excluded by the bacterial outer membrane. This work demonstrates that these iron-binding proteins damage the gram-negative outer membrane and alter bacterial outer membrane permeability. Images PMID:3169987

  1. [Bioactive effectiveness of selected disinfective agents on Gram-negative bacilli isolated from hospital environment].

    PubMed

    Pancer, Katarzyna W; Laudy, Agnieszka E; Mikulak, Ewa; Gliniewicz, Aleksandra; Staniszewska, Monika; Stypułkowska-Misiurewicz, Hanna

    2004-01-01

    In our study the susceptibility (MIC) of chosen 21 strains of Gram-negative bacilli isolated in hospitals to disinfectant agents (glucoprotamine, sodium dichloroisocyanurate, potassium persulfate), the effectiveness of these disinfectants against selected bacteria and their effectiveness to biofilm forming bacteria was determined. It was found that glucoprotamine showed the highest activity to Gram-negative bacteria. Obtained MIC values for glucoprotamine (except 1 strain of S. marcescens) were 16-64 times lower that MICs for sodium dichloroisocyanurate and 4-32 times lower that MICs for potassium persulfate. The effectiveness of disinfectants containing potassium persulfate or sodium dichloroisocyanurate was 100% tested by carrier method. Glucoprotamine was ineffective against 2 out of 9 strains (18%): E. cloacae and S. marcescens. It was found that disinfectants were more effective against Gram-negative bacteria in carrier methods than for biofilm forming bacteria. 86% of bacteria growing 5 days on a catheter were resistant to working solution of disinfectant containing glucoprotamine (5200 mg/L) or potassium persulfate (4300 mg/L); 66.6% of tested bacteria were resistant to working solution of sodium dichloroisocyanurate (1795.2 mg/L). In our study the highest effectiveness to biofilm forming bacteria showed disinfectant with sodium dichloroisocyanurate, the lowest--with glucoprotamine. PMID:15810507

  2. 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

  3. Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice

    PubMed Central

    Prestia, Kevin; Bandyopadhyay, Sheila; Slate, Andrea; Francis, Richard O.; Francis, Kevin P.; Spitalnik, Steven L.; Fidock, David A.; Brittenham, Gary M.; Hod, Eldad A.

    2014-01-01

    BACKGROUND Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra- and extravascular hemolysis. Studies suggest that iron administration increases infectious risk. STUDY DESIGN AND METHODS To assess the effects of increased iron availability on susceptibility to infection, we infected mice with model Gram-negative intracellular or extracellular pathogens (Salmonella typhimurium or Escherichia coli, respectively), accompanied by RBC transfusion, iron dextran administration, or malarial coinfection. RESULTS In our mouse models, transfusion of older RBCs exacerbates infection with both Gram-negative pathogens. Although iron dextran exacerbates E. coli infection to a similar extent as transfusion of corresponding amounts of iron, higher iron doses are required to produce comparable effects with S. typhimurium. Coinfection of mice with Plasmodium yoelii and S. typhimurium produces overwhelming Salmonella sepsis. Finally, treating mice with antibiotics abrogates the enhancing effect on E. coli infection of both older RBC transfusion and iron dextran administration. CONCLUSIONS Transfusion of older RBCs exacerbates Gram-negative infection to a similar extent as malaria coinfection or iron dextran administration. Appropriate antibiotic therapy abrogates the effect of older RBC transfusions on infection with E. coli. Iron delivery to macrophages may be an underappreciated mechanism mediating, at least some, adverse effects of RBC transfusions. PMID:24840185

  4. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.

    PubMed

    Lim, Jeong-A; Shin, Hakdong; Heu, Sunggi; Ryu, Sangryeol

    2014-06-28

    Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from 24°C to 65°C). It showed maximal activity at 50°C, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria. PMID:24690638

  5. Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species.

    PubMed Central

    Merlino, J; Siarakas, S; Robertson, G J; Funnell, G R; Gottlieb, T; Bradbury, R

    1996-01-01

    A new chromogenic plate medium, CHROMagar Orientation, was evaluated for use in the differentiation and presumptive identification of gram-negative bacilli and Enterococcus species by a multipoint inoculation (replicator) technique. In this study, 1,404 gram-negative bacilli and 74 enterococcal isolates were tested on CHROMagar Orientation. Six control American Type Culture Collection strains were also included with the testing to ensure quality control of the media. Of the Escherichia coli isolates (n = 588) tested, 99.3% produced a pink-to-red color. Only in four isolates that were O-nitrophenyl-beta-D-galactopyranoside (ONPG) negative did this result differ. Proteus mirabilis and P. vulgaris were well differentiated on this medium. P. mirabilis (n = 184) produced a clear colony with diffusible brown pigment around the periphery. By contrast, 15 of 16 P. vulgaris isolates produced bluish-green colonies with a slight brown background. All Aeromonas hydrophila isolates (n = 26) tested produced clear to pink colonies at 35 to 37 degrees C. This colony color changed to blue after 2 to 3 h of incubation at room temperature. A. hydrophila exhibited stronger color and better growth at 30 degrees C. Serratia marcescens (n = 29) demonstrated an aqua blue color that deepened to a darker blue when exposed to room temperature. All enterococcal isolates (n = 74) resulted in a blue color and gave pinpoint colonies on purity subcultures at 35 to 37 degrees C after 18 h of incubation. Similarity in color resulted in failure to discriminate accurately between Klebsiella, Enterobacter, and Citrobacter species. However, these species could be readily differentiated from other members of the family Enterobacteriaceae. Pseudomonas aeruginosa (n = 151) was easily differentiated from members of the Enterobacteriaceae but was less easily distinguishable from other gram-negative nonmembers of the Enterobacteriaceae. The medium was found to facilitate easy visual detection of mixed

  6. Potentiation Effects of Amikacin and Fosfomycin against Selected Amikacin-Nonsusceptible Gram-Negative Respiratory Tract Pathogens

    PubMed Central

    Rhomberg, Paul R.; Abuan, Tammy; Walters, Kathie-Anne; Flamm, Robert K.

    2014-01-01

    The amikacin-fosfomycin inhalation system (AFIS) is a combination of 2 antibiotics and an in-line nebulizer delivery system that is being developed for adjunctive treatment of pneumonia caused by Gram-negative organisms in patients on mechanical ventilation. AFIS consists of a combination of amikacin and fosfomycin solutions at a 5:2 ratio (amikacin, 3 ml at 100 mg/ml; fosfomycin, 3 ml at 40 mg/ml) and the PARI Investigational eFlow Inline System. In this antibiotic potentiation study, the antimicrobial activities of amikacin and fosfomycin, alone and in a 5:2 combination, were assessed against 62 Gram-negative pathogens from a worldwide antimicrobial surveillance collection (SENTRY). The amikacin MICs for 62 isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were ≥32 μg/ml (intermediate or resistant according to the Clinical and Laboratory Standards Institute [CLSI]; resistant according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST]). Each isolate was tested against amikacin (0.25 to 1,024 μg/ml), fosfomycin (0.1 to 409.6 μg/ml), and amikacin-fosfomycin (at a 5:2 ratio) using CLSI reference agar dilution methods. The median MIC values for amikacin and fosfomycin against the 62 isolates each decreased 2-fold with the amikacin-fosfomycin (5:2) combination from that with either antibiotic alone. Interactions between amikacin and fosfomycin differed by isolate and ranged from no detectable interaction to high potentiation. The amikacin-fosfomycin (5:2) combination reduced the amikacin concentration required to inhibit all 62 isolates from >1,024 to ≤256 μg/ml and reduced the required fosfomycin concentration from 204.8 to 102.4 μg/ml. These results support continued development of the amikacin-fosfomycin combination for aerosolized administration, where high drug levels can be achieved. PMID:24752275

  7. Potentiation effects of amikacin and fosfomycin against selected amikacin-nonsusceptible Gram-negative respiratory tract pathogens.

    PubMed

    Montgomery, A Bruce; Rhomberg, Paul R; Abuan, Tammy; Walters, Kathie-Anne; Flamm, Robert K

    2014-07-01

    The amikacin-fosfomycin inhalation system (AFIS) is a combination of 2 antibiotics and an in-line nebulizer delivery system that is being developed for adjunctive treatment of pneumonia caused by Gram-negative organisms in patients on mechanical ventilation. AFIS consists of a combination of amikacin and fosfomycin solutions at a 5:2 ratio (amikacin, 3 ml at 100 mg/ml; fosfomycin, 3 ml at 40 mg/ml) and the PARI Investigational eFlow Inline System. In this antibiotic potentiation study, the antimicrobial activities of amikacin and fosfomycin, alone and in a 5:2 combination, were assessed against 62 Gram-negative pathogens from a worldwide antimicrobial surveillance collection (SENTRY). The amikacin MICs for 62 isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were ≥32 μg/ml (intermediate or resistant according to the Clinical and Laboratory Standards Institute [CLSI]; resistant according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST]). Each isolate was tested against amikacin (0.25 to 1,024 μg/ml), fosfomycin (0.1 to 409.6 μg/ml), and amikacin-fosfomycin (at a 5:2 ratio) using CLSI reference agar dilution methods. The median MIC values for amikacin and fosfomycin against the 62 isolates each decreased 2-fold with the amikacin-fosfomycin (5:2) combination from that with either antibiotic alone. Interactions between amikacin and fosfomycin differed by isolate and ranged from no detectable interaction to high potentiation. The amikacin-fosfomycin (5:2) combination reduced the amikacin concentration required to inhibit all 62 isolates from >1,024 to ≤ 256 μg/ml and reduced the required fosfomycin concentration from 204.8 to 102.4 μg/ml. These results support continued development of the amikacin-fosfomycin combination for aerosolized administration, where high drug levels can be achieved. PMID:24752275

  8. Comparison of endotoxin levels and gram-negative bacteria under different conditions in microbial laboratories and a biowaste site.

    PubMed

    Hwang, Sung Ho; Park, Dong Uk; Joo, Se Ik; Park, Hyun Hee; Yoon, Chung Sik

    2011-09-01

    In this study, we assessed airborne endotoxin levels in university laboratories, hospital diagnostic laboratories, and a biowaste site. We also investigated indoor and outdoor sampling, sampling site, type of ventilation system, presence of open biowaste boxes, weather, and detection of Gram-negative bacteria (GNB). A total of 69 air samples were collected from 11 facilities in three institutions. Average total airborne endotoxin levels ranged from <0.01 to 10.02 EU m(-3), with an overall mean of 1.03 EU m(-3). Endotoxin levels were high in window-ventilated facilities, in facilities in which GNB were detected; levels were also high when it was rainy (all ps<0.05). Endotoxin levels were significantly correlated with humidity (r=0.70, p<0.01). The presence of HVAC; humidity; and the presence of open biowaste boxes affect endotoxin levels in laboratories. PMID:21726888

  9. 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

    2014-03-01

    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. PMID:24162371

  10. Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria

    PubMed Central

    Pérez-Cruz, Carla; Delgado, Lidia; López-Iglesias, Carmen; Mercade, Elena

    2015-01-01

    Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles. PMID:25581302

  11. 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

  12. Combination antibiotic therapy for multidrug-resistant Gram-negative bacteria

    PubMed Central

    2014-01-01

    Combination antibiotic therapy for Gram-negative sepsis is controversial. The present review provides a brief summary of the existing knowledge on combination therapy for severe infections with multidrug-resistant Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae. Empirical combination antibiotic therapy is recommended for severe sepsis and septic shock to reduce mortality related to inappropriate antibiotic treatment. Because definitive combination therapy has not been proven superior to monotherapy in meta-analyses, it is generally advised to de-escalate antibiotic therapy when the antibiotic susceptibility profile is known, although it cannot be excluded that some subgroups of patients might still benefit from continued combination therapy. Definitive combination therapy is recommended for carbapenemase-producing Enterobacteriaceae and should also be considered for severe infections with Pseudomonas and Acinetobacter spp. when beta-lactams cannot be used. Because resistance to broad-spectrum beta-lactams is increasing in Gram-negative bacteria and because no new antibiotics are expected to become available in the near future, the antibacterial potential of combination therapy should be further explored. In vitro data suggest that combinations can be effective even if the bacteria are resistant to the individual antibiotics, although existing evidence is insufficient to support the choice of combinations and explain the synergistic effects observed. In vitro models can be used to screen for effective combinations that can later be validated in animal or clinical studies. Further, in the absence of clinical evidence, in vitro data might be useful in supporting therapeutic decisions for severe infections with multidrug-resistant Gram-negative bacteria. PMID:24666223

  13. Extended Spectrum Beta-lactamase Detection in Gram-negative Bacilli of Nosocomial Origin

    PubMed Central

    Tsering, Dechen C; Das, Shyamasree; Adhiakari, Luna; Pal, Ranabir; Singh, Takhellambam SK

    2009-01-01

    Background: Resistance to third generation cephalosporins by acquisition and expression of extended spectrum beta lactamase (ESBL) enzymes among gram-negative bacilli is on a rise. The presence of ESBL producing organisms significantly affects the course and outcome of an infection and poses a challenge to infection management worldwide. Materials and Methods: In the period from June 2007 to 2008, we collected 1489 samples from patients suspected of nosocomial infection. The isolates were identified based on colony morphology and biochemical reaction. Gram negative bacilli resistant to third generation cephalosporins were tested for ESBL by double disc synergy test (DDST- a screening test)and then phenotypic confirmatory test. Antimicrobial susceptibility testing was done by modified Kirby Bauer disc diffusion method. Results: From the sample of 238 gram-negative bacilli, we isolated Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, Morganella morganii and Enterobacter cloacae. Following both methods, 34% isolates were ESBL-positive. The ESBL producing isolates were significantly resistant (p < 0.01) to ampicillin, piperacillin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, tetracycline, ciprofloxacin and gentamicin as compared to non-ESBL producers. Multidrug resistance was significantly (p < 0.01) higher (69.14%) in ESBL positive isolates than non-ESBL isolates (21.66%). Conclusion: High prevalence of ESBL in our hospital cannot be ignored. ESBL producers can be detected by DDST and phenotypic confirmatory test with equal efficacy. The sensitivity of screening test improved with the use of more than one antibiotic and addition of one or two antibiotics would not increase cost and labor. We recommend DDST using multiple antibiotics in all microbiology units as a routine screening test. PMID:20300397

  14. Characterization of eight beta-lactamases of Gram-negative bacteria.

    PubMed Central

    Sawai, T; Kanno, M; Tsukamoto, K

    1982-01-01

    Eight kinds of beta-lactamases produced by gram-negative bacteria were characterized by the following properties: molecular weight, isoelectric point, pH optimum, molecular activity, immunochemical reactivity, and kinetic parameters with respect to twelve kinds of common beta-lactam antibiotics. These beta-lactamases included two types of penicillinases mediated by R plasmids and six kinds of species-specific cephalosporinases. To determine a reliable value of the kinetic parameter, Km, we introduced a continuous and acidimetric assay method of beta-lactamase activity with a pH stat. PMID:6752115

  15. Chronic granulomatous disease: fatal septicemia caused by an unnamed gram-negative bacterium.

    PubMed Central

    Seger, R A; Hollis, D G; Weaver, R E; Hitzig, W H

    1982-01-01

    A 2-year-old boy with proven X-linked chronic granulomatous disease was placed under continuous co-trimoxazole prophylaxis. He remained free of infection for 4 years. At age 6.25 years, he suddenly developed a fever with no localizing signs and died 16 days later in septic shock. A gram-negative, catalase-positive, halophilic, aerobic bacterium was cultured from blood, bone marrow, and ascitic fluid. This organism could not be identified in microbiological laboratories in Europe and the United States. Its biochemical features indicate that it may belong to a species which has not previously been described. PMID:7153335

  16. 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

  17. 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

  18. BacPP: a web-based tool for Gram-negative bacterial promoter prediction.

    PubMed

    de Avila E Silva, S; Notari, D L; Neis, F A; Ribeiro, H G; Echeverrigaray, S

    2016-01-01

    Bacterial Promoter Prediction (BacPP) is a tool used to predict given sequences as promoters of Gram-negative bacteria according to the σ factor that recognizes it. The first version of BacPP was implemented in Python language in a desktop version without a friendly interface. For this reason, a web version of BacPP is now available with the purpose of improving its usability and availability. The present paper describes the implementation of the web version of this tool, focusing on its software architecture and user functionalities. The software is available at www.bacpp.bioinfoucs.com/home. PMID:27173187

  19. Multidrug Resistance in Quinolone-Resistant Gram-Negative Bacteria Isolated from Hospital Effluent and the Municipal Wastewater Treatment Plant.

    PubMed

    Vaz-Moreira, Ivone; Varela, Ana Rita; Pereira, Thamiris V; Fochat, Romário C; Manaia, Célia M

    2016-03-01

    This study is aimed to assess if hospital effluents represent an important supplier of multidrug-resistant (MDR) Gram-negative bacteria that, being discharged in the municipal collector, may be disseminated in the environment and bypassed in water quality control systems. From a set of 101 non-Escherichia coli Gram-negative bacteria with reduced susceptibility to quinolones, was selected a group of isolates comprised by those with the highest indices of MDR (defined as nonsusceptibility to at least one agent in six or more antimicrobial categories, MDR ≥6) or resistance to meropenem or ceftazidime (n = 25). The isolates were identified and characterized for antibiotic resistance phenotype, plasmid-mediated quinolone resistance (PMQR) genes, and other genetic elements and conjugative capacity. The isolates with highest MDR indices were mainly from hospital effluent and comprised ubiquitous bacterial groups of the class Gammaproteobacteria, of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas, and of the class Flavobacteriia, of the genera Chryseobacterium and Myroides. In this group of 25 strains, 19 identified as Gammaproteobacteria harbored at least one PMQR gene (aac(6')-Ib-cr, qnrB, qnrS, or oqxAB) or a class 1 integron gene cassette encoding aminoglycoside, sulfonamide, or carbapenem resistance. Most of the E. coli J53 transconjugants with acquired antibiotic resistance resulted from conjugation with Enterobacteriaceae. These transconjugants demonstrated acquired resistance to a maximum of five classes of antibiotics, one or more PMQR genes and/or a class 1 integron gene cassette. This study shows that ubiquitous bacteria, other than those monitored in water quality controls, are important vectors of antibiotic resistance and can be disseminated from hospital effluent to aquatic environments. This information is relevant to support management options aiming at the control of this public health problem. PMID

  20. Indole inhibition of N-acylated homoserine lactone-mediated quorum signalling is widespread in Gram-negative bacteria.

    PubMed

    Hidalgo-Romano, Benjamin; Gollihar, Jimmy; Brown, Stacie A; Whiteley, Marvin; Valenzuela, Ernesto; Kaplan, Heidi B; Wood, Thomas K; McLean, Robert J C

    2014-11-01

    The LuxI/R quorum-sensing system and its associated N-acylated homoserine lactone (AHL) signal is widespread among Gram-negative bacteria. Although inhibition by indole of AHL quorum signalling in Pseudomonas aeruginosa and Acinetobacter oleivorans has been reported previously, it has not been documented among other species. Here, we show that co-culture with wild-type Escherichia coli, but not with E. coli tnaA mutants that lack tryptophanase and as a result do not produce indole, inhibits AHL-regulated pigmentation in Chromobacterium violaceum (violacein), Pseudomonas chlororaphis (phenazine) and Serratia marcescens (prodigiosin). Loss of pigmentation also occurred during pure culture growth of Chro. violaceum, P. chlororaphis and S. marcescens in the presence of physiologically relevant indole concentrations (0.5-1.0 mM). Inhibition of violacein production by indole was counteracted by the addition of the Chro. violaceum cognate autoinducer, N-decanoyl homoserine lactone (C10-HSL), in a dose-dependent manner. The addition of exogenous indole or co-culture with E. coli also affected Chro. violaceum transcription of vioA (violacein pigment production) and chiA (chitinase production), but had no effect on pykF (pyruvate kinase), which is not quorum regulated. Chro. violaceum AHL-regulated elastase and chitinase activity were inhibited by indole, as was motility. Growth of Chro. violaceum was not affected by indole or C10-HSL supplementation. Using a nematode-feeding virulence assay, we observed that survival of Caenorhabditis elegans exposed to Chro. violaceum, P. chlororaphis and S. marcescens was enhanced during indole supplementation. Overall, these studies suggest that indole represents a general inhibitor of AHL-based quorum signalling in Gram-negative bacteria. PMID:25165125

  1. Multidrug-Resistant Gram-Negative Bacterial Infections in the Hospital Setting: Overview, Implications for Clinical Practice, and Emerging Treatment Options.

    PubMed

    Cerceo, Elizabeth; Deitelzweig, Steven B; Sherman, Bradley M; Amin, Alpesh N

    2016-07-01

    The increasing prevalence of infections due to multidrug-resistant (MDR) gram-negative bacteria constitutes a serious threat to global public health due to the limited treatment options available and the historically slow pace of development of new antimicrobial agents. Infections due to MDR strains are associated with increased morbidity and mortality and prolonged hospitalization, which translates to a significant burden on healthcare systems. In particular, MDR strains of Enterobacteriaceae (especially Klebsiella pneumoniae and Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii have emerged as particularly serious concerns. In the United States, MDR strains of these organisms have been reported from hospitals throughout the country and are not limited to a small subset of hospitals. Factors that have contributed to the persistence and spread of MDR gram-negative bacteria include the following: overuse of existing antimicrobial agents, which has led to the development of adaptive resistance mechanisms by bacteria; a lack of good antimicrobial stewardship such that use of multiple broad-spectrum agents has helped perpetuate the cycle of increasing resistance; and a lack of good infection control practices. The rising prevalence of infections due to MDR gram-negative bacteria presents a significant dilemma in selecting empiric antimicrobial therapy in seriously ill hospitalized patients. A prudent initial strategy is to initiate treatment with a broad-spectrum regimen pending the availability of microbiological results allowing for targeted or narrowing of therapy. Empiric therapy with newer agents that exhibit good activity against MDR gram-negative bacterial strains such as tigecycline, ceftolozane-tazobactam, ceftazidime-avibactam, and others in the development pipeline offer promising alternatives to existing agents. PMID:26866778

  2. Clinical Impact of Laboratory Implementation of Verigene BC-GN Microarray-Based Assay for Detection of Gram-Negative Bacteria in Positive Blood Cultures.

    PubMed

    Walker, Tamar; Dumadag, Sandrea; Lee, Christine Jiyoun; Lee, Seung Heon; Bender, Jeffrey M; Cupo Abbott, Jennifer; She, Rosemary C

    2016-07-01

    Gram-negative bacteremia is highly fatal, and hospitalizations due to sepsis have been increasing worldwide. Molecular tests that supplement Gram stain results from positive blood cultures provide specific organism information to potentially guide therapy, but more clinical data on their real-world impact are still needed. We retrospectively reviewed cases of Gram-negative bacteremia in hospitalized patients over a 6-month period before (n = 98) and over a 6-month period after (n = 97) the implementation of a microarray-based early identification and resistance marker detection system (Verigene BC-GN; Nanosphere) while antimicrobial stewardship practices remained constant. Patient demographics, time to organism identification, time to effective antimicrobial therapy, and other key clinical parameters were compared. The two groups did not differ statistically with regard to comorbid conditions, sources of bacteremia, or numbers of intensive care unit (ICU) admissions, active use of immunosuppressive therapy, neutropenia, or bacteremia due to multidrug-resistant organisms. The BC-GN panel yielded an identification in 87% of Gram-negative cultures and was accurate in 95/97 (98%) of the cases compared to results using conventional culture. Organism identifications were achieved more quickly post-microarray implementation (mean, 10.9 h versus 37.9 h; P < 0.001). Length of ICU stay, 30-day mortality, and mortality associated with multidrug-resistant organisms were significantly lower in the postintervention group (P < 0.05). More rapid implementation of effective therapy was statistically significant for postintervention cases of extended-spectrum beta-lactamase-producing organisms (P = 0.049) but not overall (P = 0.12). The Verigene BC-GN assay is a valuable addition for the early identification of Gram-negative organisms that cause bloodstream infections and can significantly impact patient care, particularly when resistance markers are detected. PMID:27098961

  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. Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics.

    PubMed

    Uppu, Divakara S S M; Manjunath, Goutham B; Yarlagadda, Venkateswarlu; Kaviyil, Jyothi E; Ravikumar, Raju; Paramanandham, Krishnamoorthy; Shome, Bibek R; Haldar, Jayanta

    2015-01-01

    Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-1) producing pathogens have become world's major public health threats. Development of molecular strategies that can rehabilitate the 'old antibiotics' and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs. PMID:25789871

  5. 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. PMID:26922474

  6. Rewiring Gram-Negative Bacteria Cell Surfaces with Bio-Orthogonal Chemistry via Liposome Fusion.

    PubMed

    Elahipanah, Sina; Radmanesh, Parham; Luo, Wei; O'Brien, Paul J; Rogozhnikov, Dmitry; Yousaf, Muhammad N

    2016-04-20

    The ability to tailor bacteria cell surfaces with non-native molecules is critical to advance the study of bacteria communication, cell behavior, and for next-generation therapeutics to improve livestock and human health. Such modifications would allow for novel control over cell behavior, cell-cell interactions, biofilm formation, adjuvant conjugation, and imaging. Current methods to engineer bacteria surfaces have made major advances but rely on complicated, slow, and often expensive molecular biology and metabolic manipulation methods with limited scope on the type of molecules installed onto the surface. In this report, we introduce a new straightforward method based on liposome fusion to engineer Gram-negative bacteria cells with bio-orthogonal groups that can subsequently be conjugated to a range of molecules (biomolecules, small molecules, probes, proteins, nucleic acids, ligands, and radiolabels) for further studies and programmed behavior of bacteria. This method is fast, efficient, inexpensive, and useful for installing a broad scope of ligands and biomolecules to Gram-negative bacteria surfaces. PMID:27019118

  7. Identification and Characterization of the First Cholesterol-Dependent Cytolysins from Gram-Negative Bacteria

    PubMed Central

    Hotze, Eileen M.; Le, Huynh M.; Sieber, Jessica R.; Bruxvoort, Christina; McInerney, Michael J.

    2013-01-01

    The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from the Firmicutes and Actinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which include Desulfobulbus propionicus type species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) and Enterobacter lignolyticus (formerly Enterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neither D. propionicus nor E. lignolyticus is known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators. PMID:23115036

  8. Glycopeptide Antibiotic To Overcome the Intrinsic Resistance of Gram-Negative Bacteria.

    PubMed

    Yarlagadda, Venkateswarlu; Manjunath, Goutham B; Sarkar, Paramita; Akkapeddi, Padma; Paramanandham, Krishnamoorthy; Shome, Bibek R; Ravikumar, Raju; Haldar, Jayanta

    2016-02-12

    The emergence of drug resistance along with a declining pipeline of clinically useful antibiotics has made it vital to develop more effective antimicrobial therapeutics, particularly against difficult-to-treat Gram-negative pathogens (GNPs). Many antibacterial agents, including glycopeptide antibiotics such as vancomycin, are inherently inactive toward GNPs because of their inability to cross the outer membrane of these pathogens. Here, we demonstrate, for the first time, lipophilic cationic (permanent positive charge) vancomycin analogues were able to permeabilize the outer membrane of GNPs and overcome the inherent resistance of GNPs toward glycopeptides. Unlike vancomycin, these analogues were shown to have a high activity against a variety of multidrug-resistant clinical isolates such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the murine model of carbapenem-resistant A. baumannii infection, the optimized compound showed potent activity with no observed toxicity. The notable activity of these compounds is attributed to the incorporation of new membrane disruption mechanisms (cytoplasmic membrane depolarization along with outer and inner (cytoplasmic) membrane permeabilization) into vancomycin. Therefore, our results indicate the potential of the present vancomycin analogues to be used against drug-resistant GNPs, thus strengthening the antibiotic arsenal for combating Gram-negative bacterial infections. PMID:27624964

  9. 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

  10. 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

  11. Managing and preventing outbreaks of Gram-negative infections in UK neonatal units.

    PubMed

    Anthony, Mark; Bedford-Russell, Alison; Cooper, Tracey; Fry, Carole; Heath, Paul T; Kennea, Nigel; McCartney, Maureen; Patel, Bharat; Pollard, Tina; Sharland, Mike; Wilson, Peter

    2013-11-01

    De novo guidance on the management of Gram-negative bacteria outbreaks in UK neonatal units was developed in 2012 by a Department of Health, England Antimicrobial Resistance and Healthcare Associated Infection working group. The recommendations included activation of an organisational response and establishing a control team when an outbreak is suspected; screening for the specific organism only during an outbreak; undertaking multidisciplinary reviews of cleaning routines, hand hygiene and Gram-negative bacteria transmission risks; considering deep-cleaning; cohorting colonised and infected babies preferably but not necessarily in isolation cubicles; and considering reducing beds or closing a unit to new admissions as a way of improving spacing and staff:patient ratios until the outbreak is under control. The group advised establishing mechanisms to communicate effectively across the network; informing parents of the outbreak as early as possible, and providing prewritten 'infection outbreak' information sheets. For prevention of outbreaks, the group advised meeting national staffing and cot-spacing requirements; following a Water Action Plan; using infection reduction care bundles and benchmarking; and introducing breast milk early and limiting antibiotic use. PMID:23792354

  12. Ciprofloxacin-Resistant Gram-Negative Bacilli in the Fecal Microflora of Children

    PubMed Central

    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.

    2006-01-01

    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. PMID:17005812

  13. Top-Down Strategies for the Structural Elucidation of Intact Gram-negative Bacterial Endotoxins

    PubMed Central

    O’Brien, John P.; Needham, Brittany D.; Brown, Dusty B.; Trent, M. Stephen

    2014-01-01

    Re-modelling of lipopolysaccharides, which are the primary constituent of the outer cell membrane of Gram-negative bacteria, modulates pathogenesis and resistance to microbials. Reported herein is the characterization of intact Gram-negative bacterial lipooligosaccharides (LOS) via a new strategy utilizing online liquid chromatography (LC) coupled with ultraviolet photodissociation (UVPD) mass spectrometry. Compared to collision-based MS/MS methods, UVPD and UVPD/HCD promoted a greater array of cleavages within both the glycan and lipid moieties, including C-C, C-N, C-O cleavages in the acyl chains as well as glycosidic and cross-ring cleavages, thus providing the most far-reaching structural characterization of LOS. This LC-MS/MS strategy affords a robust analytical method to structurally characterize complex mixtures of bacterial endotoxins that maintains the integrity of the core oligosaccharide and lipid A domains of LOS, providing direct feedback about the cell envelope architectures and LOS modification strategies involved in resistance host innate immune defense. PMID:25386333

  14. Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

    PubMed Central

    Uppu, Divakara S. S. M.; Manjunath, Goutham B.; Yarlagadda, Venkateswarlu; Kaviyil, Jyothi E.; Ravikumar, Raju; Paramanandham, Krishnamoorthy; Shome, Bibek R.; Haldar, Jayanta

    2015-01-01

    Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (blaNDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs. PMID:25789871

  15. 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. PMID:26804662

  16. Multidrug-resistant Gram-negative bacterial infections: are you ready for the challenge?

    PubMed

    Curcio, Daniel

    2014-02-01

    Paralleling the developments in Gram-positive bacteria, infections caused by multidrug-resistant (MDR) Gramnegative bacilli have become a growing challenge. The most important resistance problems are encountered in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp., with increasing trends observed for all major anti- Gram-negative agents (beta-lactams, fluoroquinolones and aminoglycosides). A matter of major concern is the emergence of new beta-lactamases capable of degrading the expanded-spectrum cephalosporins and/or carbapenems, such as the extended-spectrum beta-lactamases (ESBLs) and the carbapenemases (ie. KPC, NDM and other metallo-β; -lactamases). This paper reviews the evidence in the published literature of the pharmacokinetic/pharmacodynamic profile, clinical efficacy of new antimicrobial agents, against MDR- Gram-negative pathogens, such us: i-new carbapenems (doripenem, biapenem, panipenem, tonopenem, FSI-1686); ii-new cephalosporins (ceftaroline, ceftobiprole); iii-tigecycline; and iv- β- lactamases inhibitors (BLI-489, Ro 48-1220, ME 1071, aviactam [NXL104]). PMID:23489027

  17. Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens

    PubMed Central

    Briers, Yves; Walmagh, Maarten; Van Puyenbroeck, Victor; Cornelissen, Anneleen; Cenens, William; Aertsen, Abram; Oliveira, Hugo; Azeredo, Joana; Verween, Gunther; Pirnay, Jean-Paul; Miller, Stefan; Volckaert, Guido

    2014-01-01

    ABSTRACT The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). PMID:24987094

  18. QseC Inhibitors as an Antivirulence Approach for Gram-Negative Pathogens

    PubMed Central

    Curtis, Meredith M.; Russell, Regan; Moreira, Cristiano G.; Adebesin, Adeniyi M.; Wang, Changguang; Williams, Noelle S.; Taussig, Ron; Stewart, Don; Zimmern, Philippe; Lu, Biao; Prasad, Ravi N.; Zhu, Chen; Rasko, David A.; Huntley, Jason F.; Falck, John R.

    2014-01-01

    ABSTRACT Invasive pathogens interface with the host and its resident microbiota through interkingdom signaling. The bacterial receptor QseC, which is a membrane-bound histidine sensor kinase, responds to the host stress hormones epinephrine and norepinephrine and the bacterial signal AI-3, integrating interkingdom signaling at the biochemical level. Importantly, the QseC signaling cascade is exploited by many bacterial pathogens to promote virulence. Here, we translated this basic science information into development of a potent small molecule inhibitor of QseC, LED209. Extensive structure activity relationship (SAR) studies revealed that LED209 is a potent prodrug that is highly selective for QseC. Its warhead allosterically modifies lysines in QseC, impairing its function and preventing the activation of the virulence program of several Gram-negative pathogens both in vitro and during murine infection. LED209 does not interfere with pathogen growth, possibly leading to a milder evolutionary pressure toward drug resistance. LED209 has desirable pharmacokinetics and does not present toxicity in vitro and in rodents. This is a unique antivirulence approach, with a proven broad-spectrum activity against multiple Gram-negative pathogens that cause mammalian infections. PMID:25389178

  19. An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates.

    PubMed

    Mahamoud, Abdallah; Chevalier, Jacqueline; Baitiche, Milad; Adam, Elissavet; Pagès, Jean-Marie

    2011-02-01

    To date, various bacterial drug efflux pump inhibitors (EPIs) have been described. They exhibit variability in their activity spectrum with respect to antibiotic structural class and bacterial species. Among the various 4-alkylaminoquinazoline derivatives synthesized and studied in this work, one molecule, 1167, increased the susceptibility of important human-pathogenic, resistant, Gram-negative bacteria towards different antibiotic classes. This 4-(3-morpholinopropylamino)-quinazoline induced an increase in the activity of chloramphenicol, nalidixic acid, norfloxacin and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps that act in these multidrug-resistant isolates. In addition, 1167 increased the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. The rate of restoration depended on the structure of the antibiotic, suggesting that different sites in the efflux pumps may be involved. A molecule exhibiting a morpholine functional group and a propyl extension of the side chain was more active. PMID:21071494

  20. 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

  1. Evaluation of media for differentiating nonfermenting gram-negative bacteria of medical significance.

    PubMed

    Gilardi, G L

    1969-09-01

    An evaluation was made of media and tests used for differentiating nonfermenting gram-negative bacteria encountered in medical bacteriology in order to determine those diagnostic procedures most useful in identifying these bacteria. The organisms examined included Alcaligenes faecalis, A. odorans var. viridans, Moraxella duplex (Mima polymorpha var. oxidans), Acinetobacter anitratum (Herellea vaginicola), A. lwoffi (Mima polymorpha), Pseudomonas fluorescens, P. putida, P. maltophilia, P. pseudomallei, P. stutzeri, P. alcaligenes, and atypical strains of P. aeruginosa. The media and tests evaluated included Sellers' medium; Hugh and Leifson's OF medium; acid production from 10% lactose infusion agar; gluconate oxidation; starch, aesculin, and Tween 80 hydrolysis; lysine decarboxylase, arginine dihydrolase, deoxyribonuclease, and tyrosinase activity; tolerance to triphenyl tetrazolium chloride, cetrimide, cadmium sulfate, 2.5% and 6.5% sodium chloride, and pH 5.6; utilization of glucose, acetamide, and malonate. PMID:4907000

  2. Sonodynamic excitation of Rose Bengal for eradication of gram-positive and gram-negative bacteria.

    PubMed

    Nakonechny, Faina; Nisnevitch, Michael; Nitzan, Yeshayahu; Nisnevitch, Marina

    2013-01-01

    Photodynamic antimicrobial chemotherapy based on photosensitizers activated by illumination is limited by poor penetration of visible light through skin and tissues. In order to overcome this problem, Rose Bengal was excited in the dark by 28 kHz ultrasound and was applied for inactivation of bacteria. It is demonstrated, for the first time, that the sonodynamic technique is effective for eradication of gram-positive Staphylococcus aureus and gram-negative Escherichia coli. The net sonodynamic effect was calculated as a 3-4 log10 reduction in bacteria concentration, depending on the cell and the Rose Bengal concentration and the treatment time. Sonodynamic treatment may become a novel and effective form of antimicrobial therapy and can be used for low-temperature sterilization of medical instruments and surgical accessories. PMID:23509759

  3. Gram-positive and gram-negative bacterial toxins in sepsis: a brief review.

    PubMed

    Ramachandran, Girish

    2014-01-01

    Bacterial sepsis is a major cause of fatality worldwide. Sepsis is a multi-step process that involves an uncontrolled inflammatory response by the host cells that may result in multi organ failure and death. Both gram-negative and gram-positive bacteria play a major role in causing sepsis. These bacteria produce a range of virulence factors that enable them to escape the immune defenses and disseminate to remote organs, and toxins that interact with host cells via specific receptors on the cell surface and trigger a dysregulated immune response. Over the past decade, our understanding of toxins has markedly improved, allowing for new therapeutic strategies to be developed. This review summarizes some of these toxins and their role in sepsis. PMID:24193365

  4. 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.

  5. Resistance to antibiotics in gram-negative rods from clinical material of hospital and community origin.

    PubMed

    Kolár, M; Hájek, V; Sázelová, J; Krátká, J; Koukalová, D

    1995-01-01

    The authors examined the resistance to 13 antibiotics and chemotherapeutic agents by the diluting micromethod and the routine disk method in the group of 5375 gram-negative strains of 7 genera which prevailed in clinical material of an hospital (FN Olomouc) and in material of community (OHS Olomouc) provenance during the years 1992 and 1993. In the majority of cases, the resistance was more frequent in the strains isolated from the hospital material. The most remarkable differences were found in A cinetobacter sp. (9-76%), Enterobacter sp. (10-60%) and Citrobacter sp. (18-58%). In other examined species, the differences varied in the ranges 3-40% in E.coli, 2-33% in Klebsiella sp., 0-28% in P. vulgaris, 2-28% in P.mirabilis, 0-16% in M.morganii, and 0-13% in P. aeruginosa. PMID:8686556

  6. 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. PMID:25737092

  7. The HlyB/HlyD-dependent secretion of toxins by gram-negative bacteria.

    PubMed

    Koronakis, V; Stanley, P; Koronakis, E; Hughes, C

    1992-09-01

    Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer membranes of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate. It is directed by an uncleaved C-terminal targetting signal and the HlyD and HlyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the HlyB/HlyD-dependent translocation allow a discussion of the hemolysin export process in the wider context of protein translocation. PMID:1419114

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

    PubMed

    McKinney, David C; Basarab, Gregory S; Cocozaki, Alexis I; Foulk, Melinda A; Miller, Matthew D; Ruvinsky, Anatoly M; Scott, Clay W; Thakur, Kumar; Zhao, Liang; Buurman, Ed T; Narayan, Sridhar

    2015-08-13

    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

  9. Carbapenem-induced endotoxin release in gram-negative bacterial sepsis rat models.

    PubMed

    Horii, T; Kobayashi, M; Nadai, M; Ichiyama, S; Ohta, M

    1998-08-01

    The carbapenem-induced endotoxin release was evaluated using experimental models of gram-negative bacterial sepsis in Wistar rats. Infections with Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis resulted in an increase of the plasma endotoxin concentration after treatment with ceftazidime and carbapenems including imipenem, panipenem, meropenem and biapenem. Except for P. aeruginosa, the plasma endotoxin concentrations after carbapenem treatment were significantly lower than those after ceftazidime treatment. It is noteworthy that treatment of P. aeruginosa sepsis with meropenem or biapenem induced significantly more endotoxin release than other carbapenems and the endotoxin concentrations induced by these carbapenems reached those of ceftazidime treatment. The plasma endotoxin concentrations appeared to correlate with the reduction of platelet counts and the elevation of both glutamic oxaloacetic transaminase and glutamic pyruvic transaminase values. PMID:9753002

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

    PubMed

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

    1989-07-01

    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

  11. The β-barrel membrane protein insertase machinery from Gram-negative bacteria

    PubMed Central

    Noinaj, Nicholas; Rollauer, Sarah E.; Buchanan, Susan K.

    2015-01-01

    The outer membranes (OM) of Gram-negative bacteria contain a host of β-barrel outer membrane proteins (OMPs) which serve many functions for cell survival and virulence. The biogenesis of these OMPs is mediated by the β-barrel assembly machinery (BAM) complex which is composed of five components including the essential core component called BamA that mediates the insertase function within the OM. The crystal structure of BamA has recently been reported from three different species, including a full-length structure from Neisseria gonorrhoeae. Mutagenesis and functional studies identified several conformational changes within BamA that are required for function, providing a significant advancement towards unraveling exactly how BamA and the BAM complex are able to fold and insert new OMPs in the OM. PMID:25796031

  12. [Screening methods for detection of metallo-beta-lactamase producing gram negative rods].

    PubMed

    Mereuţă, Ana-Irina; Poiati, Antonia; Tuchiluş, Cristina; Dorneanu, Olivia; Nistor, Silvia; Copăcianu, Brînduşa

    2005-01-01

    Modified Hodge test and a method using a disk with imipenem plus 1000 mg of EDTA were used to determine the presence of metallo-beta-lactamase producing gram-negative rods among 166 clinical isolates from hospitals in Iaşi and Galaţi. Of 9 imipenem resistant strains found, only one Pseudomonas aeruginosa gave positive results with both tests and other two P. aeruginosa clinical isolates gave negative results with both tests. The rest of the strains (2 P. aeruginosa, 2 Acinetobacter baumanii, 1 Sphingomonas paucimobilis) did not give conclusive results. These screening methods are useful, simple and accessible to clinical laboratories. PCR is needed to confirm the presence of metallo-beta-lactamase gene in bacteria and to determine the type of the enzymes. PMID:16607806

  13. Characterization of some groups of gram-negative nonfermentative bacteria by the carbon source alkalinization technique.

    PubMed Central

    Martin, R; Riley, P S; Hollis, D G; Weaver, R E; Krichevsky, M I

    1981-01-01

    A total of 541 gram-negative nonfermentative bacterial strains comprising 26 species and unclassified groups were characterized by routine diagnostic and carbon substrate alkalinization techniques. These microorganisms were tested for the ability to cause alkalinization of a basal medium on a total of 217 substrates. We found that 58 carbon substrates had some discriminatory potential. We also performed 30 routine diagnostic tests. The results of these studies were evaluated by numerical taxonomy techniques. A cluster analysis of the results by the Jaccard coefficient method identified 30 clusters at the 45% level. We identified 39 tests that separated most of the groups. Groups of similar organisms or organisms that were difficult to identify were analyzed, and tests that were differential were identified. Because of variability within the clusters, further studies utilizing deoxyribonucleic acid-deoxyribonucleic acid homologies should be undertaken. PMID:7021587

  14. High Prevalence of Antimicrobial-resistant Gram-negative Colonization in Hospitalized Cambodian Infants

    PubMed Central

    Pol, Sreymom; Soeng, Sona; Sar, Poda; Neou, Leakhena; Chea, Phal; Day, Nicholas PJ; Cooper, Ben S.; Turner, Claudia

    2016-01-01

    Background: Antimicrobial-resistant Gram-negative infections are a significant cause of mortality in young infants. We aimed to determine characteristics of, and risk factors for, colonization and invasive infection caused by 3rd generation cephalosporin (3GC) or carbapenem-resistant organisms in outborn infants admitted to a neonatal unit (NU) in Cambodia. Methods: During the first year of operation, patients admitted to the Angkor Hospital for Children NU, Siem Reap, Cambodia, underwent rectal swabbing on admission and twice weekly until discharge. Swabs were taken also from 7 environmental sites. Swabs were cultured to identify 3GC or carbapenem-resistant Acinetobacter sp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Results: The study included 333 infants with a median age at NU admission of 10 days (range, 0–43). Colonization by ≥1 3GC-resistant organism was detected in 85.9% (286/333). Admission swabs were collected in 289 infants: 61.9% were colonized by a 3GC-resistant organism at the time of admission, and a further 23.2% were colonized during hospitalization, at a median of 4 days [95% confidence interval: 3–5]. Probiotic treatment (hazard ratio: 0.58; 95% confidence interval: 0.35–0.98) was associated with delayed colonization. Colonization by a carbapenem-resistant organism occurred in 25 (7.5%) infants. Six infants had NU-associated K. pneumoniae bacteremia; phenotypically identical colonizing strains were found in 3 infants. Environmental colonization occurred early. Conclusions: Colonization by antimicrobial-resistant Gram-negative organisms occurred early in hospitalized Cambodian infants and was associated with subsequent invasive infection. Trials of potential interventions such as probiotics are needed. PMID:27124686

  15. Ampc Beta lactamases among gram negative clinical isolates from a tertiary hospital, South India.

    PubMed

    Mohamudha Parveen, R; Harish, B N; Parija, S C

    2010-07-01

    AmpC β-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC β-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC β -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57%) strains were resistant to 3GC, among which 63(47%) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7%) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9% (51/63) of screen positive isolates were detected by Amp C disc test and 93.6% (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9%) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly. PMID:24031534

  16. 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

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

    DOE PAGESBeta

    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 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

  18. 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.

  19. 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

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

    PubMed Central

    2014-01-01

    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. PMID:25489959

  1. Raman spectroscopy of xylitol uptake and metabolism in Gram-positive and Gram-negative bacteria.

    PubMed

    Palchaudhuri, Sunil; Rehse, Steven J; Hamasha, Khozima; Syed, Talha; Kurtovic, Eldar; Kurtovic, Emir; Stenger, James

    2011-01-01

    Visible-wavelength Raman spectroscopy was used to investigate the uptake and metabolism of the five-carbon sugar alcohol xylitol by Gram-positive viridans group streptococcus and the two extensively used strains of Gram-negative Escherichia coli, E. coli C and E. coli K-12. E. coli C, but not E. coli K-12, contains a complete xylitol operon, and the viridans group streptococcus contains an incomplete xylitol operon used to metabolize the xylitol. Raman spectra from xylitol-exposed viridans group streptococcus exhibited significant changes that persisted even in progeny grown from the xylitol-exposed mother cells in a xylitol-free medium for 24 h. This behavior was not observed in the E. coli K-12. In both viridans group streptococcus and the E. coli C derivative HF4714, the metabolic intermediates are stably formed to create an anomaly in bacterial normal survival. The uptake of xylitol by Gram-positive and Gram-negative pathogens occurs even in the presence of other high-calorie sugars, and its stable integration within the bacterial cell wall may discontinue bacterial multiplication. This could be a contributing factor for the known efficacy of xylitol when taken as a prophylactic measure to prevent or reduce occurrences of persistent infection. Specifically, these bacteria are causative agents for several important diseases of children such as pneumonia, otitis media, meningitis, and dental caries. If properly explored, such an inexpensive and harmless sugar-alcohol, alone or used in conjunction with fluoride, would pave the way to an alternative preventive therapy for these childhood diseases when the causative pathogens have become resistant to modern medicines such as antibiotics and vaccine immunotherapy. PMID:21037297

  2. Emergence of carbapenemase-producing Gram-negative bacteria in Saint Petersburg, Russia.

    PubMed

    Ageevets, Vladimir A; Partina, Irina V; Lisitsyna, Eugenia S; Ilina, Elena N; Lobzin, Yuri V; Shlyapnikov, Sergei A; Sidorenko, Sergei V

    2014-08-01

    The emergence and spread of carbapenemase-producing Gram-negative bacteria represents a serious public health concern. Here we show that of 477 Gram-negative isolates collected from 18 hospitals between November 2011 and February 2013 in Saint Petersburg (Russia), minimum inhibitory concentrations (MICs) were greater than the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value of at least one carbapenem antibiotic in 101 isolates (21.2%). The bla(NDM-1) gene was detected by PCR in 17 Klebsiella pneumoniae and 1 Acinetobacter nosocomialis isolate. Multilocus sequence typing (MLST) revealed that all NDM-1-producing K. pneumoniae isolates belonged to sequence type 340 (ST340) and harboured genes encoding additional β-lactamases; presence of the bla(CTX-M-1-like) gene correlated with aztreonam resistance, whilst its absence correlated with susceptibility. The epidemiological situation in Saint Petersburg can be assessed as regional spread of NDM-1-producers. The bla(KPC-2) gene was detected in two K. pneumoniae isolates (ST258 and ST273) and one Enterobacter cloacae isolate. Two E. cloacae isolates harboured the bla(VIM-4) gene, and one K. pneumoniae (ST395) isolate harboured the bla(OXA-48) gene. In NDM-1-producers, MICs of biapenem were the lowest compared with those of other carbapenems. Most isolates were susceptible to tigecycline and polymyxin, except for one K. pneumoniae isolate that was found to be polymyxin-resistant and one E. cloacae isolate that was tigecycline-resistant. Only one patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae had a history of travel abroad (Southeast Asia). Thus, there is an actual threat of the emergence of an alarming endemic situation with NDM-1-producers in Saint Petersburg. PMID:25059447

  3. New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

    PubMed Central

    Martínez-García, Esteban; Aparicio, Tomás; de Lorenzo, Víctor; Nikel, Pablo I.

    2014-01-01

    Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes

  4. Raman Spectroscopy of Xylitol Uptake and Metabolism in Gram-Positive and Gram-Negative Bacteria▿

    PubMed Central

    Palchaudhuri, Sunil; Rehse, Steven J.; Hamasha, Khozima; Syed, Talha; Kurtovic, Eldar; Kurtovic, Emir; Stenger, James

    2011-01-01

    Visible-wavelength Raman spectroscopy was used to investigate the uptake and metabolism of the five-carbon sugar alcohol xylitol by Gram-positive viridans group streptococcus and the two extensively used strains of Gram-negative Escherichia coli, E. coli C and E. coli K-12. E. coli C, but not E. coli K-12, contains a complete xylitol operon, and the viridans group streptococcus contains an incomplete xylitol operon used to metabolize the xylitol. Raman spectra from xylitol-exposed viridans group streptococcus exhibited significant changes that persisted even in progeny grown from the xylitol-exposed mother cells in a xylitol-free medium for 24 h. This behavior was not observed in the E. coli K-12. In both viridans group streptococcus and the E. coli C derivative HF4714, the metabolic intermediates are stably formed to create an anomaly in bacterial normal survival. The uptake of xylitol by Gram-positive and Gram-negative pathogens occurs even in the presence of other high-calorie sugars, and its stable integration within the bacterial cell wall may discontinue bacterial multiplication. This could be a contributing factor for the known efficacy of xylitol when taken as a prophylactic measure to prevent or reduce occurrences of persistent infection. Specifically, these bacteria are causative agents for several important diseases of children such as pneumonia, otitis media, meningitis, and dental caries. If properly explored, such an inexpensive and harmless sugar-alcohol, alone or used in conjunction with fluoride, would pave the way to an alternative preventive therapy for these childhood diseases when the causative pathogens have become resistant to modern medicines such as antibiotics and vaccine immunotherapy. PMID:21037297

  5. 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

  6. 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

  7. In vivo efficacy of zidovudine (3'-azido-3'-deoxythymidine) in experimental gram-negative-bacterial infections.

    PubMed Central

    Keith, B R; White, G; Wilson, H R

    1989-01-01

    The therapeutic efficacy of orally administered zidovuldine (3'-azido-3'-deoxythymidine) was determined in animals infected with Escherichia coli and Salmonella dublin. The 50% effective dose (ED50) of zidovudine (9.6 to 11.8 mg/kg of body weight) compared favorably with that of trimethoprim (19.4 to 22.2 mg/kg) in mice with systemic E. coli infection. At 50 mg/kg, both zidovudine and ampicillin reduced the number of bacteria in the kidneys of mice and prevented lethal infection in mice with ascending pyelonephritis caused by E. coli. Zidovudine prevented a lethal S. dublin infection in calves over a wide dose range (8.0 to 31.0 mg/kg per day). Zidovudine levels in plasma of uninfected mice were 28.2 +/- 4.5 and 7.9 +/- 2.2 micrograms/ml at 30 and 60 min, respectively, exceeding the MICs for the bacteria used in the infections. Few zidovudine-resistant strains were observed. The in vivo data raise the possibility that zidovudine may have an antibacterial effect in patients receiving this therapy. PMID:2658792

  8. 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

  9. The Drosophila Prosecretory Transcription Factor dimmed Is Dynamically Regulated in Adult Enteroendocrine Cells and Protects Against Gram-Negative Infection.

    PubMed

    Beebe, Katherine; Park, Dongkook; Taghert, Paul H; Micchelli, Craig A

    2015-07-01

    The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner. We demonstrate that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the prosecretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets Phm, dcat-4, and the peptide hormone, Allatostatin A. Finally, we identify dimm as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. We propose that dimm provides "gain" in enteroendocrine output during the adaptive response to episodic pathogen exposure. PMID:25999585

  10. The Drosophila Prosecretory Transcription Factor dimmed Is Dynamically Regulated in Adult Enteroendocrine Cells and Protects Against Gram-Negative Infection

    PubMed Central

    Beebe, Katherine; Park, Dongkook; Taghert, Paul H.; Micchelli, Craig A.

    2015-01-01

    The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner. We demonstrate that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the prosecretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets Phm, dcat-4, and the peptide hormone, Allatostatin A. Finally, we identify dimm as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. We propose that dimm provides “gain” in enteroendocrine output during the adaptive response to episodic pathogen exposure. PMID:25999585

  11. Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

    PubMed Central

    Braun, Sascha D.; Monecke, Stefan; Thürmer, Alexander; Ruppelt, Antje; Makarewicz, Oliwia; Pletz, Mathias; Reißig, Annett; Slickers, Peter; Ehricht, Ralf

    2014-01-01

    Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and

  12. 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.

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

    PubMed

    Gniadek, Thomas J; Carroll, Karen C; Simner, Patricia J

    2016-07-01

    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

  14. Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria.

    PubMed

    Merchat, M; Bertolini, G; Giacomini, P; Villanueva, A; Jori, G

    1996-02-01

    Previous studies on the photosensitization of bacterial cells with different neutral or negatively charged porphyrins and phthalocyanines have demonstrated that, although Gram-positive bacteria are efficiently photoinactivated, Gram-negative bacteria become photosensitive only after modification of the permeability of their outer membrane. The results described in this paper show that two meso-substituted cationic porphyrins, namely tetra(4N-methyl-pyridyl)porphine tetraiodide and tetra(4N,N,N-trimethyl-anilinium)porphine, efficiently photosensitize the inactivation of Gram-negative bacteria, such as Vibrio anguillarum and Escherichia coli. A negatively charged meso-substituted porphyrin, tetra(4-sulphonatophenyl)porphine, has no appreciable photosensitizing activity towards Gram-negative bacteria, although all three porphyrins exhibit a similar subcellular distribution pattern, being mainly localized in the protoplasts or spheroplasts. Moreover, the three porphyrins show similar efficiency in the photoinactivation of the Gram-positive bacterium Entorecoccus seriolicida. PMID:8622178

  15. Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction.

    PubMed

    Davis, Michael R; Goldberg, Joanna B

    2012-01-01

    Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell(1, 2). LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli(3-5), and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence(6-8). For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods(9). LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background

  16. Production of N-acylhomoserine lactone signal molecules by gram-negative soil-borne and plant-associated bacteria.

    PubMed

    Veselova, M; Kholmeckaya, M; Klein, S; Voronina, E; Lipasova, V; Metlitskaya, A; Mayatskaya, A; Lobanok, E; Khmel, I; Chernin, L

    2003-01-01

    Quorum-sensing control mediated by N-acylhomoserine lactone (AHL) signal molecules has been established as a key feature in the regulation of various metabolic traits in many bacteria. Approximately 300 strains representing 6 genera and 18 species of soil-borne and plant-associated Gram-negative bacteria isolated in various regions of the former USSR using two reporter systems were screened for AHL production. The production was observed in 17.5% of the screened bacterial strains. Positive response was detected in all of the 14 tested strains of Erwinia herbicola, in 41 of the 239 strains of Pseudomonas species; in all 5 strains of Xanthomonas ampelina, X. campestris pv. malvacearum, pv. translucens, pv. vesicatoria and in one strain of Pantoea stewartii. AHL assay of 41 strains of X. maltophilia (syn. Stenotrophomonas maltophilia) isolated from soils with Chromobacterium violaceum reporter has revealed no strains synthesizing these signal molecules; 26 strains analyzed with Agrobacterium tumefaciens reporter showed the same result. PMID:15058194

  17. Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

    PubMed Central

    Schink, B; Stieb, M

    1983-01-01

    The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed. Images PMID:6881964

  18. Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution.

    PubMed Central

    Rådström, P; Swedberg, G; Sköld, O

    1991-01-01

    In contrast to what has been observed for many other antibiotic resistance mechanisms, there are only two known genes encoding plasmid-borne sulfonamide resistance. Both genes, sulI and sulII, encode a drug-resistant dihydropteroate synthase enzyme. In members of the family Enterobacteriaceae isolated from several worldwide sources, plasmid-mediated resistance to sulfonamides could be identified by colony hybridization as being encoded by sulI, sulII, or both. The sulI gene was in all cases found to be located in the newly defined, mobile genetic element, recently named an integron, which has been shown to contain a site-specific recombination system for the integration of various antibiotic resistance genes. The sulII gene was almost exclusively found as part of a variable resistance region on small, nonconjugative plasmids. Colony hybridization to an intragenic probe, restriction enzyme digestion, and nucleotide sequence analysis of small plasmids indicated that the sulII gene and contiguous sequences represent an independently occurring region disseminated in the bacterial population. The sulII resistance region was bordered by direct repeats, which in some plasmids were totally or partially deleted. The prevalence of sulI and sulII could thus be accounted for by their stable integration in transposons and in plasmids that are widely disseminated among gram-negative bacteria. Images PMID:1952855

  19. Rationale for use of antimicrobial combinations in treatment of gram-negative infections. A review of recent reviews.

    PubMed

    Rahal, J J

    1983-08-29

    All investigators apparently agree that the most common and compelling reason for using more than one antibiotic to treat a single organism is to achieve a bactericidal effect. Most studies, both retrospective and prospective, have demonstrated that two effective antibiotics yield better results than one in neutropenic patients and/or those with rapidly fatal underlying disease, despite the absence of consistent in vitro synergy. Bacteremias caused by Pseudomona aeruginosa or Klebsiella pneumoniae may be benefited most by synergistic combinations. This may not be true for patients with non-neoplastic disease and normal granulocyte counts, or for patients infected with other species of gram-negative bacilli. Synergistic bactericidal activity is necessary for the successful therapy of endocarditis due to P. aeruginosa, but it may not assure success. The systemic immunodeficiency of neutropenic patients may parallel a localized immunodeficiency in endocarditis, since leukocytes are not effectively mobilized to the site of infection in endocarditis. Antagonistic antibiotic combinations are likely to be particularly harmful in neutropenic patients. PMID:6351606

  20. Inhibition of various gram-positive and gram-negative bacteria growth on selenium nanoparticle coated paper towels.

    PubMed

    Wang, Qi; Larese-Casanova, Philip; Webster, Thomas J

    2015-01-01

    There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns. PMID:25926733

  1. Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

    PubMed

    Schink, B; Stieb, M

    1983-06-01

    The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed. PMID:6881964

  2. Inhibition of various gram-positive and gram- negative bacteria growth on selenium nanoparticle coated paper towels

    PubMed Central

    Wang, Qi; Larese-Casanova, Philip; Webster, Thomas J

    2015-01-01

    There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns. PMID:25926733

  3. [Non-fermentative gram-negative bacilli: their distribution to clinical materials and antibiotic susceptibility (author's transl)].

    PubMed

    Akalin, H E; Baykal, M

    1980-01-01

    A total of 7898 non-fermentative Gram-negative bacilli were isolated from various clinical materials. Pseudomonas (7526) was the most common among them. Alcaligenes faecalis (273), Acinetobacter sp. (93) and Flavobacterium (6) were the other non-fermentative Gram-negative bacilli. Most of them were found in urine and pus cultures, however they were also isolated from sputum, blood, and cerebrospinal fluid. Gentamicin was the most effective antibiotic in vitro. Fifty four per cent of Pseudomonas, 100% of Acinetobacter, and 70% of Alcaligenes faecalis were inhibited by tobramycin. PMID:7453583

  4. Prevention of Biofilm Colonization by Gram-Negative Bacteria on Minocycline-Rifampin-Impregnated Catheters Sequentially Coated with Chlorhexidine

    PubMed Central

    Jamal, Mohamed A.; Rosenblatt, Joel S.; Hachem, Ray Y.; Ying, Jiang; Pravinkumar, Egbert; Nates, Joseph L.; Chaftari, Anne-Marie P.

    2014-01-01

    Resistant Gram-negative bacteria are increasing central-line-associated bloodstream infection threats. To better combat this, chlorhexidine (CHX) was added to minocycline-rifampin (M/R) catheters. The in vitro antimicrobial activity of CHX-M/R catheters against multidrug resistant, Gram-negative Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia was tested. M/R and CHX-silver sulfadiazine (CHX/SS) catheters were used as comparators. The novel CHX-M/R catheters were significantly more effective (P < 0.0001) than CHX/SS or M/R catheters in preventing biofilm colonization and showed better antimicrobial durability. PMID:24165191

  5. In Vitro Activities of BAL9141, a Novel Broad-Spectrum Pyrrolidinone Cephalosporin, against Gram-Negative Nonfermenters

    PubMed Central

    Zbinden, R.; Pünter, V.; von Graevenitz, A.

    2002-01-01

    The activities of BAL9141 (formerly Ro 63-9141), a novel pyrrolidinone-3-ylidenemethyl cephalosporin, against 244 strains of gram-negative nonfermenters were evaluated. The overall MIC at which 50% of isolates are inhibited (MIC50) and the overall MIC90 were 2 and 64 μg/ml, respectively, which are similar to those of imipenem, lower than those of the other cephalosporins tested, amoxicillin, and the ticarcillin-clavulanic acid combination, and much higher than those of ciprofloxacin. BAL9141 shows species-dependent activity in vitro against a variety of gram-negative nonfermentative pathogens. PMID:11850276

  6. 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

  7. Antimicrobial susceptibility in gram-negative bacteremia: are nosocomial isolates really more resistant?

    PubMed Central

    McGowan, J E; Hall, E C; Parrott, P L

    1989-01-01

    Bloodstream isolates of gram-negative aerobic bacilli from nosocomial infections are more likely to be resistant to antimicrobial agents than isolates from community-acquired cases are. It is not clear, however, how much this is due to the markedly different distribution of organisms in the two groups. We compared the susceptibilities of organisms of a given species which caused community-acquired bacteremia with the susceptibilities of isolates from nosocomial cases. Nine antimicrobial agents were tested against 1,077 isolates which were obtained during a 4-year nonepidemic period. Marked differences in crude rates of resistance were noted for all isolates from nosocomial cases versus all isolates from cases acquired in the community. When results were adjusted for the different organism distributions in the two groups, statistically significant differences were found for only six drug-organism pairs; in each of these, resistance rates were higher in nosocomial isolates. However, when results were further adjusted for the effect of multiple analyses, no significant differences were seen. The major factor leading to the greater prevalence of antimicrobial resistance in our hospital organisms was the markedly different distribution of organisms in the nosocomial and community-acquired groups. For individual organisms, greater resistance in nosocomial strains was confined to certain drugs. Factors that influence differences in organism distribution may not be solely the result of antimicrobial use. PMID:2610495

  8. Molecular structure of endotoxins from Gram-negative marine bacteria: an update.

    PubMed

    Leone, Serena; Silipo, Alba; L Nazarenko, Evgeny; Lanzetta, Rosa; Parrilli, Michelangelo; Molinaro, Antonio

    2007-01-01

    Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs), or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the gamma-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups), to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments. PMID:18463721

  9. Identification of gram-negative and gram-positive bacteria by fluorescence studies

    NASA Astrophysics Data System (ADS)

    Demchak, Jonathan; Calabrese, Joseph; Tzolov, Marian

    2011-03-01

    Several type strains of bacteria including Vibrio fischeri, Azotobacter vinelandii, Enterobacter cloacae, and Corynebacterium xerosis, were cultured in the laboratory following standard diagnostic protocol based on their individual metabolic strategies. The bacterial cultures were not further treated and they were studied in their pristine state (pure culture - axenic). The fluorescent studies were applied using a continuous wave and a pulsed excitation light sources. Emission and excitation spectra were recorded for the continuous wave excitation and they all show similar spectral features with the exception of the gram positive bacteria showing vibronic structures. The vibrational modes involved in these vibronic bands have energy typical for carbon-carbon vibrations. The fluorescence is quenched in addition of water, even a very thin layer, which confirms that the observed spectral features originate from the outer parts of the bacteria. These results allow to conclude that the fluorescence spectroscopy can be used as a method for studying the membranes of the bacteria and eventually to discriminate between gram positive and gram negative bacteria. The pulsed experiments show that the fluorescence lifetime is in the sub-microsecond range. The results indicate that the observed spectra are superposition of the emission with different lifetimes.

  10. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    PubMed

    Reidl, Joachim

    2016-01-01

    Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S Typhimurium. PMID:27531914

  11. Protein-translocating outer membrane porins of Gram-negative bacteria.

    PubMed

    Yen, Ming Ren; Peabody, Christopher R; Partovi, Salar M; Zhai, Yufeng; Tseng, Yi Hsiung; Saier, Milton H

    2002-05-01

    Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins. PMID:11988218

  12. Emergence of unusual nonfermenting Gram-negative nosocomial pathogens in a Saudi hospital.

    PubMed

    Asaad, Ahmed Morad; Al-Ayed, Mohamed Said Zayed; Qureshi, Mohamed Ansar

    2013-01-01

    This study aimed to determine the frequency of isolation and prevalence of drug resistance in nonfermenting Gram-negative bacilli (NFGNB) other than Pseudomonas aeruginosa and predisposing factors for the acquisition of nosocomial infections caused by these emerging pathogens in a Saudi tertiary care hospital. A total of 125 nonduplicating NFGNB nosocomial strains were isolated, of these, 68 (54.4%) were Acinetobacter baumannii, 26 (20.8%) Stenotrophomonas maltophilia, 14 (11.2%) Alcaligenes faecalis, 12 (9.6%) Chryseobacterium indologenes, and 5 (4%) Ralstonia pickettii. MICs of 11 antibiotics were determined using the reference broth microdilution method. With the exception of colistin that inhibited 100% of A. baumannii isolates, trimethoprim/sulfamethoxazole that inhibited 100% of S. maltophilia isolates, and carbapenems that inhibited 100% of A. faecalis isolates, none of the tested antimicrobial agents inhibited 100% of the other NFGNB spp. Our results emphasize that clinicians and microbiologists should consider A. faecalis, C. indologenes, and R. pickettii as emerging nosocomial pathogens. In addition, local resistance data are essential for helping physicians in deciding an appropriate antibiotic for empirical therapy of infections with these emerging and unusual NFGNB. PMID:24270139

  13. Pectinatus brassicae sp. nov., a Gram-negative, anaerobic bacterium isolated from salty wastewater.

    PubMed

    Zhang, Wen-wu; Fang, Ming-xu; Tan, Hai-qin; Zhang, Xin-qi; Wu, Min; Zhu, Xu-fen

    2012-09-01

    A novel Gram-negative, non-spore-forming, strictly anaerobic, heterotrophic bacterium, strain TY(T), was isolated from salty pickle wastewater. Cells were rod-shaped with comb-like flagella, slightly curved and very variable in length. Optimal growth occurred at 28 °C and pH 6.5. Cells were resistant to up to 50 g NaCl l(-1). Strain TY(T) produced acid from glycerol, sucrose, glucose, fructose and mannitol. The main fermentation products from glucose were acetic and propionic acids. Tests for acid phosphatase and naphthol-AS-BI-phosphohydrolase activities were positive. The major fatty acids were C(14 : 0) DMA (18.7 %), C(15 : 0) (15.4 %), anteiso-C(18 : 1) (15.2 %), C(11 : 0) (13.3 %) and summed feature 5 (C(17 : 1)ω7c and/or C(17 : 2)) (11.0 %). The DNA G+C content was 35.9 mol%. 16S rRNA gene sequence-based phylogenetic analysis indicated that strain TY(T) represented a novel species of the genus Pectinatus (sequence similarity to other members of the genus ranged from 93.2 to 94.8 %). Based on its phenotypic, genotypic and phylogenetic characteristics, strain TY(T) is proposed to represent a novel species, named Pectinatus brassicae sp. nov. (type strain TY(T) = JCM 17499(T) = DSM 24661(T)). PMID:22058316

  14. 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

    2014-03-01

    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). PMID:25409355

  15. 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

    2014-03-01

    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). PMID:25409356

  16. 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

    2013-09-01

    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). PMID:24890957

  17. Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria.

    PubMed

    Tang, Hong; Zhang, Peng; Kieft, Thomas L; Ryan, Shannon J; Baker, Shenda M; Wiesmann, William P; Rogelj, Snezna

    2010-07-01

    The antimicrobial activity of chitosan and chitosan derivatives has been well established. However, although several mechanisms have been proposed, the exact mode of action is still unclear. Here we report on the investigation of antibacterial activity and the antibacterial mode of action of a novel water-soluble chitosan derivative, arginine-functionalized chitosan, on the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli. Two different arginine-functionalized chitosans (6% arginine-substituted and 30% arginine-substituted) each strongly inhibited P. fluorescens and E. coli growth. Time-dependent killing efficacy experiments showed that 5000 mg l(-1) of 6%- and 30%-substituted chitosan-arginine killed 2.7 logs and 4.5 logs of P. fluorescens, and 4.8 logs and 4.6 logs of E. coli in 4h, respectively. At low concentrations, the 6%-substituted chitosan-arginine was more effective in inhibiting cell growth even though the 30%-substituted chitosan-arginine appeared to be more effective in permeabilizing the cell membranes of both P. fluorescens and E. coli. Studies using fluorescent probes, 1-N-phenyl-naphthylamine (NPN), nile red (NR) and propidium iodide (PI), and field emission scanning electron microscopy (FESEM) suggest that chitosan-arginine's antibacterial activity is, at least in part, due to its interaction with the cell membrane, in which it increases membrane permeability. PMID:20060936

  18. Molecular Structure of Endotoxins from Gram-negative Marine Bacteria: An Update

    PubMed Central

    Leone, Serena; Silipo, Alba; L.Nazarenko, Evgeny; Lanzetta, Rosa; Parrilli, Michelangelo; Molinaro, Antonio

    2007-01-01

    Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs), or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the γ-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups), to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments. PMID:18463721

  19. Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods.

    PubMed

    Bombicino, Karina A; Almuzara, Marisa N; Famiglietti, Angela M R; Vay, Carlos

    2007-01-01

    To evaluate the activity of pyrrolidonyl arylamidase (PYR) for the differentiation and identification of nonfermenting gram negative rods (NFGNR), 293 isolates were tested. A 24 h culture of each test organism was prepared. From this a 108-109 cfu/mL suspension was added to 0.25 mL of sterile physiologic solution. A PYR disk was then added and the test was incubated for 30 minutes at 35-37 degrees C, at environmental atmosphere. Reading was done by adding 1 drop of cinnamaldehyde reagent. Strains of Acinetobacter baumannii, Acinetobacter haemolyticus, Alcaligenes faecalis, Bergeyella zoohelcum, Bordetella bronchiseptica, Bordetella hinzii, Brevundimonas diminuta, Brevundimonas vesicularis, Brucella ovis, Brucella spp., Brucella suis, Burkholderia cepacia complex, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Oligella ureolytica, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas Vb3, Psychrobacter phenylpyruvicus, and Stenotrophomonas maltophilia were PYR negative. On the other hand Achromobacter piechaudii, Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia gladioli, Chryseobacterium gleum-indologenes, Comamonas testosroni, Cupriavidus pauculus, Delftia acidovorans, Elizabethkingia meningoseptica, Myroides spp., Ochrobactrum anthropi, Pseudomonas oryzihabitans, Ralstonia pickettii, Rhizobium radiobacter, Shewanella spp., Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Weeksella virosa were PYR positive. Finally, Acinetobacter lwoffii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Roseomonas spp., and Sphingomonas paucimobilis-parapaucimobilis were PYR variable. PYR testing should be considered as a useful tool to facilitate the identification of NFGNR. PMID:16822636

  20. Biofilms Formed by Gram-Negative Bacteria Undergo Increased Lipid A Palmitoylation, Enhancing In Vivo Survival

    PubMed Central

    Chalabaev, Sabina; Chauhan, Ashwini; Novikov, Alexey; Iyer, Pavithra; Szczesny, Magdalena; Beloin, Christophe; Caroff, Martine

    2014-01-01

    ABSTRACT Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria. PMID:25139899

  1. Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria.

    PubMed

    Rahnamaeian, Mohammad; Cytryńska, Małgorzata; Zdybicka-Barabas, Agnieszka; Dobslaff, Kristin; Wiesner, Jochen; Twyman, Richard M; Zuchner, Thole; Sadd, Ben M; Regoes, Roland R; Schmid-Hempel, Paul; Vilcinskas, Andreas

    2015-05-01

    Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics. PMID:25833860

  2. Endotoxin neutralization with rabbit antisera to Escherichia coli J5 and other gram-negative bacteria.

    PubMed Central

    Warren, H S; Novitsky, T J; Bucklin, A; Kania, S A; Siber, G R

    1987-01-01

    To study the mechanisms of protection against endotoxin challenge offered by antisera to smooth and rough gram-negative organisms, we have developed an assay to quantitate endotoxin neutralization based on inhibition of the Limulus amoebocyte lysate test. Dilutions of different bacterial lipopolysaccharides (LPSs) were incubated with hyperimmune rabbit sera against Escherichia coli O113, E. coli O18, and rough mutants E. coli J5 and Salmonella minnesota Re595 and were then combined with limulus lysate. The gelation reaction induced by LPS in the lysate was monitored spectrophotometrically, and the concentration of LPS resulting in a 50% lysate response was determined and correlated with antibody titers measured by enzyme-linked immunosorbent assay. Antisera to smooth organisms neutralized homologous LPS markedly and heterologous LPSs only minimally relative to neutralization by preimmune serum. Neutralization of homologous LPS occurred immediately without preincubation of serum and LPS. Antisera to rough mutants neutralized more heterologous LPS than did antisera to smooth organisms. However, this heterologous neutralization required preincubation of serum and LPS and did not appear to be correlated with antibody concentrations. We conclude that antisera to LPS rapidly neutralize the biological activity of the homologous LPS, as detected by limulus lysate, and that neutralization is at least in part antibody mediated. Antisera to rough-mutant organisms slowly neutralized the activity of heterologous LPSs, but this effect appeared not to be correlated with concentrations of antibody to the LPS of the rough mutant, as measured by enzyme-linked immunosorbent assay. PMID:3298063

  3. ADANSONIAN ANALYSIS AND DEOXYRIBONUCLEIC ACID BASE COMPOSITION OF SOME GRAM-NEGATIVE BACTERIA

    PubMed Central

    Colwell, R. R.; Mandel, M.

    1964-01-01

    Colwell, R. R. (Georgetown University, Washington, D.C.), and M. Mandel. Adansonian analysis and deoxyribonucleic acid base composition of some gram-negative bacteria. J. Bacteriol. 87:1412–1422. 1964.—The deoxyribonucleic acid (DNA) base compositions and S values for a minimum of 134 coded properties were determined for representative cultures of the genera Pseudomonas, Xanthomonas, Aeromonas, Vibrio, Aerobacter, Escherichia, Alcaligenes, and Flavobacterium. Those cultures having a high degree of similarity by the criterion of numerical taxonomy were found to have similar DNA base compositions. The relative affinities of clusters of cultures suggest taxonomic relations. Eleven species of Xanthomonas might be a single species, and V. metschnikovii was shown to be more closely related to enteric bacteria than to other vibrios which, in turn, were found to be like pseudomonads. Aeromonas was found to be intermediate in similarity to enterics and pseudomonads and divisible into at least two, but possibly three, species. F. aquatile was unlike any of the other organisms studied, and its DNA also differed greatly in composition from other representatives of the genus. PMID:14188722

  4. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?

    PubMed

    Falagas, Matthew E; Bliziotis, Ioannis A

    2007-06-01

    The evolving problem of antimicrobial resistance in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae has led to the emergence of clinical isolates susceptible to only one class of antimicrobial agents and eventually to pandrug-resistant (PDR) isolates, i.e. resistant to all available antibiotics. We reviewed the available evidence from laboratory and clinical studies that reported on polymyxin-resistant and/or PDR P. aeruginosa, A. baumannii or K. pneumoniae clinical isolates. Eleven laboratory studies reported on isolates with resistance to polymyxins, three of which (including two surveillance studies) also included data regarding PDR isolates. In addition, two clinical studies (from Central and Southern Europe) reported on the clinical characteristics and outcomes of patients infected with PDR isolates. These data suggest that polymyxin-resistant or PDR P. aeruginosa, A. baumannii and K. pneumoniae clinical isolates are currently relatively rare. However, they have important global public health implications because of the therapeutic problems they pose. The fears for the dawn of a post-antibiotic era appear to be justified, at least for these three Gram-negative bacteria. We must increase our efforts to preserve the activity of available antibiotics, or at least expand as much as possible the period of their use, whilst intense research efforts should be focused on the development and introduction into clinical practice of new antimicrobial agents. PMID:17306965

  5. Therapy of Infections due to Carbapenem-Resistant Gram-Negative Pathogens

    PubMed Central

    Lee, Chang-Seop

    2014-01-01

    The prevalence of carbapenem-resistant gram-negative bacterial pathogens (CRGNs) has increased dramatically during the last 10 years, but the optimal treatment for CRGN infections is not well established due to the relative scarcity of robust clinical data. The polymyxins remain the most consistently active agents against CRGNs in vitro. Tigecycline, based on its in vitro antibacterial spectrum, could also be considered as a therapeutic option in the treatment of infections caused by certain CRGNs. Other agents, including aminoglycosides, rifampin, trimethoprim-sulfamethoxazole, fosfomycin and fluoroquinolones, could be considered as monotherapy or combination therapy against CRGNs in appropriate contexts, as combination therapy with two or more in vitro active drugs appears to be more effective than monotherapy based on some clinical data. Several promising new agents are in late-stage clinical development, including ceftolozane-tazobactam, ceftazidime-avibactam and plazomicin. Given the shortage of adequate treatment options, containment of CRGNs should be pursued through implementation of adequate infection prevention procedures and antimicrobial stewardship to reduce the disease burden and prevent future outbreaks of CRGNs. PMID:25298904

  6. 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.

  7. 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. PMID:26991296

  8. 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.

  9. 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.

  10. Characterization of Gram-negative psychrotrophic bacteria isolated from Italian bulk tank milk.

    PubMed

    Decimo, Marilù; Morandi, Stefano; Silvetti, Tiziana; Brasca, Milena

    2014-10-01

    Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD-PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat-resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19). PRATICAL APPLICATION: The study provides new data on the enzymatic activity of Gram-negative psychrotrophic bacteria, useful in developing strategies to control the proteo-lipolytic spoilage of raw and processed milk that causes gelation, off-flavors, and loss of sensory quality and shelf life. PMID:25224662

  11. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

    PubMed Central

    Howard, S P; Buckley, J T

    1985-01-01

    The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously. Images PMID:3972770

  12. Altered glucose kinetics in diabetic rats during Gram-negative infection

    SciTech Connect

    Lang, C.H.; Dobrescu, C.; Bagby, G.J.; Spitzer, J.J. )

    1987-08-01

    The present study examined the purported exacerbating effect of sepsis on glucose metabolism in diabetes. Diabetes was induced in rats by an intravenous injection of 70 or 45 mg/kg streptozotocin. The higher dose produced severe diabetes, whereas the lower dose of streptozotocin produced a miler, latent diabetes. After a chronic diabetic state had developed for 4 wk, rats had catheters implanted and sepsis induced by intraperitoneal injections of live Escherichia coli. After 24 h of sepsis the blood glucose concentration was unchanged in nondiabetics and latent diabetics, but glucose decreased from 15 to 8 mM in the septic severe diabetic group. This decrease in blood glucose was not accompanied by alterations in the plasma insulin concentration. Glucose turnover, assessed by the constant intravenous infusion of (6-{sup 3}H)- and (U-{sup 14}C)glucose, was elevated in the severe diabetic group, compared with either latent diabetics or nondiabetics. Sepsis increased the rate of glucose disappearance in nondiabetic rats but had no effect in either group of diabetic animals. Sepsis also failed to alter the insulinogenic index, used to estimate the insulin secretory capacity, in diabetic rats. Thus the present study suggests that the imposition of nonlethal Gram-negative sepsis on severe diabetic animals does not further impair glucose homeostasis and that the milder latent diabetes was not converted to a more severe diabetic state by the septic challenge.

  13. Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

    PubMed Central

    Rahnamaeian, Mohammad; Cytryńska, Małgorzata; Zdybicka-Barabas, Agnieszka; Dobslaff, Kristin; Wiesner, Jochen; Twyman, Richard M.; Zuchner, Thole; Sadd, Ben M.; Regoes, Roland R.; Schmid-Hempel, Paul; Vilcinskas, Andreas

    2015-01-01

    Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics. PMID:25833860

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

    PubMed

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

    1999-08-01

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

  15. Active surveillance for multidrug-resistant Gram-negative bacteria in the intensive care unit.

    PubMed

    Abbott, Iain J; Jenney, Adam W J; Spelman, Denis W; Pilcher, David V; Sidjabat, Hanna E; Richardson, Leisha J; Paterson, David L; Peleg, Anton Y

    2015-10-01

    A short-term program of performing serial active screening cultures (ASC) in the intensive care unit was instituted to establish a method for the detection of antibiotic-resistant Gram-negative bacteria (GNB) and the local rates of colonisation. Of all submitted ASC, 25.9% (30/116 collected swabs) isolated an antibiotic-resistant GNB. ChromID ESBL agar (bioMérieux, France) identified the majority of these organisms, with the additional antibiotic-impregnated media [MacConkey agar (MCA) with ciprofloxacin, MCA with gentamicin and MCA with ceftazidime] adding limited benefit. Compared to swabs performed on admission, 37.8% (14/37) of patients cultured a new antibiotic-resistant isolate on discharge. Serial screening in intensive care has the ability to identify patients with unrecognised colonisation with antibiotic-resistant GNB; however, the increase in the laboratory workload and logistical challenges in the collection of the surveillance swabs may limit this program's expansion. PMID:26308128

  16. National Committee for Clinical Laboratory Standards agar dilution susceptibility testing of anaerobic gram-negative bacteria.

    PubMed Central

    Brown, W J

    1988-01-01

    One hundred nine recent clinical isolates of anaerobic gram-negative bacteria were tested in triplicate by the National Committee for Clinical Laboratory Standards agar dilution procedure for their susceptibility to 32 antimicrobial agents. All isolates were inhibited by imipenem, but there were significant numbers of strains resistant to other beta-lactam drugs, and therefore the in vitro response to these antimicrobial agents cannot be predicted. This was particularly true for the bile-resistant or Bacteroides fragilis group. beta-Lactamase production was detected in 82% of the bacteroides with the nitrocefin test. Clavulanic acid combined with amoxicillin and ticarcillin and sulbactam combined with ampicillin resulted in synergistic activity against all beta-lactamase-positive organisms. Ceftizoxime was the most active of the cephalosporins. Two percent of the isolates were resistant to chloramphenicol and metronidazole. Clindamycin resistance was detected in 38% of the B. fragilis group, which is a marked increase from the 4% detected 10 years ago at this institution. PMID:3364956

  17. Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens.

    PubMed

    Botos, Istvan; Majdalani, Nadim; Mayclin, Stephen J; McCarthy, Jennifer Gehret; Lundquist, Karl; Wojtowicz, Damian; Barnard, Travis J; Gumbart, James C; Buchanan, Susan K

    2016-06-01

    Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand β-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane. PMID:27161977

  18. 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

  19. 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.

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

    PubMed Central

    Keasey, Sarah L.; Schmid, Kara E.; Lee, Michael S.; Meegan, James; Tomas, Patricio; Minto, Michael; Tikhonov, Alexander P.; Schweitzer, Barry; Ulrich, Robert G.

    2009-01-01

    Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising ∼70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria. PMID:19112181

  1. 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

  2. Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry.

    PubMed

    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

  3. Chlorhexidine resistance in a Gram-negative bacterium isolated from an aquatic source.

    PubMed

    Sekavec, Jeffrey G; Moore, William T; Gillock, Eric T

    2013-01-01

    Aeromonas hydrophila is a Gram-negative bacterium of considerable importance in both clinical, especially nosocomial infections, and zoonotic respects, both aquatic and terrestrial infections. In addition to the ability to thrive in a wide range of conditions, A. hydrophila is resistant to numerous antibiotics and antimicrobials. In conjunction with Kansas State University and the Kansas Water Office, water samples from various locations within Kansas were screened for organisms resistant to chlorhexidine. Chlorhexidine is the active agent in many surgical scrubs, prescription mouthwashes, and other bactericidal and bacteriostatic substances. Aeromonas hydrophila capable of growth in therapeutic levels of chlorhexidine was detected in one of the water samples. The isolate was determined not to be harboring a plasmid, lending to a course of inquiry founded on the premise that the source of chlorhexidine resistance resides within the chromosome. Investigation into efflux pumps (EP) is underway with select resistance-nodulation-cell division (RND) pumps, especially the AheABC EP, being exculpated of responsibility for chlorhexidine resistance following testing with a select EP inhibitor. Inquiry into the major facilitator superfamily (MFS) EPs is underway with specific examination of the EmrB/QacA EPs, as chlorhexidine resistance in Staphylococcus aureus carrying the QacA gene has been observed. PMID:24007437

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

    PubMed

    Lobos, J H; Leib, T K; Su, T M

    1992-06-01

    A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-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 CO2, 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. PMID:1622258

  5. 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

  6. Short-Term Starvation of Immune Deficient Drosophila Improves Survival to Gram-Negative Bacterial Infections

    PubMed Central

    Brown, Anthony E.; Baumbach, Janina; Cook, Peter E.; Ligoxygakis, Petros

    2009-01-01

    Background Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. Methodology/Principal Findings We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria. Conclusions/Significance Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity. PMID:19221590

  7. 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

  8. 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

  9. Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis.

    PubMed

    Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S; Le Brun, Anton P; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H

    2016-08-23

    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

  10. Assessment of the Levels of Airborne Bacteria, Gram-Negative Bacteria, and Fungi in Hospital Lobbies

    PubMed Central

    Park, Dong-Uk; Yeom, Jeong-Kwan; Lee, Won Jae; Lee, Kyeong-Min

    2013-01-01

    Aims: We assessed the levels of airborne bacteria, Gram-negative bacteria (GNB), and fungi in six hospital lobbies, and investigated the environmental and hospital characteristics that affected the airborne microorganism levels. Methods: An Andersen single-stage sampler equipped with appropriate nutrition plate agar was used to collect the samples. The three types of microorganisms were repeatedly collected at a fixed location in each hospital (assumed to be representative of the entire hospital lobby) from 08:00 through 24:00, with a sampling time of less than 5 min. Temperature and relative humidity were simultaneously monitored. Results: Multiple regression analysis was used to identify the major factors affecting microorganism levels. The average levels of bacteria (7.2 × 102 CFU/m3), GNB (1.7 × 10 CFU/m3), and fungi (7.7 × 10 CFU/m3) indicated that all hospital lobbies were generally contaminated. Season was the only factor that significantly affected the levels of all microorganisms (p < 0.0001), where contamination was the highest during the summer, significantly higher than during the winter. Other significant factors varied by microorganism, as follows: airborne bacteria (number of people in the lobby, sampling time), GNB (scale of hospital), and fungi (humidity and air temperature). Conclusions: Hospital lobby air was generally contaminated with microorganisms, including bacteria, GNB, and fungi. Environmental factors that may significantly influence the airborne concentrations of these agents should be managed to minimize airborne levels. PMID:23435586

  11. Infection-related hemolysis and susceptibility to Gram-negative bacterial co-infection.

    PubMed

    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

  12. Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

    PubMed Central

    Landeta, Cristina; Blazyk, Jessica L.; Hatahet, Feras; Meehan, Brian M.; Eser, Markus; Myrick, Alissa; Bronstain, Ludmila; Minami, Shoko; Arnold, Holly; Ke, Na; Rubin, Eric J.; Furie, Barbara C.; Furie, Bruce; Beckwith, Jon; Dutton, Rachel; Boyd, Dana

    2015-01-01

    In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors. Thus, bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a novel target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes E. coli DsbB (EcDsbB) or M. tuberculosis VKOR (MtbVKOR). MtbVKOR can replace EcDsbB although the two are not homologues. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic E. coli growth. Furthermore, these compounds inhibit all but one of the DsbBs of nine other gram-negative pathogenic bacteria tested. PMID:25686372

  13. 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.

  14. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    PubMed Central

    2016-01-01

    ABSTRACT Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S. Typhimurium. PMID:27531914

  15. Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria.

    PubMed

    Osei Sekyere, J; Govinden, U; Essack, S Y

    2015-11-01

    The minimal antibiotic options for carbapenemase-producing Gram-negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI-TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem-resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase-producing isolates. MALDI-TOF MS and/or molecular methods like microarray, loop-mediated isothermal amplification and real-time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies. PMID:26251303

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

    PubMed

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

    2016-03-17

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

  17. Facing the challenge of multidrug-resistant gram-negative bacilli in Australia.

    PubMed

    Harris, Patrick; Paterson, David; Rogers, Benjamin

    2015-03-16

    Multidrug-resistant (MDR) gram-negative bacilli (GNB) are now globally widespread and present a major challenge to modern medical practice. Resistance to common antibiotics such as ceftriaxone is becoming more frequent in Australia, primarily mediated by extended-spectrum β-lactamase enzymes in common organisms such as Escherichia coli, and may occur in both hospital- and community-acquired infections. Carbapenem-resistant Enterobacteriaceae have emerged rapidly in recent years and are well established in many countries in the Asia-Pacific region. Although rare at present in Australia, they have caused significant nosocomial outbreaks. GNB have numerous mechanisms by which they can develop antibiotic resistance. Genes that encode extended-spectrum β-lactamases or carbapenemases are frequently co-located with multiple other resistance determinants on highly transmissible genetic structures such as plasmids. A key risk factor for infection with MDR GNB is travel to countries with high rates of resistance, especially with health care exposure. With limited prospects for new antibiotics in late-stage development that are active against MDR GNB, our national response to these challenges will require a multifaceted approach, including widespread implementation of antimicrobial stewardship, enhanced surveillance, targeted screening of at-risk patients and improved infection control practices. In the longer term, restriction of agricultural use of antibiotic classes critical to human medicine, removal of barriers to new drug development, and technological advances in rapid microbiological diagnostics will be required. PMID:25758692

  18. Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates.

    PubMed Central

    Wireman, J; Liebert, C A; Smith, T; Summers, A O

    1997-01-01

    Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components. PMID:9361435

  19. 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

  20. 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 Central

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

    2014-01-01

    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. PMID:24759724

  1. Interaction of antimicrobial peptide temporin L with lipopolysaccharide in vitro and in experimental rat models of septic shock caused by gram-negative bacteria.

    PubMed

    Giacometti, Andrea; Cirioni, Oscar; Ghiselli, Roberto; Mocchegiani, Federico; Orlando, Fiorenza; Silvestri, Carmela; Bozzi, Argante; Di Giulio, Antonio; Luzi, Carla; Mangoni, Maria Luisa; Barra, Donatella; Saba, Vittorio; Scalise, Giorgio; Rinaldi, Andrea C

    2006-07-01

    Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used beta-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and beta-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-alpha levels, resulting in the highest survival rates. PMID:16801429

  2. Evaluation of osmoprotectants and carriers for formulating Gram-negative biocontrol agents active against potato dry rot in storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The production of a dry formulation containing viable cells of a Gram-negative biological control agent is a challenging yet vital step in developing the agent into a commercial product. Pseudomonas fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 reduce Fusarium dry rot of potatoes (cau...

  3. 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...

  4. Discovery of a Novel Class of Boron-Based Antibacterials with Activity against Gram-Negative Bacteria

    PubMed Central

    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.; Plattner, Jacob J.

    2013-01-01

    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. PMID:23295920

  5. Bacteremia and resistant gram-negative pathogens among under-fives in Tanzania

    PubMed Central

    2013-01-01

    Background Antibiotic resistance is one of the most serious public health concerns worldwide and is increasing at an alarming rate, making daily treatment decisions more challenging. This study is aimed at identifying local bacterial isolates and their antimicrobial susceptibility patterns to avoid irrational antibiotic use, especially in settings where unguided management occurs and febrile illnesses are predominant. Material and methods A hospital-based prospective cross-sectional study was conducted from September 2011 to February 2012. Febrile children were serially recruited and demographic and clinical data were collected using a standardized data collection tool. A blood culture was performed and identification of the isolates was undertaken using in-house biochemical tests. Susceptibility to common antibiotics was investigated using the disc diffusion methods. Results Of the 1081 children admitted during the study period, 317 (29.3%) met the inclusion criteria and were recruited, of whom 195 (61.5%) and 122 (38.5%) were male and female respectively. The median age was 18 months with an interquartile range of 9 to 36 months. Of the 317 children, 251 (79.2%) were below or equal to 36 months of age. The prevalence of bacteremia was 6.6%. A higher prevalence of bacteraemia was observed in children below 36 months than in those ≥ 36 months (7.5% vs. 3.0%, p = 0.001). Predictors of bacteraemia were an axillary temperature of >38.5 °C (OR =7, 95% CI = 2.2 - 14.8, p-value = 0.0001), a positive malaria slide (OR =5, 95% CI = 3.0 - 21.2, p-value = 0.0001) and a high neutrophils’ count (OR =21 95% CI = 5.6 - 84, p-value = 0.0001). Escherichia coli and Klebsiella pneumoniae accounted for 7 (33.3%) and 6 (28.6%) of all the isolates respectively. Others gram-negatives bacteria were Citrobacter spp 2 (9.5%), Enterobacter spp 1 (4.25%), Pseudomonas spp 2 (9.5%), Proteus spp 1 (4.25%) and Salmonella spp 1 (4.25%). These isolates

  6. Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

    PubMed Central

    Chavada, Ruchir; Maley, Michael

    2015-01-01

    Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

  7. 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

  8. 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

  9. The Relationship between Extensively Drug-Resistant Tuberculosis and Multidrug-Resistant Gram-Negative Bacilli

    PubMed Central

    Zhao, Jiang-nan; Zhang, Xian-xin; He, Xiao-chun; Yang, Guo-ru; Zhang, Xiao-qi; Li, Huai-chen

    2015-01-01

    Objective The relationship between extensively drug-resistant tuberculosis (XDR-TB) and multidrug-resistant Gram-negative bacilli (MDR-GNB) is unclear. Identification of the relationship between XDR-TB and MDR-GNB would have important implications for patient care. Methods We conducted a retrospective study reviewing the records of patients admitted with a confirmed pulmonary TB from 2011 to 2014. To identify the relationship between XDR-TB and MDR-GNB, univariable comparison and multivariable logistic regression were performed. Results Among 2962 pulmonary TB patients, 45(1.5%) patients had a diagnosis of XDR-TB. A total of 165 MDR-GNB strains were detected in 143 (4.8%) pulmonary TB patients. XDR-TB patients had a significantly higher occurrence of MDR-GNB than non-XDR-TB patients (24.4% vs. 4.5%; P<0.001). Age (OR 1.02, 95% CI 1.01–1.03), hypoalbuminemia (OR 1.48, 95% CI 1.18–1.85), chronic renal failure (OR 6.67, 95% CI 1.42–31.47), chronic hepatic insufficiency (OR 1.99, 95% CI 1.15–3.43), presence of XDR-TB (OR 6.56, 95% CI 1.61–26.69), and duration of TB diagnostic delay (OR 1.01, 95% CI 1.00–1.02) were the independent risk factors for MDR-GNB infection. Conclusions Patients with XDR-TB have a significantly higher risk of being affected by MDR-GNB pathogen. The underlying mechanism association warrant further studies. PMID:26230499

  10. In vitro activity of cefoperazone-sulbactam combination against gram negative bacilli.

    PubMed

    Poudyal, N; Gyawali, N; Gurung, R; Bhattarai, N R; Baral, R; Khanal, B; Shrestha, S; Amatya, R; Bhattacharya, S K

    2012-03-01

    Cefoperazone is a â-lactam antimicrobial and Sulbactam is an irreversible â-lactamase inhibitor. The objective of this study was to know the susceptibility pattern of gram negative bacilli (GNB) towards cefoperazone-sulbactum. All GNB isolated from different clinical samples during the period of May, 2010 to Aug, 2010 were tested for susceptibility to cefoperazone-sulbactum, meropenem, ceftazidime, cefotaxime, ceftriaxone, chloromphenicol, cotrimoxazole, ampicillin, amikacin, nalidixic acid, ciprofloxacin, carbenicillin and piperacillin using standard Kirby-Bauer disc diffusion antimicrobial susceptibility testing method. The susceptibilities were recorded according to CLSI guidelines. A total of 406 GNB were isolated (urine: 66.7%, pus: 19.2%, and blood: 7.9%). Escherichia coli (54.4%) was most frequently isolated organisms followed by Acinetobacter species (17.7%), Klebsiella pneumoniae (9.1%) and Pseudomonas species (6.1%). Overall, 11.8% of isolates showed resistance to cefoperazone-sulbactam. Frequencies of isolates showing resistance to meropenem and amikacin were 14.7% and 26.25% respectively. Only 3.9% of Escherichia coli isolates showed resistance to cefoperazone-sulbactam. For other organisms, their lowest frequency ranging from 0-20%, exhibited resistance to meropenem. In Pseudomonas spp, in-vitro activity of amikacin was also better as only 11.1% isolates showed resistance to it. This study demonstrated the in-vitro synergistic effect of cefoperazonerazone-sulbactam and meropenem having good activity against GNB compared to the activity of other commonly tested antimicrobials. Cefoperazone-sulbactam can be recommended for the clinical practice against GNB exhibiting resistant to other antimicrobials as it is cheaper alternative to meropenem. Our results also focused on the continuous surveillance of the trends and features of resistance of common antimicrobials. PMID:23441486

  11. 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

  12. The structural diversity of carbohydrate antigens of selected gram-negative marine bacteria.

    PubMed

    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

  13. Thermodynamic Analysis of the Lipopolysaccharide-Dependent Resistance of Gram-Negative Bacteria against Polymyxin B

    PubMed Central

    Howe, Jörg; Andrä, Jörg; Conde, Raquel; Iriarte, Maite; Garidel, Patrick; Koch, Michel H. J.; Gutsmann, Thomas; Moriyón, Ignacio; Brandenburg, Klaus

    2007-01-01

    Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS. PMID:17237210

  14. 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. PMID:3529797

  15. The Forgotten One: Lemierre’s Syndrome Due to Gram-Negative Rods Prevotella Bacteremia

    PubMed Central

    Wani, Priyanka; Antony, Nishaal; Wardi, Miraie; Rodriguez-Castro, Carlos E.; Teleb, Mohamed

    2016-01-01

    Patient: Male, 22 Final Diagnosis: Lemierre’s syndrome Symptoms: Dyspnea • chest pain • swelling Medication: — Clinical Procedure: Thoracentesis Specialty: Infectious Diseases Objective: Rare co-existance of disease or pathology Background: Lemierre’s syndrome (LS) is a rare syndrome caused by an acute oropharyngeal infection with metastatic spreading. It was described in 1939 as jugular vein septic thrombophlebitis associated with retropharyngeal infection. Different organisms can cause LS, such as Fusobacterium species, Peptostreptococcus, group B and C, Streptococcus, Staphylococcus, and Enterococcus species, but the most commonly isolated pathogen is Fusobacterium necrophorum, a common oral flora. Management depends on the initial presentation, type of pathogen isolated, and proper selection of antibiotics. Case Report: We report a case of a 22-year-old man with no past medical history, who presented with left jaw pain and progressive left facial area swelling associated with dyspnea. A final diagnosis of LS was made based on criteria of computed tomography (CT) of the neck and the clinical symptoms. The patient was started on broad-spectrum antibiotics. Subsequent imaging of the chest showed pleural effusion with septic emboli. He underwent thoracentesis and chest tube placement. Final blood cultures were remarkable for gram-negative rods – Prevotella anaerobes – which supported the diagnosis of LS. His condition improved, including the dyspnea, and he was discharged on the proper antibiotics coverage with outpatient follow-up. Conclusions: LS is a rare condition associated with metastatic infection spreading. This syndrome can be associated with further complications, such as pleural effusions and/or empyemas. Early recognition is important to prevent fatal complications and provide adequate antibiotics coverage. We report only the third case in the medical literature of Prevotella-induced LS with a secondary complication of pleural effusion

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

    PubMed

    Monogue, Marguerite L; Thabit, Abrar K; Hamada, Yukihiro; Nicolau, David P

    2016-08-01

    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

  17. 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. PMID:26659214

  18. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli

    PubMed Central

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  19. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

    PubMed

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  20. Cost-Effective and Rapid Presumptive Identification of Gram-Negative Bacilli in Routine Urine, Pus, and Stool Cultures: Evaluation of the Use of CHROMagar Orientation Medium in Conjunction with Simple Biochemical Tests

    PubMed Central

    Ohkusu, Kiyofumi

    2000-01-01

    The algorithm for a new identification system was designed on the basis of colony color and morphology on CHROMagar Orientation medium in conjunction with simple biochemical tests such as indole (IND), lysine decarboxylase (LDC), and ornithine decarboxylase (ODC) utilization tests with gram-negative bacilli isolated from urine samples as well as pus, stool, and other clinical specimens by the following colony characteristics, biochemical reactions, and serological results: pinkish to red, IND positive (IND+), Escherichia coli; metallic blue, IND+, LDC+, and ODC negative (ODC−), Klebsiella oxytoca; IND+, LDC−, and ODC+, Citrobacter diversus; IND+ or IND−, LDC−, and ODC−, Citrobacter freundii; IND−, LDC+, and ODC+, Enterobacter aerogenes; IND−, LDC−, and ODC+, Enterobacter cloacae; IND−, LDC+, and ODC−, Klebsiella pneumoniae; diffuse brown and IND+, Morganella morganii; IND−, Proteus mirabilis; aqua blue, Serratia marcescens; bluish green and IND+, Proteus vulgaris; transparent yellow-green, serology positive, Pseudomonas aeruginosa; clear and serology positive, Salmonella sp.; other colors and reactions, the organism was identified by the full identification methods. The accuracy and cost-effectiveness of this new system were prospectively evaluated. During an 8-month period, a total of 345 specimens yielded one or more gram-negative bacilli. A total of 472 gram-negative bacillus isolates were detected on CHROMagar Orientation medium. For 466 of the isolates (98.7%), no discrepancies in the results were obtained on the basis of the identification algorithm. The cost of identification of gram-negative bacilli during this period was reduced by about 70%. The results of this trial for the differentiation of the most commonly encountered gram-negative pathogens in clinical specimens with the new algorithm were favourable in that it permitted reliable detection and presumptive identification. In addition, this rapid identification system not only

  1. Treatment of Gram-negative pneumonia in the critical care setting: is the beta-lactam antibiotic backbone broken beyond repair?

    PubMed

    Bassetti, Matteo; Welte, Tobias; Wunderink, Richard G

    2016-01-01

    Beta-lactam antibiotics form the backbone of treatment for Gram-negative pneumonia in mechanically ventilated patients in the intensive care unit. However, this beta-lactam antibiotic backbone is increasingly under pressure from emerging resistance across all geographical regions, and health-care professionals in many countries are rapidly running out of effective treatment options. Even in regions that currently have only low levels of resistance, the effects of globalization are likely to increase local pressures on the beta-lactam antibiotic backbone in the near future. Therefore, clinicians are increasingly faced with a difficult balancing act: the need to prescribe adequate and appropriate antibiotic therapy while reducing the emergence of resistance and the overuse of antibiotics. In this review, we explore the burden of Gram-negative pneumonia in the critical care setting and the pressure that antibiotic resistance places on current empiric therapy regimens (and the beta-lactam antibiotic backbone) in this patient population. New treatment approaches, such as systemic and inhaled antibiotic alternatives, are on the horizon and are likely to help tackle the rising levels of beta-lactam antibiotic resistance. In the meantime, it is imperative that the beta-lactam antibiotic backbone of currently available antibiotics be supported through stringent antibiotic stewardship programs. PMID:26821535

  2. Molecular dynamic study of MlaC protein in Gram-negative bacteria: conformational flexibility, solvent effect and protein-phospholipid binding.

    PubMed

    Huang, Yu-Ming M; Miao, Yinglong; Munguia, Jason; Lin, Leo; Nizet, Victor; McCammon, J Andrew

    2016-08-01

    The composition of the outer membrane in Gram-negative bacteria is asymmetric, with the lipopolysaccharides found in the outer leaflet and phospholipids in the inner leaflet. The MlaC protein transfers phospholipids from the outer to inner membrane to maintain such lipid asymmetry in the Mla pathway. In this work, we have performed molecular dynamics simulations on apo and phospholipid-bound systems to study the dynamical properties of MlaC. Our simulations show that the phospholipid forms hydrophobic interactions with the protein. Residues surrounding the entrance of the binding site exhibit correlated motions to control the site opening and closing. Lipid binding leads to increase of the binding pocket volume and precludes entry of the water molecules. However, in the absence of the phospholipid, water molecules can freely move in and out of the binding site when the pocket is open. Dehydration occurs when the pocket closes. This study provides dynamic information of the MlaC protein and may facilitate the design of antibiotics against the Mla pathway of Gram-negative bacteria. PMID:27111825

  3. Antibiotic susceptibility pattern in urinary isolates of gram negative bacilli with special reference to AmpC β-lactamase in a tertiary care hospital

    PubMed Central

    Patel, Mitesh H.; Trivedi, Grishma R.; Patel, Sachin M.; Vegad, Mahendra M.

    2010-01-01

    Introduction: Resistance to higher antimicrobial agent is commonly seen in gram negative bacilli. This issue is a challenging problem to the medical practitioners in addition to it is financial impact on the health care system. Objectives: To document the prevalence of multi drug resistant gram negative bacilli isolated from urine of patients attending the Urology Department of Tertiary care Hospital of western India in year 2008. Results: Out of total 328 isolates, 118 (35.98%) E.coli, 72 (21.95 %) Klebsiella, 64 (19.51%) Pseudomonas aeruginosa, 30 (9.15%) Acinetobacter, 18 (5.49%) Proteus vulgaris, 18 (5.49%) Proteus mirabilis, 6 (1.83%) Providencia rettgerii, 2 (0.61%) Citrobacter freundii. Out of these isolates, 228 (69.51%) were β-lactamase positive, while 100 (30.51%) were β-lactamase negative. Out of 228 β-lactamase positive, 104 (45.61%) were AmpC β-lactamase positive. Conclusions: Stringent protocol such as Antibiotic policy and Hospital infection control program are mandatory to curb these microbes in a tertiary care hospital. PMID:20842250

  4. RND-type drug efflux pumps from Gram-negative bacteria: molecular mechanism and inhibition

    PubMed Central

    Venter, Henrietta; Mowla, Rumana; Ohene-Agyei, Thelma; Ma, Shutao

    2015-01-01

    Drug efflux protein complexes confer multidrug resistance on bacteria by transporting a wide spectrum of structurally diverse antibiotics. Moreover, organisms can only acquire resistance in the presence of an active efflux pump. The substrate range of drug efflux pumps is not limited to antibiotics, but it also includes toxins, dyes, detergents, lipids, and molecules involved in quorum sensing; hence efflux pumps are also associated with virulence and biofilm formation. Inhibitors of efflux pumps are therefore attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. Recent successes on the structure determination and functional analysis of the AcrB and MexB components of the AcrAB-TolC and MexAB-OprM drug efflux systems as well as the structure of the fully assembled, functional triparted AcrAB-TolC complex significantly contributed to our understanding of the mechanism of substrate transport and the options for inhibition of efflux. These data, combined with the well-developed methodologies for measuring efflux pump inhibition, could allow the rational design, and subsequent experimental verification of potential efflux pump inhibitors (EPIs). In this review we will explore how the available biochemical and structural information can be translated into the discovery and development of new compounds that could reverse drug resistance in Gram-negative pathogens. The current literature on EPIs will also be analyzed and the reasons why no compounds have yet progressed into clinical use will be explored. PMID:25972857

  5. Impact of microbiology cascade reporting on antibiotic de-escalation in cefazolin-susceptible Gram-negative bacteremia.

    PubMed

    Johnson, L S; Patel, D; King, E A; Maslow, J N

    2016-07-01

    Cascade reporting (CR) involves reporting the susceptibilities of broad-spectrum agents only when the organism is resistant to more narrow-spectrum agents. The purpose of this study is to evaluate the impact of CR on antibiotic de-escalation practices and to characterize the impact of CR on clinical outcomes. CR rules were implemented in the microbiology laboratory at Atlantic Health System (AHS) in June 2013. A retrospective chart review was conducted at two community teaching hospitals in adult patients who had a blood culture positive for a Gram-negative organism susceptible to cefazolin and who were empirically treated with broad-spectrum beta-lactam (BSBL) antibiotics. De-escalation practices were compared in the pre-CR (July 2012-December 2012) and post-CR (July 2013-December 2013) periods. The primary endpoint was the percentage of patients whose BSBL agent was de-escalated to agents listed on the post-CR antibiotic susceptibility report within 48 h of the final report. Secondary endpoints include the difference in pre-CR and post-CR periods in terms of hospital length of stay, in-hospital mortality, 30-day readmission, Clostridium difficile infections, and re-initiation of a BSBL agent within 7 days. A total of 73 patients were included; 31 in the pre-CR and 42 in the post-CR period. Patients had similar baseline characteristics. Therapy was de-escalated in 48 % of pre-CR vs 71 % of post-CR patients (p = 0.043). No significant differences were observed in secondary endpoints between patients in the pre-CR and post-CR periods. CR resulted in significant improvements in de-escalation practices without affecting safety outcomes. PMID:27130036

  6. Travel-related carbapenemase-producing Gram-negative bacteria in Alberta, Canada: the first 3 years.

    PubMed

    Peirano, Gisele; Ahmed-Bentley, Jasmine; Fuller, Jeff; Rubin, Joseph E; Pitout, Johann D D

    2014-05-01

    We describe here the characteristics of Alberta, Canada, patients with infections or colonizations with carbapenemase-producing Gram-negative bacteria during 2010 to 2013 that were linked to recent travel outside Canada. Antimicrobial susceptibility was determined by broth microdilution, and isolates were characterized using PCR, sequencing, and multilocus sequencing typing. A broth mating study was used to assess the transferability of resistance plasmids, which were subsequently characterized. All the patients (n=12) included in our study had contact with a health care system while abroad. Most of the patients presented with urinary tract infections (UTIs) and were admitted to hospitals within weeks after their return to Alberta. Secondary spread occurred in 1 case, resulting in the death of another patient. The carbapenemase-producing bacteria (n=17) consisted of Escherichia coli (sequence type 101 [ST101], ST365, ST405, and ST410) with NDM-1, Klebsiella pneumoniae (ST15, ST16, ST147, ST258, ST340, ST512, and ST972) with NDM-1, OXA-181, KPC-2, and KPC-3, Acinetobacter baumannii with OXA-23, Providencia rettgeri with NDM-1, Enterobacter cloacae with KPC-2, and Citrobacter freundii with NDM-1. The blaNDM-1 gene was associated with various narrow- (i.e., IncF) and broad- (i.e., IncA/C and IncL/M) host-range plasmids with different addiction factors. Our results show that NDM-producing K. pneumoniae, belonging to a variety of sequence types with different plasmid scaffolds, are regularly imported from India into Alberta. Clinical microbiology laboratories should remain vigilant in detecting bacteria with carbapenemases. PMID:24599977

  7. Travel-Related Carbapenemase-Producing Gram-Negative Bacteria in Alberta, Canada: the First 3 Years

    PubMed Central

    Peirano, Gisele; Ahmed-Bentley, Jasmine; Fuller, Jeff; Rubin, Joseph E.

    2014-01-01

    We describe here the characteristics of Alberta, Canada, patients with infections or colonizations with carbapenemase-producing Gram-negative bacteria during 2010 to 2013 that were linked to recent travel outside Canada. Antimicrobial susceptibility was determined by broth microdilution, and isolates were characterized using PCR, sequencing, and multilocus sequencing typing. A broth mating study was used to assess the transferability of resistance plasmids, which were subsequently characterized. All the patients (n = 12) included in our study had contact with a health care system while abroad. Most of the patients presented with urinary tract infections (UTIs) and were admitted to hospitals within weeks after their return to Alberta. Secondary spread occurred in 1 case, resulting in the death of another patient. The carbapenemase-producing bacteria (n = 17) consisted of Escherichia coli (sequence type 101 [ST101], ST365, ST405, and ST410) with NDM-1, Klebsiella pneumoniae (ST15, ST16, ST147, ST258, ST340, ST512, and ST972) with NDM-1, OXA-181, KPC-2, and KPC-3, Acinetobacter baumannii with OXA-23, Providencia rettgeri with NDM-1, Enterobacter cloacae with KPC-2, and Citrobacter freundii with NDM-1. The blaNDM-1 gene was associated with various narrow- (i.e., IncF) and broad- (i.e., IncA/C and IncL/M) host-range plasmids with different addiction factors. Our results show that NDM-producing K. pneumoniae, belonging to a variety of sequence types with different plasmid scaffolds, are regularly imported from India into Alberta. Clinical microbiology laboratories should remain vigilant in detecting bacteria with carbapenemases. PMID:24599977

  8. The Increasing Challenge of Multidrug-Resistant Gram-Negative Bacilli

    PubMed Central

    Giuffrè, Mario; Geraci, Daniela M.; Bonura, Celestino; Saporito, Laura; Graziano, Giorgio; Insinga, Vincenzo; Aleo, Aurora; Vecchio, Davide; Mammina, Caterina

    2016-01-01

    Abstract Colonization and infection by multidrug-resistant gram-negative bacilli (MDR GNB) in neonatal intensive care units (NICUs) are increasingly reported. We conducted a 5-year prospective cohort surveillance study in a tertiary NICU of the hospital “Paolo Giaccone,” Palermo, Italy. Our objectives were to describe incidence and trends of MDR GNB colonization and the characteristics of the most prevalent organisms and to identify the risk factors for colonization. Demographic, clinical, and microbiological data were prospectively collected. Active surveillance cultures (ASCs) were obtained weekly. Clusters of colonization by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were analyzed by conventional and molecular epidemiological tools. During the study period, 1152 infants were enrolled in the study. Prevalences of colonization by MDR GNB, ESBL-producing GNB and multiple species/genera averaged, respectively, 28.8%, 11.7%, and 3.7%. Prevalence and incidence density of colonization by MDR GNB and ESBL-producing GNB showed an upward trend through the surveillance period. Rates of ESBL-producing E coli and K pneumoniae colonization showed wide fluctuations peaking over the last 2 years. The only independent variables associated with colonization by MDR GNB and ESBL-producing organisms and multiple colonization were, respectively, the days of NICU stay (odds ratio [OR] 1.041), the days of exposure to ampicillin–sulbactam (OR 1.040), and the days of formula feeding (OR 1.031). Most clusters of E coli and K pneumoniae colonization were associated with different lineages. Ten out of 12 clusters had an outborn infant as their index case. Our study confirms that MDR GNB are an increasing challenge to NICUs. The universal once-a-week approach allowed us to understand the epidemiology of MDR GNB, to timely detect new clones and institute contact precautions, and to assess risk factors. Collection of these data can be an

  9. Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism.

    PubMed Central

    Tsai, C C; McArthur, W P; Baehni, P C; Hammond, B F; Taichman, N S

    1979-01-01

    The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 4 degrees C, heat sensitive (56 degrees C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 4 degrees C but did bind to dead PMN membrane components at both 4 and 37 degrees C. The addition of bovine serum albumin (51 mg/ml) to PMN-Y4 leukotoxin cultures inhibited the release of lactate dehydrogenase from the PMNs, but did not prevent the death of the cells as indicated by electron microscopy. Lysosomal markers were released in parallel to the cytoplasmic enzyme lactate dehydrogenase from Y4 leukotoxin-treated PMNs. The addition of 0.02 M ethylenedinitrilotetraacetic acid to these cultures inhibited release of lysosomal markers but enhanced the release of lactate dehydrogenase. These results suggested that a soluble leukotoxin with specificity for only human PMNs and monocytes can be liberated from viable Y4. What role this leukotoxin plays in the pathogenicity of the Y4 microorganism is not yet known. However, this leukotoxin is one of the first materials from a plaque

  10. Thickness and Elasticity of Gram-Negative Murein Sacculi Measured by Atomic Force Microscopy

    PubMed Central

    Yao, X.; Jericho, M.; Pink, D.; Beveridge, T.

    1999-01-01

    Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 × 107 N/m2 was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 × 108 to 4 × 108 N/m2 and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intact E. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927–953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial

  11. ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition.

    PubMed

    Klebba, Phillip E

    2016-01-01

    The rotational surveillance and energy transfer (ROSET) model of TonB action suggests a mechanism by which the electrochemical proton gradient across the Gram-negative bacterial inner membrane (IM) promotes the transport of iron through ligand-gated porins (LGP) in the outer membrane (OM). TonB associates with the IM by an N-terminal hydrophobic helix that forms a complex with ExbBD. It also contains a central extended length of rigid polypeptide that spans the periplasm and a dimeric C-terminal-ββαβ-domain (CTD) with LysM motifs that binds the peptidoglycan (PG) layer beneath the OM bilayer. The TonB CTD forms a dimer with affinity for both PG- and TonB-independent OM proteins (e.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded β-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of iron-loaded LGP and then the internalization of iron through their trans-outer membrane β-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake. PMID:26787763

  12. Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

    PubMed

    Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

    2016-09-01

    Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. PMID:27620957

  13. Functional polymorphisms in the gene encoding macrophage migration inhibitory factor are associated with Gram-negative bacteremia in older adults.

    PubMed

    Das, Rituparna; Subrahmanyan, Lakshman; Yang, Ivana V; van Duin, David; Levy, Rebecca; Piecychna, Marta; Leng, Lin; Montgomery, Ruth R; Shaw, Albert; Schwartz, David A; Bucala, Richard

    2014-03-01

    Macrophage migration inhibitory factor (MIF) is an immune mediator encoded in a functionally polymorphic locus. We found the genotype conferring low expression of MIF to be enriched in a cohort of 180 patients with gram-negative bacteremia, compared with 229 healthy controls (odds ratio [OR], 2.4; P = .04), an association that was more pronounced in older adults (OR, 4.6; P = .01). Among older subjects, those with low expression of MIF demonstrated 20% reduced MIF production from lipopolysaccharide-stimulated peripheral blood monocytes and 30% lower monocyte surface Toll-like receptor 4, compared with those with high expression. Our work suggests that older adults with low expression of MIF may be predisposed to hyporesponsiveness to lipopolysaccharide and gram-negative bacterial infection. PMID:24158957

  14. Functional Polymorphisms in the Gene Encoding Macrophage Migration Inhibitory Factor Are Associated With Gram-Negative Bacteremia in Older Adults

    PubMed Central

    Das, Rituparna; Subrahmanyan, Lakshman; Yang, Ivana V.; van Duin, David; Levy, Rebecca; Piecychna, Marta; Leng, Lin; Montgomery, Ruth R.; Shaw, Albert; Schwartz, David A.; Bucala, Richard

    2014-01-01

    Macrophage migration inhibitory factor (MIF) is an immune mediator encoded in a functionally polymorphic locus. We found the genotype conferring low expression of MIF to be enriched in a cohort of 180 patients with gram-negative bacteremia, compared with 229 healthy controls (odds ratio [OR], 2.4; P = .04), an association that was more pronounced in older adults (OR, 4.6; P = .01). Among older subjects, those with low expression of MIF demonstrated 20% reduced MIF production from lipopolysaccharide-stimulated peripheral blood monocytes and 30% lower monocyte surface Toll-like receptor 4, compared with those with high expression. Our work suggests that older adults with low expression of MIF may be predisposed to hyporesponsiveness to lipopolysaccharide and gram-negative bacterial infection. PMID:24158957

  15. Diversity of culturable Gram-negative bacteria isolated from irrigation water of two rice crop regions in Southern Brazil.

    PubMed

    Reche, Maria Helena Lima Ribeiro; Reali, Catiusca; Pittol, Michele; de Athayde Saul, Danilo; Macedo, Vera Regina Mussoi; Valiati, Victor Hugo; Machado, Vilmar; Fiuza, Lidia Mariana

    2016-06-01

    In this study, we assessed the diversity of Gram-negative bacteria found in water used for irrigation of rice crops in two growing areas of southern Brazil. Samples were collected from the main irrigation channel and field drain area. Twenty-two bacterial species were found in Cachoeirinha and 28 in Camaquã. In both areas, the most frequent bacterial families were Enterobacteriaceae and Aeromonadaceae. Differences in microbial diversity were observed in both study areas. Thirty-five Gram-negative species were identified; however, only 15 were common in both locations. In addition, there were found pathogenic and drug-resistant species, such as Acinetobacter sp., Brucella spp., and Chryseobacterium meningosepticum. This study demonstrates the existence of a number of pathogenic species in aquatic ecosystems analyzed in three consecutive crop years, especially water used for rice production. PMID:27197729

  16. Purification of equine neutrophil lysozyme and its antibacterial activity against gram-positive and gram-negative bacteria.

    PubMed

    Pellegrini, A; Waiblinger, S; Von Fellenberg, R

    1991-01-01

    Lysozyme from equine neutrophil granulocytes was isolated in a pure form by fast performance liquid chromatography, i.e. ion-exchange chromatography and reversed-phase chromatography. The lysozyme lysed Micrococcus luteus, Bacillus subtilis and Staphylococcus lentus and was also bactericidal against the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Bordetella bronchiseptica, and Serratia marcescens. Staphylococcus aureus and Staphylococcus epidermidis were not lysed. The lysozyme was only very slightly bactericidal for S. epidermidis and S. aureus. Equine neutrophil lysozyme was found to be bactericidal for Gram-positive as well as for Gram-negative bacteria without further treatment. Equine and chicken egg white lysozymes were found to be immunologically related when examined using specific antisera against each of them. Both lysozymes also had very similar specific enzymatic activities against M. luteus membranes. PMID:1803722

  17. The clinical impact of multidrug-resistant gram-negative bacilli in the management of septic shock

    PubMed Central

    Pop-Vicas, Aurora; Opal, Steven M

    2014-01-01

    Multi-antibiotic drug-resistant (MDR) gram-negative bacilli are becoming a major threat to the standard care of septic patients. Empiric antimicrobial drug regimens to cover likely bacterial pathogens have to be altered in keeping with the spread of MDR pathogens in the health care setting and in the community. Reliable antibiotics for broad spectrum coverage for sepsis such as extended spectrum β-lactam antibiotics, carbapenems, and fluoroquinolones can no longer be counted upon to provide activity against a range of common, virulent pathogens that cause sepsis. In some regions of Asia, South America, and Eastern Europe in particular, MDR pathogens have become a major concern, necessitating the use of potentially toxic and costly antibiotic combinations as initial antibiotic therapy for septic shock. In this brief review, we will focus on the emergence of MDR gram-negative pathogens, resistance mechanisms, and suggest some management and prevention strategies against MDR pathogens. PMID:24200870

  18. The role of the outer membrane of Gram-negative bacteria in antibiotic resistance: Ajax' shield or Achilles' heel?

    PubMed

    Page, Malcolm G P

    2012-01-01

    There has been an enormous increase in our knowledge of the fundamental steps in the biosynthesis and assembly of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria as is peptidoglycan. Porins, efflux pumps and other transport proteins of the outer membrane are also present. It is clear that there are numerous essential proteins that have the potential to be targets for novel antimicrobial agents. Progress, however, has been slow. Much of the emphasis has been on cytoplasmic processes that were better understood earlier on, but have the drawback that two penetration barriers, with different permeability properties, have to be crossed. With the increased understanding of the late-stage events occurring in the periplasm, it may be possible to shift focus to these more accessible targets. Nevertheless, getting drugs across the outer membrane will remain a challenge to the ingenuity of the medicinal chemist. PMID:23090596

  19. Korormicin, a novel antibiotic specifically active against marine gram-negative bacteria, produced by a marine bacterium.

    PubMed

    Yoshikawa, K; Takadera, T; Adachi, K; Nishijima, M; Sano, H

    1997-11-01

    A novel antibiotic named korormicin was isolated from the marine bacterium, Pseudoalteromonas sp. F-420. This strain was isolated from the surface of a macro alga Halimeda sp. collected from Palau (the Republic of Belau). The planar structure of korormicin was determined by the result of 2D NMR studies and mass spectral data. Korormicin had specific inhibitory activity against marine Gram-negative bacteria, but was inactive against terrestrial microorganisms. PMID:9592569

  20. 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

  1. 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. PMID:26577143

  2. Influenza-induced type I interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice

    PubMed Central

    Lee, Benjamin; Robinson, Keven M.; McHugh, Kevin J.; Scheller, Erich V.; Mandalapu, Sivanarayana; Chen, Chen; Di, Y. Peter; Clay, Michelle E.; Enelow, Richard I.; Dubin, Patricia J.

    2015-01-01

    Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection. PMID:26001778

  3. Evaluation of antimicrobial activity of the endophytic actinomycete R18(6) against multiresistant Gram-negative bacteria.

    PubMed

    Carvalho, Tiele; Van Der Sand, Sueli

    2016-03-01

    Endophytic actinomycetes are promising sources of antimicrobial substances. This study evaluates the activity of metabolites produced by the endophytic actinomycete R18(6) against Gram-negative bacteria multiresistant to antimicrobials. R18(6) isolate was grown in submerged cultures under different conditions: carbon source, temperature, pH and incubation time to optimize antimicrobials production. The actinomycete grown in base medium supplemented with 1% glucose, pH 6.5 and incubation at 30 ºC for 96 h with shaking at 100 rpm, exhibited the highest activity against the used Gram-negative bacteria. Minimum inhibitory concentration (MIC) of the crude extract produced by the microorganism varied between 1/32 and 1/256. It had bactericide or bacteriostatic activity, depending on the Gram-negative organism. The active extract was stable at high temperatures, and unstable in medium containing proteolytic enzymes. Micromorphology of R18(6) was investigated by optical and scan microscopy, revealing that it was morphologically similar to the genusStreptomyces. PMID:26871499

  4. The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria

    PubMed Central

    Billings, Emily A.; Lee, Chang Sup; Owen, Katherine A.; D’Souza, Ryan S.; Ravichandran, Kodi S.; Casanova, James E.

    2016-01-01

    The detection of microbes and initiation of an innate immune response occur through pattern recognition receptors (PRRs), which are critical for the production of inflammatory cytokines and activation of the cellular microbicidal machinery. In particular, the production of reactive oxygen species (ROS) by the NADPH oxidase complex is a critical component of the macrophage bactericidal machinery. We previously characterized brain-specific angiogenesis inhibitor 1 (BAI1), a member of the adhesion family of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs), as a PRR that mediates the selective phagocytic uptake of Gram-negative bacteria by macrophages. We showed that BAI1 promoted phagosomal ROS production through activation of the Rho family guanosine triphosphatase (GTPase) Rac1, thereby stimulating NADPH oxidase activity. Primary BAI1-deficient macrophages exhibited attenuated Rac GTPase activity and reduced ROS production in response to several Gram-negative bacteria, resulting in impaired microbicidal activity. Furthermore, in a peritoneal infection model, BAI1-deficient mice exhibited increased susceptibility to death by bacterial challenge because of impaired bacterial clearance. Together, these findings suggest that BAI1 mediates the clearance of Gram-negative bacteria by stimulating both phagocytosis and NADPH oxidase activation, thereby coupling bacterial detection to the cellular microbicidal machinery. PMID:26838550

  5. 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

  6. 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

  7. 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

  8. 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

  9. Neither Single nor a Combination of Routine Laboratory Parameters can Discriminate between Gram-positive and Gram-negative Bacteremia

    PubMed Central

    Ratzinger, Franz; Dedeyan, Michel; Rammerstorfer, Matthias; Perkmann, Thomas; Burgmann, Heinz; Makristathis, Athanasios; Dorffner, Georg; Loetsch, Felix; Blacky, Alexander; Ramharter, Michael

    2015-01-01

    Adequate early empiric antibiotic therapy is pivotal for the outcome of patients with bloodstream infections. In clinical practice the use of surrogate laboratory parameters is frequently proposed to predict underlying bacterial pathogens; however there is no clear evidence for this assumption. In this study, we investigated the discriminatory capacity of predictive models consisting of routinely available laboratory parameters to predict the presence of Gram-positive or Gram-negative bacteremia. Major machine learning algorithms were screened for their capacity to maximize the area under the receiver operating characteristic curve (ROC-AUC) for discriminating between Gram-positive and Gram-negative cases. Data from 23,765 patients with clinically suspected bacteremia were screened and 1,180 bacteremic patients were included in the study. A relative predominance of Gram-negative bacteremia (54.0%), which was more pronounced in females (59.1%), was observed. The final model achieved 0.675 ROC-AUC resulting in 44.57% sensitivity and 79.75% specificity. Various parameters presented a significant difference between both genders. In gender-specific models, the discriminatory potency was slightly improved. The results of this study do not support the use of surrogate laboratory parameters for predicting classes of causative pathogens. In this patient cohort, gender-specific differences in various laboratory parameters were observed, indicating differences in the host response between genders. PMID:26522966

  10. 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. PMID:23827568

  11. [Utility of pyrrolidonyl-arylamidase detection for typing Enterobacteriaceae and non-fermenting Gram-negative bacteria].

    PubMed

    Nicola, F; Centorbi, H; Bantar, C; Smayevsky, J; Bianchini, H

    1995-01-01

    Detection of pyrrolidonyl-aryl-amidase activity (PYR) is an important tool to identify gram-positive cocci, such as staphylococci, enterococci, streptococci, and other related genera. However, only few studies evaluating its usefulness with gram-negative rods have been published. Thus, a prospective study including 542 and 215 unique clinical isolates of Enterobacteriaceae and non-fermentative gram-negative rods, respectively, was undertaken. Strains were identified by conventional methods. PYR test was performed using a commercial kit, according to the manufacturer recommendations. Positive results were uniformly obtained for the PYR test with the following species: Citrobacter spp, Klebsiella spp, Enterobacter aerogenes, Enterobacter agglomerans group, Serratia marcescens and S. odorifera. On the other hand, negative results were uniformly displayed by E. coli (including inactive E. coli), Protease group, Salmonellia spp, Shigella spp, Acinetobacter spp, Burkholderia (Pseudomonas) cepacia and Flavobacterium spp. Variable results were shown in Pseudomonas aeruginosa, Stenotrophomonas (xanthomonas) malthophilia, Kluyvera cryocrescens, and Enterobacter cloacae. PYR test proved to be a reliable and simple tool to rapidly distinguish certain species belonging to Enterobacteriaceae (ie. Citrobacter freundii from Salmonella spp, and inactive E. coli from K. ozaenae). Further studies, including a wide diversity of species, are required to assess usefulness of the PYR test for the identification of non-fermentative gram-negative rods. PMID:8850133

  12. Hydroxy fatty acids in fresh snow samples from northern Japan: long-range atmospheric transport of Gram-negative bacteria by Asian winter monsoon

    NASA Astrophysics Data System (ADS)

    Tyagi, P.; Yamamoto, S.; Kawamura, K.

    2015-08-01

    Hydroxy fatty acids (FAs) in fresh snow from Sapporo, one of the heaviest snowfall regions in the world, have been studied to ascertain the airborne bacterial endotoxin concentrations and their biomass. The presence of β-hydroxy FAs (C9-C28), constituents of Gram-negative bacteria (GNB), suggests long-range transport of soil microbes. Likewise, the occurrence of α- and ω-hydroxy FAs (C9-C30 and C9-C28, respectively) in snow reveals their contribution from epicuticular waxes and soil microorganisms. Estimated endotoxin and GNB mass can aid in assessing their possible impacts on the diversity and functioning of aquatic and terrestrial ecosystems, as well as lethal effects on pedestrians through dispersal of microbes. Air mass back trajectories together with hydroxy FAs unveil their sources from Siberia, Russian Far East and North China by the Asian monsoon. This study highlights the role of fresh snow that reduces the human health risk of GNB and endotoxin by scavenging from the air.

  13. Hydroxy fatty acids in fresh snow samples from northern Japan: long-range atmospheric transport of Gram-negative bacteria by Asian winter monsoon

    NASA Astrophysics Data System (ADS)

    Tyagi, P.; Yamamoto, S.; Kawamura, K.

    2015-12-01

    Hydroxy fatty acids (FAs) in fresh snow from Sapporo, one of the heaviest snowfall regions in the world, have been studied to ascertain the airborne bacterial endotoxin concentrations and their biomass. The presence of β-hydroxy FAs (C9-C28), constituents of the Gram-negative bacterium (GNB), suggests long-range transport of soil microbes. Likewise, the occurrence of α- and ω-hydroxy FAs (C9-C30 and C9-C28, respectively) in snow reveals their contribution from epicuticular waxes and soil microorganisms. Estimated endotoxin and GNB mass can aid in assessing their possible impacts on the diversity and functioning of aquatic and terrestrial ecosystems, as well as lethal effects on pedestrians through dispersal of microbes. Air mass back trajectories together with hydroxy FAs reveal their sources from Siberia, the Russian Far East and northern China by the Asian monsoon. This study highlights the role of fresh snow that reduces the human health risk of GNB and endotoxin by the scavenging from air.

  14. 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

  15. [Investigation of the presence of New Delhi metallo-beta-lactamase-1 (NDM-1) by PCR in carbapenem-resistant gram-negative isolates].

    PubMed

    Yanık, Keramettin; Emir, Dilek; Eroğlu, Cafer; Karadağ, Adil; Güney, Akif Koray; Günaydın, Murat

    2013-04-01

    Bacteria producing New Delhi metallo-beta-lactamase-1 (NDM-1) exhibit high level resistance to beta-lactams including carbapenems. This broad-spectrum resistance limits treatment options for infections caused by NDM-1 producers. NDM-1 was first isolated from an Indian patient in Sweden; since then, NDM-1 producing isolates have been identified in many countries including Turkey. In this study, we investigated the presence of NDM-1 by PCR method in various gram-negative isolates recovered from clinical specimens in tertiary care hospitals in Samsun, Turkey. A total of 210 carbapenem-resistant gram-negative isolates (132 Acinetobacter baumannii, 54 Pseudomonas aeruginosa, 5 Pseudomonas putida, 8 Enterobacter cloacae, 3 Enterobacter aerogenes, 3 Klebsiella pneumoniae, 2 Providencia rettgeri, 2 Escherichia coli and 1 Citrobacter freundii) were included in the study. Identification and antibiotic susceptibility testing of the isolates were performed by using Vitek-2 Compact (bioMerieux, France) and BD Phoenix (BD Diagnostic Systems, MD) automated systems. The results of antibiotic susceptibility testing were interpreted according to the CLSI recommendations. In our study, NDM-1 gene was not detected in any of the clinical isolates by PCR. There was only one case study that reported the presence of NDM-1 in clinical isolates from Turkey [Poirel L et al. Antimicrob Agents Chemother 2012;56:2784]. Our data, together with the others, indicated that the existence of NDM-1 in clinical isolates is not common in Turkey. However, since NDM-1 is a plasmid-encoded enzyme, there is always a risk of spread of this resistance through the bacterial strains in our country. Therefore, continuous surveillance and investigation of carbapenem-resistant isolates with resistance patterns suggestive of NDM-1 may enable to identify NDM-1 producing isolates. Meanwhile special care should be given on rational antibiotic use and establishment of appropriate infection control policies to prevent

  16. 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. PMID:26498989

  17. MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria.

    PubMed

    Magnus, Marcin; Pawlowski, Marcin; Bujnicki, Janusz M

    2012-12-01

    Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction. PMID:22705560

  18. Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound.

    PubMed

    Rapoport, N; Smirnov, A I; Pitt, W G; Timoshin, A A

    1999-02-15

    The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls. PMID:9989932

  19. 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

  20. Antimicrobial susceptibility and beta-lactamase production of selected gram-negative bacilli from two Croatian hospitals: MYSTIC study results.

    PubMed

    Bedenic, B; Goic-Barisic, I; Budimir, A; Tonkic, M; Mihajkevic, L J; Novak, A; Sviben, M; Plecko, V; Punda-Polic, V; Kalenic, S

    2010-06-01

    The meropenem yearly Susceptibility Test Information Collection (MYSTIC) programme is a global, longitudinal resistance surveillance network that monitors the activity of meropenem and compares its activity with other broadspectrum antimicrobial agents. We now report the antimicrobial efficacy of meropenem compared to other broad-spectrum agents within the selective Gram-negative pathogen groups from two Croatian Hospitals investigated between 2002-2007. A total of 1510 Gram-negative pathogens were tested and the minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI.There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in both medical centers. High resistance rates of K. pneumoniae to ceftazidime (18%), cefepime (17%) and gentamicin (39%) are raising concern. Acinetobacter baumannii turned out to be the most resistant Gram-negative bacteria with 81% resistant to ceftazidime, 73% to cefepime, 69% to gentamicin and 71% to ciprofloxacin. Almost 20% of Pseudomonas aeruginosa strains were resistant to imipenem, 13% to meropenem, 69% to gentamicin and 38% to ciprofloxacin.The prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli was 10% and in K. pneumoniae 49%. PCR and sequencing of the amplicons revealed the presence of SHV-5 in nine E. coli strains and additional tem-1 beta-lactamase five strains. Five K. pneumoniae strains were positive for bla(SHV-5 )gene. Eight ESBL positive Enterobacter spp. strains were found to produce tem and CtX-m beta-lactamases. Plasmid-mediated AmpC beta-lactamases were not found among K. pneumoniae, E. coli and Enterobacter spp. Three A. baumannii strains from Zagreb University Center were identified by multiplex PCR as OXA-58 like producers. Six A. baumannii strains from Split University Center were found to possess an ISAba1 insertion sequence upstream of bla(OXA-51 )gene. According to our results

  1. Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

    PubMed

    Stathopoulos, C; Hendrixson, D R; Thanassi, D G; Hultgren, S J; St Geme, J W; Curtiss, R

    2000-07-01

    Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence. PMID:10967286

  2. 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.

  3. Nosocomial Infections: Multicenter surveillance of antimicrobial resistance profile of Staphylococcus aureus and Gram negative rods isolated from blood and other sterile body fluids in Iran

    PubMed Central

    Poorabbas, Bahman; Mardaneh, Jalal; Rezaei, Zahra; Kalani, Mehdi; Pouladfar, Gholamreza; Alami, Mohammad Hasan; Soltani, Jafar; Shamsi-Zadeh, Ahmad; Abdoli-Oskooi, Shahram; Saffar, Mohammed Jafar; Alborzi, Abdolvahab

    2015-01-01

    Background and Objective: Antibiotic resistance is increasing, especially in healthcare-associated infections causing significant public health concerns worldwide. National information is required to make appropriate policies, update list of essential drugs for treatment, and evaluate the effects of intervention strategies. A nationwide surveillance of antimicrobial resistant bacteria in nosocomial infections was established in Iran in 2008, so that the data obtained through the surveillance would enable us to construct a database. Materials and Methods: Seven major teaching hospitals in Shiraz, Tabriz, Sari, Mashhad, Sanandaj, Ahwaz and Isfahan participated in this study. A total of 858 strains isolated from blood and other sterile body fluids were tested. Identification at the species level was performed with conventional biochemical methods and the API system. Susceptibility tests were done using disk diffusion method. The methicillin-resistance in S. aureus (MRSA) was determined by the oxacillin agar screen plate and respective MIC values were assessed using the E-test strips. The confirmatory disk diffusion methods were applied for phenotypic identification of extended-spectrum β- lactamase (ESBL) production for E. coli and K. pneumoniae, according to CLSI guidelines. Results: Cultivation and re-identification of the strains yielded 858 isolates, consisting of 224 S. aureus, 148 Klebsiella spp., 105 Serratia spp., 146 E. coli, 67 Acinetobacter spp., 38 Enterobacter spp., 95 Pseudomonas spp., 71 P.aeruginosa. 35 Stenotrophomonas sp., and 8 other organisms. MRSA was detected in 37.5% of the isolates. No vancomycin-resistant or vancomycin-intermediate resistant S. aureus was detected. With the exception of Acinetobacter and Stenotrophomonas, 85% of the Gram-negative isolates were found to be susceptible in vitro to imipenem. Overall, about 61% of K. pneumoniae and 35% of E. coli isolates were ESBL producing. Conclusion: Multidrug resistant isolates of Gram-negative

  4. Optimization of Routine Identification of Clinically Relevant Gram-Negative Bacteria by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and the Bruker Biotyper

    PubMed Central

    Ford, Bradley A.

    2013-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) might complement and one day replace phenotypic identification of bacteria in the clinical microbiology laboratory, but there is no consensus standard regarding the requirements for its validation prior to clinical use in the United States. The objective of this study was to assess the preanalytical variables influencing Gram-negative identification by use of the Bruker Biotyper MALDI-TOF MS system, including density of organism spotting on a stainless steel target plate and the direct overlay of organisms with formic acid. A heavy smear with formic acid overlay was either superior or equivalent to alternative smear conditions. Microbiological preanalytical variables were also assayed, such as culture medium, growth temperature, and use of serial subculture. Postanalytical analysis included the application of modified species-level identification acceptance criteria. Biotyper identifications were compared with those using traditional phenotypic methods, and discrepancies were resolved with 16S rRNA gene sequencing. Compared to the recommended score cutoffs of the manufacturer, the application of optimized Biotyper score cutoffs for species-level identification increased the rate of identification by 6.75% for the enteric Gram-negative bacteria and 4.25% for the nonfermenting Gram-negative bacteria. Various incubation temperatures, growth medium types, and repeat subcultures did not result in misidentification. We conclude that the Bruker MALDI Biotyper is a robust system for the identification of Gram-negative organisms in the clinical laboratory and that meaningful performance improvements can be made by implementing simple pre- and postanalytical techniques. PMID:23426923

  5. Association between infections caused by multidrug-resistant gram-negative bacteria and mortality in critically ill patients

    PubMed Central

    Paramythiotou, Elisabeth; Routsi, Christina

    2016-01-01

    The incidence of gram-negative multidrug-resistant (MDR) bacterial pathogens is increasing in hospitals and particularly in the intensive care unit (ICU) setting. The clinical consequences of infections caused by MDR pathogens remain controversial. The purpose of this review is to summarize the available data concerning the impact of these infections on mortality in ICU patients. Twenty-four studies, conducted exclusively in ICU patients, were identified through PubMed search over the years 2000-2015. Bloodstream infection was the only infection examined in eight studies, respiratory infections in four and variable infections in others. Comparative data on the appropriateness of empirical antibiotic treatment were provided by only seven studies. In ten studies the presence of antimicrobial resistance was not associated with increased mortality; on the contrary, in other studies a significant impact of antibiotic resistance on mortality was found, though, sometimes, mediated by inappropriate antimicrobial treatment. Therefore, a direct association between infections due to gram-negative MDR bacteria and mortality in ICU patients cannot be confirmed. Sample size, presence of multiple confounders and other methodological issues may influence the results. These data support the need for further studies to elucidate the real impact of infections caused by resistant bacteria in ICU patients. PMID:27152254

  6. Sensitive assay, based on hydroxy fatty acids from lipopolysaccharide lipid A, for Gram-negative bacteria in sediments.

    PubMed Central

    Parker, J H; Smith, G A; Fredrickson, H L; Vestal, J R; White, D C

    1982-01-01

    Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the lipid A of the lipopolysaccharide in sediments to provide an estimate of the gram-negative bacteria. The method was sensitive to picomolar amounts of hydroxy fatty acids. The recovery of lipopolysaccharide hydroxy fatty acids from organisms added to sediments was quantitative. The lipids were extracted from the sediments with single-phase chloroform-methanol extraction. The lipid-extraction residue was hydrolyzed in 1 N HCl, and the hydroxy fatty acids of the lipopolysaccharide were recovered in chloroform for analysis by gas-liquid chromatography. This method proved to be about fivefold more sensitive than the classical phenol-water or trichloroacetic acid methods when applied to marine sediments. By examination of the patterns of hydroxy fatty acids, it was also possible to help define the community structure of the sedimentary gram-negative bacteria. PMID:6817712

  7. Trichokonins from Trichoderma pseudokoningii SMF2 induce resistance against Gram-negative Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage.

    PubMed

    Li, Hai-Yun; Luo, Yan; Zhang, Xiu-Sheng; Shi, Wei-Ling; Gong, Zhi-Ting; Shi, Mei; Chen, Lei-Lei; Chen, Xiu-Lan; Zhang, Yu-Zhong; Song, Xiao-Yan

    2014-05-01

    Peptaibols, mainly produced by Trichoderma, play a pivotal role in controlling plant disease caused by fungi, virus, and Gram-positive bacteria. In the current study, we evaluated the control effect of Trichokonins, antimicrobial peptaibols from Trichoderma pseudokoningii SMF2, on soft rot disease of Chinese cabbage caused by a Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum and analyzed the mechanism involved. Trichokonins treatment (0.3 mg L(-1) ) enhanced the resistance of Chinese cabbage against Pcc infection. However, Trichokonins could hardly inhibit the growth of Pcc in vitro, even at high concentration (500 mg L(-1) ). Therefore, the direct effect of Trichokonins on Pcc may not the main reason why Trichokonins could control soft rot of Chinese cabbage. Trichokonin treatment led to an obvious increase in the production of reactive oxygen species hydrogen peroxide and superoxide radical, a significant enhance of the activities of pathogenesis-related enzymes catalase, polyphenoloxidase and peroxidase, and upregulation of the expression of salicylic acid - responsive pathogenesis-related protein gene acidic PR-1a in Chinese cabbage. These results indicate that Trichokonins induce resistance in Chinese cabbage against Pcc infection through the activation of salicylic acid signaling pathway, which imply the potential of Trichoderma and peptaibols in controlling plant disease caused by Gram-negative bacteria. PMID:24655217

  8. Ultrasound-induced inactivation of gram-negative and gram-positive bacteria in secondary treated municipal wastewater.

    PubMed

    Drakopoulou, S; Terzakis, S; Fountoulakis, M S; Mantzavinos, D; Manios, T

    2009-06-01

    The effect of 24kHz, high energy ultrasound in the presence and absence of titanium dioxide particles on the destruction of different bacteria groups was studied. Applying a total of 1500W/L for 60min (this corresponds to 5400kJ/L specific nominal energy), the mean destruction of gram-negative bacteria such as total coliforms, faecal coliforms and Pseudomonas spp. was 99.5%, 99.2% and 99.7%, respectively. More recalcitrant to sonolytic inactivation were the gram-positive bacteria Clostridium perfringens and faecal streptococci with a mean removal of 66% and 84%, respectively. The presence of 5g/L TiO(2) generally enhanced the destruction of gram-negative bacteria, yielding three to five logs reduction. On the other hand, the relatively weak sonochemical inactivation of gram-positive bacteria was only slightly affected by the presence of solid particles. Inactivation was found to follow first-order kinetics regarding bacteria population and was not affected significantly by the wastewater quality. Ultrasound irradiation at 4000kJ/L specific nominal energy and in the presence of 5g/L TiO(2) achieved less than 10(3) CFU/100mL total coliforms, thus meeting USEPA quality standards for wastewater reuse. PMID:19131265

  9. Performances of VITEK 2 Colorimetric Cards for Identification of Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    Wallet, Frédéric; Loïez, Caroline; Renaux, Emilie; Lemaitre, Nadine; Courcol, René J.

    2005-01-01

    Thepurpose of this study was to evaluate the new VITEK 2 identification cards that use colorimetric reading to identify gram-positive and gram-negative bacteria (GP and GN cards, respectively) in comparison to fluorimetric cards (ID-GPC and ID-GNB, respectively). A total of 580 clinical isolates and stock collection strains belonging to 116 taxa were included in the study. Of the 249 gram-positive strains tested with both the ID-GPC and GP cards, 218 (87.5%) and 235 (94.4%) strains were correctly identified (to the genus and species level), respectively. Of the 331 gram-negative strains tested with the ID-GNB and GN cards, 295 (89.1%) and 321 (97%) strains were correctly identified, respectively. Another focus of the study was to apply the percentages of correct identifications obtained in this study to the list of bacteria isolated in our laboratory (32,739 isolates) in the year 2004. We obtained 97.9% correct identifications with the colorimetric cards and 93.9% with fluorescent cards. PMID:16145083

  10. Therapy of acute and chronic gram-negative osteomyelitis with ciprofloxacin. Report from a Swedish Study Group.

    PubMed

    1988-08-01

    Oral ciprofloxacin was used at doses ranging from 500 mg to 1500 mg twice daily for 15 to 476 (mean 139) days for treatment of acute or chronic osteomyelitis in 38 patients, and acute arthritis in two. Clinical efficacy could be evaluated in 34 patients; 22 had resolution of their osteomyelitis, five improved and there were seven failures. Pseudomonas aeruginosa was the causative agent in 28 patients. It was eradicated in 22 patients, persisted but remained sensitive to ciprofloxacin in three and persisted with emergence of resistance to ciprofloxacin in three. Nineteen other pathogens, five Gram-negative and 14 Gram-positive, were isolated. Of those, one strain of Staphylococcus aureus, two of Staph. epidermidis and three of Streptococcus faecalis remained sensitive to ciprofloxacin during treatment. In one patient, Slr. faecalis persisted with emergence of resistance to ciprofloxacin. Ten adverse events related to ciprofloxacin treatment were observed in nine patients; two phototoxic reactions, two cases of impaired colour vision, and one each of exanthema, abdominal pain, malaise, drug fever, peripheral neuropathy and eosinophilia. In three patients the adverse events led to treatment discontinuation. In conclusion, ciprofloxacin seems to offer an oral alternative to injectible antibiotics in patients with osteomyelitis caused by Gram-negative bacteria, including Ps. aeruginosa. PMID:3053554

  11. Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways

    PubMed Central

    Rabehi, Lila; Irinopoulou, Théano; Cholley, Béatrice; Haeffner-Cavaillon, Nicole; Carreno, Marie-Paule

    2001-01-01

    Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1α (IL-1α), IL-1ra, and tumor necrosis factor alpha (TNF-α) production, whereas blocking of the ERK1/2 pathways inhibited IL-1α, IL-1β, and IL-1ra but not TNF-α production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1α production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production. PMID:11402003

  12. 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. PMID:24164211

  13. In Vitro Analysis of Activities of 16 Antimicrobial Agents against Gram-Negative Bacteria from Six Teaching Hospitals in China.

    PubMed

    Chen, Hongbin; Wang, Zhanwei; Li, Henan; Wang, Qi; Zhao, Chunjiang; He, Wenqiang; Wang, Xiaojuan; Zhang, Feifei; Wang, Hui

    2015-01-01

    To evaluate the in vitro antimicrobial activities of biapenem, arbekacin, and cefminox against different gram-negative bacterial isolates in China, a total of 100 non-duplicated Escherichia coli, 100 Acinetobacter baumannii, 100 Pseudomonas aeruginosa, and 99 Klebsiella pneumoniae isolates were collected from 6 teaching hospitals in China in 2012. The minimal inhibitory concentrations (MICs) of biapenem, arbekacin, cefminox and 13 other antibiotics were determined by the broth microdilution method. The carbapenems (biapenem, meropenem, and imipenem) exhibited high antimicrobial activity against E. coli (98%) and K. pneumoniae (≥95%), followed by colistin and amikacin. The MIC50 and MIC90 of biapenem against E. coli were ≤0.06 mg/L and 0.25 mg/L, respectively. For K. pneumoniae, the MIC50 and MIC90 of biapenem were 0.25 mg/L and 1.0 mg/L, respectively. The MIC50 and MIC90 of cefminox against E. coli were 1.0 mg/L and 4.0 mg/L, respectively. The resistance rates of A. baumannii to most of the antibiotics were more than 50%, except for colistin. Amikacin was the most active antibiotic against P. aeruginosa (97%), followed by colistin (93%). The MIC50 and MIC90 of arbekacin against P. aeruginosa were 2.0 mg/L and 8.0 mg/L, respectively. In conclusion, carbapenems, colistin, amikacin, and arbekacin exhibited high antimicrobial activities against gram-negative bacteria, except A. baumannii. PMID:25672407

  14. 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

  15. Antibacterial Activity of Stenotrophomonas maltophilia Endolysin P28 against both Gram-positive and Gram-negative Bacteria

    PubMed Central

    Dong, Hongling; Zhu, Chaoyang; Chen, Jingyi; Ye, Xing; Huang, Yu-Ping

    2015-01-01

    Maltocin P28 is a phage-tail like bacteriocin produced by Stenotrophomonas maltophilia P28. The ORF8 of maltocin P28 gene cluster is predicted to encode an endolysin and we name it endolysin P28. Sequence analysis revealed that it contains the lysozyme_like superfamily conserved domain. Endolysin P28 has the four consensus motifs as that of Escherichia coli phage lambda gpR. In this study, endolysin P28 was expressed in E. coli BL21 (DE3) and purified with a C-terminal oligo-histidine tag. The antibacterial activity of endolysin P28 increased as the temperature rose from 25 to 45°C. Thermostability assays showed that endolysin P28 was stable up to 50°C, while its residual activity was reduced by 55% after treatment at 70°C for 30 min. Acidity and high salinity could enhance its antibacterial activity. Endolysin P28 exhibited a broad antibacterial activity against 14 out of 16 tested Gram-positive and Gram-negative bacteria besides S. maltophilia. Moreover, it could effectively lyse intact Gram-negative bacteria in the absence of ethylenediaminetetraacetic acid as an outer membrane permeabilizer. Therefore, the characteristics of endolysin P28 make it a potential therapeutic agent against multi-drug-resistant pathogens. PMID:26635765

  16. Liposomal nanoformulations of rhodamine for targeted photodynamic inactivation of multidrug resistant gram negative bacteria in sewage treatment plant.

    PubMed

    Vimaladevi, Mohan; Divya, Kurunchi Chellapathi; Girigoswami, Agnishwar

    2016-09-01

    The antimicrobial photodynamic therapy is an alternative method for killing bacterial cells in view of the rising problem of antibiotic resistance microorganisms. The present study examined the effect of a water soluble photosensitizer, Rhodamine 6G (R6G) in stealth liposomes on multidrug resistant Pseudomonas aeruginosa in the presence of visible light. Liposomes were prepared with cholesterol and phospholipids that extracted from hen eggs in a cost effective way and characterized by light microscopy, particle size analyzer, electron microscopy, steady state spectrophotometry and spectrofluorometry. The photoefficacies of R6G in polymer encapsulated liposomes and positively charged liposomes are much higher compared to the free R6G (R6G in water) in terms of singlet oxygen quantum yield. This high potential of producing more reactive oxygen species (ROS) by liposomal nanoformulated R6G leads to efficient photodynamic inactivation of multidrug resistant gram negative bacteria in waste water. Though the singlet oxygen quantum yield of polymer coated liposomal R6G was higher than the cationic liposomal formulation, a faster decrease in bacterial survival was observed for positively charged liposomal R6G treated bacteria due to electrostatic charge interactions. Therefore, it can be concluded that the positively charged liposomal nanoformulations of laser dyes are efficient for photodynamic inactivation of multiple drug resistant gram negative microorganisms. PMID:27371913

  17. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    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

  18. 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

  19. Sensitive EDTA-Based Microbiological Assays for Detection of Metallo-β-Lactamases in Nonfermentative Gram-Negative Bacteria

    PubMed Central

    Marchiaro, Patricia; Mussi, María A.; Ballerini, Viviana; Pasteran, Fernando; Viale, Alejandro M.; Vila, Alejandro J.; Limansky, Adriana S.

    2005-01-01

    The worldwide spread of metallo-β-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different β-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM. PMID:16272499

  20. Oral Gram-negative anaerobic bacilli as a reservoir of β-lactam resistance genes facilitating infections with multiresistant bacteria.

    PubMed

    Dupin, Clarisse; Tamanai-Shacoori, Zohreh; Ehrmann, Elodie; Dupont, Anais; Barloy-Hubler, Frédérique; Bousarghin, Latifa; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

    2015-02-01

    Many β-lactamases have been described in various Gram-negative bacilli (Capnocytophaga, Prevotella, Fusobacterium, etc.) of the oral cavity, belonging to class A of the Ambler classification (CepA, CblA, CfxA, CSP-1 and TEM), class B (CfiA) or class D in Fusobacterium nucleatum (FUS-1). The minimum inhibitory concentrations of β-lactams are variable and this variation is often related to the presence of plasmids or other mobile genetic elements (MGEs) that modulate the expression of resistance genes. DNA persistence and bacterial promiscuity in oral biofilms also contribute to genetic transformation and conjugation in this particular microcosm. Overexpression of efflux pumps is facilitated because the encoding genes are located on MGEs, in some multidrug-resistant clinical isolates, similar to conjugative transposons harbouring genes encoding β-lactamases. All these facts lead us to consider the oral cavity as an important reservoir of β-lactam resistance genes and a privileged place for genetic exchange, especially in commensal strictly anaerobic Gram-negative bacilli. PMID:25465519

  1. Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

    PubMed Central

    Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2012-01-01

    Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

  2. Late-acting dominant lethal genetic systems and mosquito control

    PubMed Central

    Phuc, Hoang Kim; Andreasen, Morten H; Burton, Rosemary S; Vass, Céline; Epton, Matthew J; Pape, Gavin; Fu, Guoliang; Condon, Kirsty C; Scaife, Sarah; Donnelly, Christl A; Coleman, Paul G; White-Cooper, Helen; Alphey, Luke

    2007-01-01

    Background Reduction or elimination of vector populations will tend to reduce or eliminate transmission of vector-borne diseases. One potential method for environmentally-friendly, species-specific population control is the Sterile Insect Technique (SIT). SIT has not been widely used against insect disease vectors such as mosquitoes, in part because of various practical difficulties in rearing, sterilization and distribution. Additionally, vector populations with strong density-dependent effects will tend to be resistant to SIT-based control as the population-reducing effect of induced sterility will tend to be offset by reduced density-dependent mortality. Results We investigated by mathematical modeling the effect of manipulating the stage of development at which death occurs (lethal phase) in an SIT program against a density-dependence-limited insect population. We found late-acting lethality to be considerably more effective than early-acting lethality. No such strains of a vector insect have been described, so as a proof-of-principle we constructed a strain of the principal vector of the dengue and yellow fever viruses, Aedes (Stegomyia) aegypti, with the necessary properties of dominant, repressible, highly penetrant, late-acting lethality. Conclusion Conventional SIT induces early-acting (embryonic) lethality, but genetic methods potentially allow the lethal phase to be tailored to the program. For insects with strong density-dependence, we show that lethality after the density-dependent phase would be a considerable improvement over conventional methods. For density-dependent parameters estimated from field data for Aedes aegypti, the critical release ratio for population elimination is modeled to be 27% to 540% greater for early-acting rather than late-acting lethality. Our success in developing a mosquito strain with the key features that the modeling indicated were desirable demonstrates the feasibility of this approach for improved SIT for disease

  3. Ventriculo-peritoneal shunt independence following successful treatment of Gram negative (E. coli) ventriculitis: Case report and review of the literature.

    PubMed

    Hussain, Rahim A; Sainuddin, Sajid; Bhatti, I; Leach, P

    2016-08-01

    We report a case of tumour-related hydrocephalus in a child treated with a ventriculo-peritoneal shunt which subsequently became infected with gram negative bacteria (Escherichia coli). After successful treatment of the infection the patient became shunt independent and has remained so for over 2 years. Gram negative ventriculitis is associated with diminished cerebro-spinal fluid production and we discuss the literature to date regarding this phenomenon. PMID:26449688

  4. Gram-negative Biomass in Clay Minerals Analogs: Testing Habitability Potential for the 2011 Mars Science Laboratory Mission

    NASA Astrophysics Data System (ADS)

    Bonaccorsi, R.; McKay, C. P.

    2009-12-01

    Landing sites of next missions to Mars i.e., the US 2011 Mars Science Laboratory (MSL11) and the ESA2016 Pasteur ExoMars, will include phyllosilicate outcrops as targets for investigating the geological and biological history of that planet. In this context, we present a study assessing the living biomass and habitability potential in mineralogical Mars analogs such as phyllosilicates and hematite-rich deposits encompassing a broad arid-hyper-arid climate range (annual rainfall <0.2 to ~700mm/y). Samples from the Atacama Desert (Chile), the Death Valley (CA), and the California Coast (USA) were analyzed for microbial lipopolysaccharide (LPS) as proxy for Gram-negatives biomass with the Limulus-Amebocite-Lysate (LAL) assay. Mineral phases were identified using X-Ray-Diffraction (XRD). These samples resulted to contain phyllosilicate phases similar to those identified, or inferred [1], on the surface of Mars by the OMEGA-Mars/Express [e.g., 2], the Mars Reconnaissance Orbiter (MRO) instruments (HiRISE and CRISM) [3]. Basic observations were: 1) there is no systematic pattern in biomass content of clays vs. non-clays (oxidized) materials from the study sites; 2) Atacama desiccation polygons (muscovite and kaolinite) and contiguous hematite-rich hyper-arid deposits contain the lowest biomass, i.e., ~104to-105 cells/g, respectively; 3) the hyper-arid clays contain three-order magnitude lower Gram-negative biomass than those (montmorillonite, illite, and chlorite) from the arid Death Valley site (~107cells/g); and 4) finally, the Gram-negative (~107cells/g) of clay minerals-rich materials from the arid site is about the same than that (~1.5 to ~3.0 x 107cells/g) of water-saturated massive deposits (kaolinite, illite, and vermiculite) from the wetter California coast. Results from this investigation will help testing for the habitability potential of phyllosilicate deposits sampled by the MSL11 Mission. REFERENCES:[1] Bibring et al., 2006, Science 312:400-404; [2] Wang et

  5. 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

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

    PubMed Central

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

    2010-01-01

    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. 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 chronic and burn wounds. PMID:20939612

  7. Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.

    PubMed

    Castro, A; Bemer, V; Nóbrega, A; Coutinho, A; Truffa-Bachi, P

    1998-02-01

    Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections. PMID:9521057

  8. Contamination of an operating theatre by Gram-negative bacteria. Examination of water supplies, cleaning methods and wound infections

    PubMed Central

    Thomas, Mair E. M.; Piper, Elizabeth; Maurer, Isobel M.

    1972-01-01

    This paper describes a search for Gram-negative bacteria in an operating theatre and the steps taken to reduce the level of environmental contamination. A high rate of infection in clean wounds prompted a bacteriological survey. Potential sources of infection found, and the measures employed are described in the hope that others may be encouraged to examine familiar equipment critically and to improve hygiene even in old premises. The choice, design, use and care of cleaning and sterilizing equipment were open to criticism. In particular, a currently popular floor-scrubbing machine provided a breeding ground for Pseudomonas aeruginosa and was distributing it in the theatre environment. ImagesPlate 1 PMID:4622481

  9. 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

  10. Development of a multiplex polymerase chain reaction to detect five common Gram-negative bacteria of aquatic animals.

    PubMed

    Tsai, M-A; Ho, P-Y; Wang, P-C; E, Y-J; Liaw, L-L; Chen, S-C

    2012-07-01

    A multiplex polymerase chain reaction (m-PCR) technique was developed as a rapid and accurate diagnostic tool for identifying five major Gram-negative bacilli -Vibrio vulnificus, V. parahaemolyticus, Aeromonas hydrophila, Chryseobacterium meningosepticum and Edwardsiella tarda- that cause major diseases in cultured aquatic animals in Taiwan. The expected amplicons for V. vulnificus, V. parahaemolyticus, A. hydrophila, C. meningosepticum and E. tarda were 410, 368, 685, 180 and 230bp, respectively. The assay was shown to be specific for the target pathogens. The sensitivities of detection were estimated to be 20.5fg∼200pg of genomic DNA or 10(2) ∼10(4) colony-forming units (cfu) of bacterial isolates when adopted as PCR templates. The m-PCR was capable of simultaneously amplifying target fragments from bacterial genome DNA mixed with the DNA extracted from viscera and tissues taken from fish without affecting the performance of the method. PMID:22571515

  11. Tackling antibiotic resistance in febrile neutropenia: current challenges with and recommendations for managing infections with resistant Gram-negative organisms.

    PubMed

    Nouér, Simone A; Nucci, Marcio; Anaissie, Elias

    2015-10-01

    Multidrug resistant (MDR) Gram-negative bacteria (GNB) have emerged as important pathogens and a serious challenge in the management of neutropenic patients worldwide. The great majority of infections are caused by the Enterobacteriaceae (especially Escherichia coli and Klebsiella spp.) and Pseudomonas aeruginosa, and less frequently Acinetobacter spp. and Stenotrophomonas maltophilia. A broader-spectrum empiric antibiotic regimen is usually recommended in patients with a history of prior bloodstream infection caused by a MDR GNB, in those colonized by a MDR GNB, and if MDR GNBs are frequently isolated in the initial blood cultures. In any situation, de-escalation to standard empiric regimen is advised if infection with MDR GNB is not documented. PMID:26115679

  12. Paraoxonases as Potential Antibiofilm Agents: Their Relationship with Quorum-Sensing Signals in Gram-Negative Bacteria ▿

    PubMed Central

    Camps, Jordi; Pujol, Isabel; Ballester, Frederic; Joven, Jorge; Simó, Josep M.

    2011-01-01

    The property of many bacteria to form biofilms constitutes a major health problem. Bacteria living in biofilms have a very high resistance to antibiotics. Biofilms may develop at a certain locations with the participation of secreted molecules, termed quorum-sensing signals, when a sufficient density of bacterial growth occurs. In Gram-negative bacteria, acyl homoserine lactones (AHL) have been identified as major quorum-sensing signals. The paraoxonases (PONs) constitute a family of enzymes comprising 3 members (PON1, PON2, and PON3) that have lactonase activity and are able to hydrolyze AHL. In this minireview, we summarize some existing basic knowledge on PON genetics, biochemistry, and function and describe recent research that reports evidence of the important roles that they may play in the organism's defense against biofilm formation. Finally, we propose some lines of future research that could be very productive. PMID:21199929

  13. Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity

    PubMed Central

    Otręebska-Machaj, Ewa; Chevalier, Jacqueline; Handzlik, Jadwiga; Szymańska, Ewa; Schabikowski, Jakub; Boyer, Gérard; Bolla, Jean-Michel; Kieć-Kononowicz, Katarzyna; Pagès, Jean-Marie; Alibert, Sandrine

    2016-01-01

    Multidrug resistant (MDR) bacteria are an increasing health problem with the shortage of new active antibiotic agents. Among effective mechanisms that contribute to the spread of MDR Gram-negative bacteria are drug efflux pumps that expel clinically important antibiotic classes out of the cell. Drug pumps are attractive targets to restore the susceptibility toward the expelled antibiotics by impairing their efflux activity. Arylhydantoin derivatives were investigated for their potentiation of activities of selected antibiotics described as efflux substrates in Enterobacter aerogenes expressing or not AcrAB pump. Several compounds increased the bacterial susceptibility toward nalidixic acid, chloramphenicol and sparfloxacin and were further pharmacomodulated to obtain a better activity against the AcrAB producing bacteria. PMID:27199950

  14. Candidatus Renichlamydia lutjani, a Gram-negative bacterium in internal organs of blue striped snapper Lutjanus kasmira from Hawaii.

    USGS Publications Warehouse

    Corsaro, Daniele; Work, Thierry M.

    2012-01-01

    The blue-striped snapper Lutjanus kasmira (Perciformes, Lutjanidae) are cosmopolitan in the Indo-Pacific but were introduced into Oahu, Hawaii, USA, in the 1950s and have since colonized most of the archipelago. Studies of microparasites in blue-striped snappers from Hawaii revealed chlamydia-like organisms (CLO) infecting the spleen and kidney, characterized by intracellular basophilic granular inclusions containing Gram-negative and Gimenez-positive bacteria similar in appearance to epitheliocysts when seen under light microscopy. We provide molecular evidence that CLO are a new member of Chlamydiae, i.e. Candidatus Renichlamydia lutjani, that represents the first reported case of chlamydial infection in organs other than the gill in fishes.

  15. Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Zheng, Amy; Panja, Subrata; Woodson, Sarah A

    2016-06-01

    The Sm-protein Hfq facilitates interactions between small non-coding RNA (sRNA) and target mRNAs. In enteric Gram-negative bacteria, Hfq is required for sRNA regulation, and hfq deletion results in stress intolerance and reduced virulence. By contrast, the role of Hfq in Gram-positive is less established and varies among species. The RNA binding and RNA annealing activity of Hfq from Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, and Staphylococcus aureus were compared using minimal RNAs and fluorescence spectroscopy. The results show that RNA annealing activity increases with the number of arginines in a semi-conserved patch on the rim of the Hfq hexamer and correlates with the previously reported requirement for Hfq in sRNA regulation. Thus, the amino acid sequence of the arginine patch can predict the chaperone function of Hfq in sRNA regulation in different organisms. PMID:27049793

  16. Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

    PubMed Central

    2014-01-01

    Background The continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps. Methods The qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method. Results All the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics. Conclusion These results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria

  17. Gram-negative and gram-positive antibacterial properties of the whole plant extract of willow herb (Epilobium angustifolium).

    PubMed

    Bartfay, Wally J; Bartfay, Emma; Johnson, Julia Green

    2012-01-01

    The emergence of new pathogens and the increase in the number of multidrug-resistant strains in well-established pathogens during the past decade represent a growing public health concern globally. With the current lack of research and development of new antibiotics by large pharmaceutical companies due to poor financial returns, new alternatives need to be explored including natural herbal or plant-based extracts with reported antibacterial properties. Willow herb (Epilobium angustifolium) preparations have been used in traditional aboriginal and folk medicine preparations externally as an antiphlogistic to treat prostate and gastrointestinal disorders and as an antiseptic to treat infected wounds. The authors hypothesized that a whole plant extract of willow herb would exhibit antimicrobial properties on a variety of both Gram-positive and gram-negative bacteria in culture. The authors found that, in comparison to growth controls, willow herb extract significantly inhibited the growth of Micrococcus luteus (p < .01), Staphylococcus aureus (p < .05), Escherichia coli (p < .001), and Pseudomonas aeruginosa (p < .001). They also found that willow herb extract inhibited the growth of bacteria in culture more effectively than vancomycin (p < .05) or tetracycline (p < .004). These results provide preliminary support for the traditional folkloric claim that the plant willow herb possesses antibacterial properties against a variety of gram-positive and gram-negative bacteria. Given that whole plant extract was utilized for this study, further investigations are warranted to determine which specific part of the plant (i.e., leaves, stem, roots, and flowers) possess the antibacterial properties. PMID:21208973

  18. In vitro antimicrobial activity of "last-resort" antibiotics against unusual nonfermenting Gram-negative bacilli clinical isolates.

    PubMed

    Jacquier, Herve; Le Monnier, Alban; Carbonnelle, Etienne; Corvec, Stephane; Illiaquer, Marina; Bille, Emmanuelle; Zahar, Jean-Ralph; Jauréguy, Françoise; Fihman, Vincent; Tankovic, Jacques; Cattoir, Vincent

    2012-08-01

    In this prospective multicentric study, we assessed the in vitro antimicrobial activity of carbapenems (imipenem, meropenem, and doripenem), tigecycline, and colistin against 166 unusual nonfermenting Gram-negative bacilli (NF-GNB) clinical isolates collected from nine French hospitals during a 6-month period (from December 1, 2008, to May 31, 2009). All NF-GNB isolates were included, except those phenotypically identified as Pseudomonas aeruginosa or Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of antimicrobial agents were determined by using the E-test technique. The following microorganisms were identified: Stenotrophomonas maltophilia (n=72), Pseudomonas spp. (n=30), Achromobacter xylosoxidans (n=25), Acinetobacter spp. (n=18), Burkholderia cepacia complex (n=9), Alcaligenes faecalis (n=7), and Delftia spp. (n=5). All isolates of Acinetobacter spp., A. faecalis, and Delftia spp. were susceptible to the three carbapenems. Imipenem exhibited the lowest MICs against Pseudomonas spp., and meropenem, as compared with imipenem and doripenem, displayed an interesting antimicrobial activity against A. xylosoxidans and B. cepacia complex isolates. Conversely, no carbapenem exhibited any activity against S. maltophilia. Except for S. maltophilia isolates, tigecycline and colistin exhibited higher MICs than carbapenems, but covered most of the microorganisms tested in this study. To our knowledge, no prior study has compared antimicrobial activity of these five antibiotics, often considered as "last-resort" treatment options for resistant Gram-negative infections, against unusual NF-GNB clinical isolates. Further studies should be carried out to assess the potential clinical use of these antibiotics for the treatment of infections due to these microorganisms. PMID:22335615

  19. French regional surveillance program of carbapenemase-producing Gram-negative bacilli: results from a 2-year period.

    PubMed

    Pantel, A; Boutet-Dubois, A; Jean-Pierre, H; Marchandin, H; Sotto, A; Lavigne, J-P

    2014-12-01

    In February 2011, the CARB-LR group was created as a sentinel laboratory-based surveillance network to control the emergence of carbapenem-resistant Gram-negative bacilli (CR GNB) in a French Southern Region. We report the epidemiological results of a 2-year study. All the Gram-negative bacilli isolates detected in the different labs (hospital and community settings) of a French Southern Region and with reduced susceptibility to ertapenem and/or imipenem were characterised with regard to antibiotic resistance, bla genes content, repetitive sequence-based polymerase chain reaction (rep-PCR) profiles and multilocus sequence typing (MLST). A total of 221 strains were analysed. Acinetobacter baumannii was the most prevalent carbapenemase-producing bacteria, with a majority of OXA-23 producers (n = 37). One isolate co-produced OXA-23 and OXA-58 enzymes. Klebsiella pneumoniae was the most frequent carbapenemase-producing Enterobacteriaceae (CPE) (OXA-48 producer: n = 29, KPC producer: n = 1), followed by Escherichia coli (OXA-48 producer: n = 8, KPC producer: n = 1) and Enterobacter cloacae (OXA-48 producer, n = 1). One isolate of Pseudomonas aeruginosa produced a VIM-1 carbapenemase. A clonal diversity of carbapenemase-producing K. pneumoniae and E. coli was noted with different MLSTs. On the other hand, almost all OXA-23-producing A. baumannii strains belonged to the widespread ST2/international clone II. The link between the detection of CR GNB and a foreign country was less obvious, suggesting the beginning of a local cross-transmission. The number of CR GNB cases in our French Southern Region has sharply increased very recently due to the diffusion of OXA-48 producers. PMID:25037867

  20. ['In vitro' activity of different antimicrobial agents on Gram-negative nonfermentative bacilli, excluding Pseudomonas aeruginosa and Acinetobacter spp].

    PubMed

    Vay, C A; Almuzara, M N; Rodríguez, C H; Pugliese, M L; Lorenzo Barba, F; Mattera, J C; Famiglietti, A M R

    2005-01-01

    Gram-negative nonfermentative bacilli (NFB) are widely spread in the environment. Besides of difficulties for identification, they often have a marked multiresistance to antimicrobial agents, including those active against Pseudomonas aeruginosa. The objective of this study was to evaluate the 'in vitro' activity of different antimicrobial agents on 177 gram-negative nonfermentative bacilli isolates (excluding Pseudomonas aeruginosa and Acinetobacter spp.) isolated from clinical specimens. Minimum inhibitory concentrations (MIC) were determined according to the Mueller Hinton agar dilution method against the following antibacterial agents: ampicillin, piperacillin, piperacillin-tazobactam, sulbactam, cefoperazone, cefoperazone-sulbactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, colistin, gentamicin, amikacin, trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, rifampin, norfloxacin, ciprofloxacin and minocycline. Seven isolates: Sphingobacterium multivorum (2), Sphingobacteriumspiritivorum (1), Empedobacterbrevis (1), Weeksella virosa (1), Bergeyella zoohelcum (1) and Oligella urethralis (1), were tested for amoxicillin-clavulanic acid and ampicillin-sulbactam susceptibility, and susceptibility to cefoperazone or sulbactam was not determined. Multiresistance was generally found in Stenotrophomonas maltophilia, Burkholderia cepacia, Chryseobacterium spp., Myroides spp., Achromobacter xylosoxidans, and Ochrobactrum anthropi isolates. On the other hand, Pseudomonas stutzeri, Shewanella putrefaciens-algae, Sphingomonas paucimobilis, and Pseudomonas oryzihabitans, Bergeyella zoohelcum, Weeksella virosa and Oligella urethralis were widely susceptible to the antibacterial agents tested. As a result of the wide variation in antimicrobial susceptibility shown by different species, a test on susceptibility to different antibacterial agents is essential in order to select an adequate therapy. The marked multiresistance evidenced by some species

  1. Hydrogen Peroxide Linked to Lysine Oxidase Activity Facilitates Biofilm Differentiation and Dispersal in Several Gram-Negative Bacteria▿

    PubMed Central

    Mai-Prochnow, Anne; Lucas-Elio, Patricia; Egan, Suhelen; Thomas, Torsten; Webb, Jeremy S.; Sanchez-Amat, Antonio; Kjelleberg, Staffan

    2008-01-01

    The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria. PMID:18502869

  2. Antibacterial activities of Beilschmiedia obscura and six other Cameroonian medicinal plants against multi-drug resistant Gram-negative phenotypes

    PubMed Central

    2014-01-01

    Background The rapid spread of bacteria expressing multi-drug resistance propels the search for new antibacterial agents. The present study was designed to evaluate the antibacterial activities of the methanol extracts from Beilschmiedia obscura and six other Cameroonian plants against a panel of twenty nine Gram-negative bacteria including Multi-drug resistant (MDR) phenotypes. Methods The phytochemical investigations of the extracts were carried out according to the standard methods and the liquid micro-dilution assay was used for all antibacterial assays. Results Phytochemical analysis showed the presence of alkaloids in all studied extracts. Other chemical classes of secondary metabolites such as anthocyanines, anthraquinones flavonoids, saponins, tannins, sterols and triterpenes were selectively detected in the extracts. The extract from the fruits of Beilschmiedia obscura, Pachypodanthium staudtii leaves and Peperomia fernandopoiana (whole plant) displayed the best spectrum of activity with MIC values ranging from 16 to 1024 μg/mL against at least 65% and above of the tested bacteria. The extract from Beilschmiedia obscura was the most active with MIC values below 100 μg/mL against ten of the tested bacteria. This extract also showed MBC values below 1024 μg/mL against 55.17% of the studied microorganisms. Phenylalanine arginine β-naphthylamide (PAβN) significantly modulated the activities of extracts from the leaves and fruits of Pachypodanthium staudtii and Beilschmiedia obscura respectively, by increasing their inhibitory activity against Klebsiella pneumoniae KP55 strain at least four fold. Conclusion The overall results of the present investigation provide information for the possible use of the methanol extracts of the studied plant species, especially B. obscura to fight infectious diseases caused by Gram-negative bacteria including MDR phenotypes. PMID:25023038

  3. The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods

    PubMed Central

    Laudy, Agnieszka E.; Mrowka, Agnieszka; Krajewska, Joanna; Tyski, Stefan

    2016-01-01

    Background Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. Methodology The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAβN (Phe-Arg-β-naphtylamide) were measured. Results The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAβN, were significantly (≥4-fold) reduced, decreasing to 25–1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAβN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains’ susceptibility to antibiotics. Conclusions The effects of PAβN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains. PMID:26771525

  4. Fatty acid and hydroxy acid adaptation in three gram-negative hydrocarbon-degrading bacteria in relation to carbon source.

    PubMed

    Soltani, Mohamed; Metzger, Pierre; Largeau, Claude

    2005-12-01

    The lipids of three gram-negative bacteria, Acinetobacter calcoaceticus, Marinobacter aquaeolei, and Pseudomonas oleovorans grown on mineral media supplemented with ammonium acetate or hydrocarbons, were isolated, purified, and their structures determined. Three pools of lipids were isolated according to a sequential procedure: unbound lipids extracted with organic solvents, comprising metabolic lipids and the main part of membrane lipids, OH--labile lipids (mainly ester-bound in the lipopolysaccharides, LPS) and H+-labile lipids (mainly amide-bound in the LPS). Unsaturated FA composition gave evidence for an aerobic desaturation pathway for the synthesis of these acids in A. calcoaceticus and M. aquaeolei, a nonclassic route in gram-negative bacteria. Surprisingly, both aerobic and anaerobic pathways are operating in the studied strain of P. oleovorans. The increase of the proportion of saturated FA observed for the strain of P. oleovorans grown on light hydrocarbons would increase the temperature transition of the lipids for maintaining the inner membrane fluidity. An opposite phenomenon occurs in A. calcoaceticus and M. aquaeolei grown on solid or highly viscous C19 hydrocarbons. The increases of FA < C18 when the bacteria were grown on n-nonadecane, or of iso-FA in cultures on isononadecane would decrease the transition temperature of the lipids, to maintain the fluidity of the inner membranes. Moreover, P. oleovorans grown on hydrocarbons greatly decreases the proportion of P-hydroxy acids of LPS, thus likely maintaining the physical properties of the outer membrane. By contrast, no dramatic change in hydroxy acid composition occurred in the other two bacteria. PMID:16477811

  5. [Yearly changes in antibacterial activities of cefozopran against various clinical isolates between 1996 and 2001--II. Gram-negative bacteria].

    PubMed

    Suzuki, Yumiko; Nishinari, Chisato; Endo, Harumi; Hiramatsu, Nobuyoshi; Akiyama, Kazumitsu; Koyama, Tsuneo

    2003-08-01

    The in vitro antibacterial activities of cefozopran (CZOP), an agent of cephems, against various clinical isolates obtained between 1996 and 2001 were yearly evaluated and compared with those of other cephems, oxacephems and carbapenems. A total of 3,245 strains in 32 species of Gram-negative bacteria were isolated from the clinical materials annually collected from January to December, and consisted of Moraxella subgenus Branhamella catarrhalis, Escherichia coli, Citrobacter freundii, Citrobacter koseri, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus mirabillis, Proteus vulgaris, Morganella morganii, Providencia spp. (P. alcalifaciens, P. rettgeri, P. stuartii), Pseudomonas aeruginosa, Pseudomonas putida, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, Acinetobactor baumannii, Acinetobactor lwoffii, Bacteroides fragilis group (B. fragilis, B. vulgatus, B. distasonis, B. ovatus, B. thetaiotaomicron), and Prevotella spp. (P. melaninogenica, P. intermedia, P. bivia, P. oralis, P. denticola). CZOP possessed stable antibacterial activities against M. (B.) catarrhalis, E. coli, C. freundii, C. koseri, K. pneumoniae, K. oxytoca, E. aerogenes, E. cloacae, S. marcescens, P. mirabilis, P. vulgaris, M. morganii, Providencia spp., P. aeruginosa, and A. lwoffii throughout 6 years. The MIC90 of CZOP against those strains were consistent with those obtained from the studies performed until the new drug application approval. On the other hand, the MIC90 of CZOP against H. influenzae yearly obviously increased with approximately 64-time difference during the study period. The MIC90 of cefpirome, cefepime, and flomoxef against H. influenzae also yearly tended to rise. The present results demonstrated that CZOP had maintained the antibacterial activity against almost Gram-negative strains tested. However, the decrease in antibacterial activities of CZOP against B. cepacia, and H

  6. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability.

    PubMed

    Wu, Huixing; Kuzmenko, Alexander; Wan, Sijue; Schaffer, Lyndsay; Weiss, Alison; Fisher, James H; Kim, Kwang Sik; McCormack, Francis X

    2003-05-01

    The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A-null mice and was increased in SP-D-overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D-mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane. PMID:12750409

  7. In vitro activity of tigecycline and comparators against gram-negative bacteria isolated from a tertiary hospital in Alexandria, Egypt.

    PubMed

    Mohamed, Nelly M; Youssef, Alaa A F

    2011-12-01

    The emergence of infections caused by multidrug-resistant Gram-negative bacteria, in particular Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, has necessitated the search for alternative therapy by either introducing new agents or renewing interest in old agents. This study compares the in vitro activity of tigecycline (TIG), recently introduced to Egyptian market, to other potentially active antimicrobials as Colistin (COL), imipenem (IPM), levofloxacin (LEV), and piperacillin/tazobactam (PIP/TAZ) against 67 Gram-negative clinical isolates obtained from El- Meery Hospital in Alexandria, Egypt. El-Meery Hospital is a 1,500-bed tertiary teaching hospital where TIG has not been previously used. Based on MIC(90)s, TIG was found to be a comparator to IPM and COL (MIC(90)= 8 μg/ml). LEV and PIP/TAZ were less active than TIG exhibiting high MIC(90)s. TIG inhibited 100% of Escherichia coli and K. pneumoniae and 60% of Ps. aeruginosa and A. baumannii isolates. In time-kill studies against IPM-resistant isolates, TIG showed bactericidal activity after 6 hours of contact against the Enterobacteriaceae isolates and after 3 hours for the tested Ps. aeruginosa isolates at 4× and 8× MIC. Against A. baumannii, TIG exerted a bacteriostatic effect. TIG demonstrated variable ability to suppress biofilm formation affecting mainly E. coli and A. baumannii isolates. These results point TIG to be a promising agent in treatment of infections caused by strains for which adequate therapy has been limited. As far as we know, this is the first report evaluating the in vitro activity of TIG against Egyptian clinical isolates. PMID:21875338

  8. Predominance of multi-resistant gram-negative bacteria colonizing chronic lower limb ulcers (CLLUs) at Bugando Medical Center

    PubMed Central

    2014-01-01

    Background Infections, trauma, malignances and poorly controlled diabetes are common causes of chronic lower limb ulcerations in developing countries. Infected wound with multi-drug resistant bacteria usually are associated with increased morbidity and mortality. We report the distribution of bacteria pathogens colonizing the chronic lower limb ulcers and their drug susceptibility pattern from Bugando Medical Centre (BMC) a tertiary hospital in Tanzania. Findings Three hundred non-repetitive wound swabs were aseptically collected from 300 patients with chronic lower limb ulcers using sterile swabs and processed following standard operative procedures. Isolates were identified using in house biochemical testing and in case of ambiguous confirmation was done using API 20NE and API 20E. Susceptibility was determined using disc diffusion test following clinical laboratory standard Institute guidelines (CLSI). Of 300 swabs from patients with chronic lower limbs ulcers, 201 (67.7%) had positive aerobic culture within 48 hours of incubation. Of 201 isolates, 180(89.6%) were gram-negative bacteria. Out of 180 gram negative bacteria, resistance was detected for ampicillin (95%, n = 171), amoxicillin/clavulanate (83.9%, n = 151), trimethoprim-sulphamethoxazole (78.9%, n = 142), ceftriaxone (46.7%, n = 84), ceftazidime (45.6%, n = 82), gentamicin (39.4%, n = 71), ciprofloxacin (17.8%, n = 32) and meropenem 28(15.6%, n = 25). A total of 41 (35%) of enterobacteriaceae were found to be extended spectrum beta-lactamases (ESBL) producers while of 18 Staphylococcus aureus, 8(44.4%) were found to be methicillin resistant Staphylococcus aureus (MRSA). Conclusion There is high prevalence of ESBL and MRSA isolates in surgical wards at BMC. We recommend infection control and antibiotic stewardship programs in these wards to minimize spread of multi-resistant organisms. PMID:24708843

  9. 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. PMID:26182074

  10. Endoplasmic reticulum chaperone gp96 in macrophages is essential for protective immunity during Gram-negative pneumonia.

    PubMed

    Anas, Adam A; de Vos, Alex F; Hoogendijk, Arie J; van Lieshout, Miriam H P; van Heijst, Jeroen W J; Florquin, Sandrine; Li, Zihai; van 't Veer, Cornelis; van der Poll, Tom

    2016-01-01

    Klebsiella pneumoniae is among the most common Gram-negative bacteria that cause pneumonia. Gp96 is an endoplasmic reticulum chaperone that is essential for the trafficking and function of Toll-like receptors (TLRs) and integrins. To determine the role of gp96 in myeloid cells in host defence during Klebsiella pneumonia, mice homozygous for the conditional Hsp90b1 allele encoding gp96 were crossed with mice expressing Cre-recombinase under control of the LysM promoter to generate LysMcre-Hsp90b1-flox mice. LysMcre-Hsp90b1-flox mice showed absence of gp96 protein in macrophages and partial depletion in monocytes and granulocytes. This was accompanied by almost complete absence of TLR2 and TLR4 on macrophages. Likewise, integrin subunits CD11b and CD18 were not detectable on macrophages, while being only slightly reduced on monocytes and granulocytes. Gp96-deficient macrophages did not release pro-inflammatory cytokines in response to Klebsiella and displayed reduced phagocytic capacity independent of CD18. LysMcre-Hsp90b1-flox mice were highly vulnerable to lower airway infection induced by K. pneumoniae, as reflected by enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1-flox mice was characterized by strongly impaired recruitment of granulocytes into the lungs, accompanied by attenuated production of pro-inflammatory cytokines, while the inflammatory response during late-stage pneumonia was not dependent on the presence of gp96. Blocking CD18 did not reproduce the impaired host defence of LysMcre-Hsp90b1-flox mice during Klebsiella pneumonia. These data indicate that macrophage gp96 is essential for protective immunity during Gram-negative pneumonia by regulating TLR expression. PMID:26365983

  11. Classification of gram-positive and gram-negative foodborne pathogenic bacteria with hyperspectral microscope imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Optical method with hyperspectral microscope imaging (HMI) has potential for identification of foodborne pathogenic bacteria from microcolonies rapidly with a cell level. A HMI system that provides both spatial and spectral information could be an effective tool for analyzing spectral characteristic...

  12. Dissemination of carbapenemases producing Gram negative bacteria in the Middle East

    PubMed Central

    Zahedi bialvaei, Abed; Samadi kafil, Hossein; Ebrahimzadeh Leylabadlo, Hamed; Asgharzadeh, Mohammad; Aghazadeh, Mohammad

    2015-01-01

    The emergence and spread of carbapenemase-producing bacteria, that hydolyze most β-lactams, including carbapenems, are a major concern of public health system worldwide, particularly in the Middle East area. Since the plasmids harboring resistance genes could be spread across other bacterial populations, detection of carbapenemase-producing organisms has become more problematic. These organisms produce different types of enzymes including the most prevalent types including KPC, VIM, IMP, NDM, and OXA-48. Carbapenemase producers are mostly identified among Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. This study reviewed almost all papers, which conducted in the Middle East. In order to decrease the spread of resistance, the regional cooperation has been emphasized by the Middle East countries. The highest resistance, which is mediated by KPC has been observed in Afghanistan, Saudi Arabia and Jordan followed by NDM in Pakistan and OXA in Turkey and Pakistan. It is important to mention that the spread of these types have been reported sporadically in the other countries of this area. This review described the widespread carbapenemases in the Middle East area, which have been identified in an alarming rate. PMID:26719779

  13. 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

  14. 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

  15. Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria.

    PubMed

    Husain, Fohad M; Ahmad, Iqbal; Khan, Mohammad S; Ahmad, Ejaz; Tahseen, Qudisa; Khan, Mohd Shahnawaz; Alshabib, Nasser A

    2015-01-01

    Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Mentha piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia coli biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor. PMID:26029178

  16. Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria

    PubMed Central

    Husain, Fohad M.; Ahmad, Iqbal; Khan, Mohammad S.; Ahmad, Ejaz; Tahseen, Qudisa; Khan, Mohd Shahnawaz; Alshabib, Nasser A.

    2015-01-01

    Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Mentha piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia coli biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor. PMID:26029178

  17. Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154.

    PubMed Central

    Palissa, H; von Döhren, H; Kleinkauf, H; Ting, H H; Baldwin, J E

    1989-01-01

    The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level beta-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including alpha-aminoadipic acid and cysteine. The Km for alpha-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a beta-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of beta-lactams in bacteria. Images PMID:2793834

  18. Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples.

    PubMed

    Haslan, Ezgi; Kimiran-Erdem, Ayten

    2013-09-01

    In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p < 0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria. PMID:23250628

  19. Evaluation of the ID 32E for the identification of Gram-negative glucose-fermenting and glucose-non-fermenting bacilli.

    PubMed

    O'Hara, Caroline Mohr; Miller, J. Michael

    1999-05-01

    OBJECTIVE: To evaluate the ID 32E bacterial identification system for accuracy in the identification of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii/Iwoffii. METHODS: Stock cultures of 497 Enterobacteriaceae and 27 commonly encountered non-enteric Gram-negative rods were tested in the ID 32E system. For each isolate, the resulting 11-digit profile number was converted to an identification using the APILAB Plus software (version 3.2.2). This identification was then compared to the reference identification obtained using conventional biochemicals. RESULTS: Of the 524 isolates tested, 405 (77.3%) were identified correctly; 52 (9.9%) were identified incorrectly. Sixty-seven (12.8%) identifications were either doubtful or unacceptable, and were not limited to any particular genus or species, with the exception of Ewingella americana and Serratia plymuthica, which did not grow well enough in the strip at 35 degrees C to be correctly identified. All five isolates of Acinetobacter Iwoffii were misidentified as Alcaligenes spp. CONCLUSIONS: With this challenge set of organisms, the ID 32E correctly identified 77.3% of the isolates tested. For commonly encountered isolates, the accuracy approached 90%. We conclude that the ID 32E offers an alternative for the identification of common clinical isolates. PMID:11856267

  20. Pattern recognition receptors and interleukin-8 mediate effects of Gram-positive and Gram-negative bacteria on lung epithelial cell function

    PubMed Central

    Sorrentino, R; de Souza, P M; Sriskandan, S; Duffin, C; Paul-Clark, M J; Mitchell, J A

    2008-01-01

    Background and purpose: Lung epithelial cells express pattern recognition receptors, which react to bacteria. We have evaluated the effect of Gram-positive and Gram-negative bacteria on interleukin-8 (CXCL8) release from epithelial cells and the integrity of the epithelial barrier. Experimental approach: Primary cultures of human airway epithelial cells and the epithelial cell line A549 were used, and CXCL8 release was measured after exposure to Gram-negative or Gram-positive bacteria. Epithelial barrier function was assessed in monolayer cultures of A549 cells. Results: Gram-positive bacteria Staphylococcus aureus or Streptococcus pneumoniae, induced release of CXCL8 from human airway epithelial cells. These bacteria also disrupted barrier function in A549 cells, an effect mimicked by CXCL8 and blocked by specific binding antibodies to CXCL8. Gram–negative bacteria Escherichia coli or Pseudomonas aeruginosa induced greater release of CXCL8 than Gram-positive bacteria. However, Gram-negative bacteria did not affect epithelial barrier function directly, but prevented disruption induced by Gram-positive bacteria. These effects of Gram-negative bacteria on barrier function were mimicked by FK565, an agonist of the nucleotide-binding oligomerization domain 1 (NOD1) receptor, but not by the Toll-like receptor (TLR) 4 agonist bacterial lipopolysaccharide. Neither the Gram-negative bacteria nor FK565 blocked CXCL8 release. Conclusions: These data show differential functional responses induced by Gram-negative and Gram-positive bacteria in human lung epithelial cells. The NOD1 receptors may have a role in preventing disruption of the epithelial barrier in lung, during inflammatory states. PMID:18536738

  1. Direct identification of major Gram-negative pathogens in respiratory specimens by respiFISH® HAP Gram (-) Panel, a beacon-based FISH methodology.

    PubMed

    Koncan, R; Parisato, M; Sakarikou, C; Stringari, G; Fontana, C; Favuzzi, V; Ligozzi, M; Lo Cascio, G

    2015-10-01

    Rapid detection of microorganisms in respiratory specimens is of paramount importance to drive the proper antibiotic regimen to prevent complications and transmission of infections. In the present study, the respiFISH® HAP Gram (-) Panel (miacom diagnostics GmbH, Duesseldorf, Germany) for the etiological diagnosis of hospital-acquired pneumonia was compared with the traditional culture method for the detection of major Gram-negative pathogens in respiratory specimens. respiFISH® combined the classical fluorescence in situ hybridization (FISH) technology with fluorescence-labeled DNA molecular beacons as probes. From September 2011 to January 2012, 165 samples were analyzed: the sensitivity and specificity were 94.39 and 87.93%, respectively. Only six pathogens (3.6%) were not identified with respiFISH®, while seven specimens (3%) provided false-positive results. This beacon-based identification shortens the time to result by at least one work day, providing species-level identification within half an hour. Considering the high sensitivity and specificity and the significant time saving, the introduction of bbFISH® assays could effectively complement traditional systems in microbiology laboratories. PMID:26219682

  2. The Comparison of the Combined Toxicity between Gram-negative and Gram-positive Bacteria: a Case Study of Antibiotics and Quorum-sensing Inhibitors.

    PubMed

    Wang, Dali; Lin, Zhifen; Ding, Xiruo; Hu, Jingyun; Liu, Ying

    2016-02-01

    Quorum-sensing inhibitors (QSIs) are being used increasingly in diverse fields, and are likely to end up in the environment, where they may encounter the antibiotics and consequently cause joint effects on biological systems. However, the potential joint effects of QSIs and antibiotics have received little attention. In this study, the joint effects of antibiotics, represented by sulfonamides (SAs) and penicillin, as well as three potential QSIs, were investigated using both Gram-negative (Escherichia coli, E. coli) and Gram-positive bacteria (Bacillus subtilis, B. subtilis). It was found that E. coli tend to be more sensitive to the individual drugs than B. subtilis, whereas the joint effects on the two bacteria showed no difference regarding the same combination of antibiotics and QSIs. In general, SAs presented additive effects with γ-Valerolactone and 2-Pyrrolidinone, but antagonistic effects with L-(+)-Prolinol; penicillin exhibited antagonistic effects with all three QSIs. Moreover, it was found that the rate of resistance in E. coli against the individual antibiotics was reduced through the addition of the QSIs, which suggests a promising use of the QSIs in the bacterial infection treatment. This study also offers a valuable reference for the risk assessment of the antibiotics and QSIs in the real environment. PMID:27491790

  3. Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

    PubMed Central

    Simon, R; Hötte, B; Klauke, B; Kosier, B

    1991-01-01

    On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different. Images PMID:1847366

  4. Rapid Discrimination of Gram-Positive and Gram-Negative Bacteria in Liquid Samples by Using NaOH-Sodium Dodecyl Sulfate Solution and Flow Cytometry

    PubMed Central

    Wada, Atsushi; Kono, Mari; Kawauchi, Sawako; Takagi, Yuri; Morikawa, Takashi; Funakoshi, Kunihiro

    2012-01-01

    Background For precise diagnosis of urinary tract infections (UTI), and selection of the appropriate prescriptions for their treatment, we explored a simple and rapid method of discriminating gram-positive and gram-negative bacteria in liquid samples. Methodology/Principal Findings We employed the NaOH-sodium dodecyl sulfate (SDS) solution conventionally used for plasmid extraction from Escherichia coli and the automated urine particle analyzer UF-1000i (Sysmex Corporation) for our novel method. The NaOH-SDS solution was used to determine differences in the cell wall structures between gram-positive and gram-negative bacteria, since the tolerance to such chemicals reflects the thickness and structural differences of bacterial cell walls. The UF-1000i instrument was used as a quantitative bacterial counter. We found that gram-negative bacteria, including E. coli, in liquid culture could easily be lysed by direct addition of equal volumes of NaOH-SDS solution. In contrast, Enterococcus faecalis, which is a gram-positive bacterium, could not be completely lysed by the solution. We then optimized the reaction time of the NaOH-SDS treatment at room temperature by using 3 gram-positive and 4 gram-negative bacterial strains and determined that the optimum reaction time was 5 min. Finally, in order to evaluate the generalizability of this method, we treated 8 gram-positive strains and 8 gram-negative strains, or 4 gram-positive and 4 gram-negative strains incubated in voluntary urine from healthy volunteers in the same way and demonstrated that all the gram-positive bacteria were discriminated quantitatively from gram negative bacteria using this method. Conclusions/Significance Using our new method, we could easily discriminate gram-positive and gram-negative bacteria in liquid culture media within 10 min. This simple and rapid method may be useful for determining the treatment course of patients with UTIs, especially for those without a prior history of UTIs. The method

  5. Geographical Variability in the Likelihood of Bloodstream Infections Due to Gram-Negative Bacteria: Correlation with Proximity to the Equator and Health Care Expenditure

    PubMed Central

    Carmeli, Yehuda; Perencevich, Eli; Tuite, Ashleigh R.; Mermel, Leonard A.

    2014-01-01

    Objective Infections due to Gram-negative bacteria exhibit seasonal trends, with peak infection rates during warmer months. We hypothesized that the likelihood of a bloodstream infection due to Gram-negative bacteria increases with proximity to the equator. We tested this hypothesis and identified geographical, climatic and social factors associated with this variability. Design We established a network of 23 international centers in 22 cities. Setting: De-identified results of positive blood cultures from 2007–2011 and data sources for geographic, climatic and socioeconomic factors were assembled for each center. Participants Patients at the 23 centers with positive blood cultures. Main outcome Due to variability in the availability of total culture volumes across sites, our primary outcome measure was the fraction of positive blood cultures that yielded Gram-negative bacteria; sources of variability in this outcome measure were explored using meta-regression techniques. Results The mean fraction of bacteremia associated with Gram-negative bacteria was 48.4% (range 26.4% to 61.8%). Although not all sites displayed significant seasonality, the overall P-value for seasonal oscillation was significant (P<0.001). In univariate meta-regression models, temperature, latitude, latitude squared, longitude, per capita gross domestic product and percent of gross domestic product spent on healthcare were all associated with the fraction of bacteremia due to Gram-negative bacteria. In multivariable models, only percent of gross domestic product spent on healthcare and distance from the equator (ie. latitude squared) were significantly associated with the fraction of bacteremia due to Gram-negative bacteria. Conclusions The likelihood of bacteremia due to Gram-negative bacteria varies markedly between cities, in a manner that appears to have both geographic (latitude) and socioeconomic (proportion gross domestic product devoted to health spending) determinants. Thus, the

  6. Antimicrobial activity of cationic gemini surfactant containing an oxycarbonyl group in the lipophilic portion against gram-positive and gram-negative microorganisms.

    PubMed

    Tatsumi, Taiga; Imai, Yoshitane; Kawaguchi, Kakuhiro; Miyano, Naoko; Ikeda, Isao

    2014-01-01

    We evaluated the antimicrobial activities of a cationic Gemini surfactant, trans-1,4-bis[2-(alkanoyloxy)ethyldimethylammonio]-2-butene dichloride [II-m-2(t-butene)] and its derivatives against Gram-positive and Gram-negative microorganisms. The II-m-2(t-butene) compound was previously shown to have good surface activity and biodegradability. A dodecanoyloxy derivative (m = 12) of II-m-2(t-butene) showed excellent antimicrobial activity against Gram-positive Streptococcus aureus [minimum inhibitory concentration (MIC): 7.8 μg/mL] and Gram-negative Escherichia coli (MIC: 31.2 μg/mL). PMID:24420061

  7. Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure.

    PubMed Central

    Gellin, G; Langlois, B E; Dawson, K A; Aaron, D K

    1989-01-01

    The antibiotic resistance patterns of gram-negative fecal bacteria from pigs in three herds with different histories of antibiotic exposure were examined. In general, smaller proportions of antibiotic-resistant or multiply resistant fecal isolates (P less than 0.05) were obtained from pigs in a herd not exposed to antimicrobial agents for 154 months than from pigs in a herd continuously exposed to antimicrobial agents at subtherapeutic doses or from pigs in a herd exposed only to therapeutic doses of antimicrobial agents. The proportions of antibiotic-resistant and multiply resistant strains were greater among isolates from pigs in the therapeutic herd than in the non-antibiotic-exposed herd (P less than 0.05). The proportion of antibiotic-resistant isolates in the non-lactose-fermenting population was greater than that in the lactose-fermenting population, regardless of herd. The results suggest that any form of antimicrobial exposure will increase the prevalence of antimicrobial resistance and multiple resistance of fecal bacteria. PMID:2802608

  8. alpha-Macroglobulins are present in some gram-negative bacteria: characterization of the alpha2-macroglobulin from Escherichia coli.

    PubMed

    Doan, Ninh; Gettins, Peter G W

    2008-10-17

    alpha-Macroglobulins (alphaMs) are large glycoproteins that have been identified in a wide range of vertebrate and invertebrate species and are mostly thiol ester containing proteinase inhibitors. A recent analysis of bacterial genomes ( Budd, A., Blandin, S., Levashina, E. A., and Gibson, T. J. (2004) Genome Biol. 5, R38 ) identified many alpha-macroglobulin-like sequences that appear to have been acquired by Gram-negative bacteria from their metazoan hosts. We report the first expression and characterization of such a bacterial alpha-macroglobulin, that from Escherichia coli. This is also the first alpha-macroglobulin to be characterized that is predicted to be membrane-anchored. We found that the 183-kDa protein contains an intact thiol ester, is monomeric, and is localized to the periplasmic space. Reaction with proteinase results in limited cleavage within a bait region, rapid activation of the thiol ester, cross-linking to the attacking proteinase or other available nucleophiles, and partial protection of the proteinase against macromolecular substrates. Given these properties and the co-occurrence of the alphaM gene with one for a repair transglycosylase, this suggests a possible role for bacterial alphaMs in cell defense following host attack. Such a role would make bacterial alphaMs appropriate novel targets for antibiotic drugs. PMID:18697741

  9. Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex.

    PubMed

    Kronvall, G; Hanson, H S; von Stedingk, L V; Törnqvist, E; Falsen, E

    2000-03-01

    A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species. PMID:10752687

  10. 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

    2014-01-01

    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. PMID:24759068

  11. Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria

    PubMed Central

    Bishop, Russell E.; Gibbons, Henry S.; Guina, Tina; Trent, M.Stephen; Miller, Samuel I.; Raetz, Christian R.H.

    2000-01-01

    Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His6-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg2+ limitation and host innate immune defenses. PMID:11013210

  12. Extended spectrum beta lactamase producing gram negative bacilli in a tertiary referral hospital of Assam--experience with two methods.

    PubMed

    Nath, Reema; Saikia, Lahari; Mahanta, J

    2006-10-01

    Extended Spectrum Beta Lactamases (ESBL) are enzymes produced in some gram negative bacilli that mediate resistance to extended spectrum cephalosporins. 683 clinical isolates of Escherisia coli and Klebsiella pneumoniae were studied for their capacity to produce ESBL. Isolates showing resistance to at least two of the third generation cephalosporins were studied for ESBL production by Jarlier technique and combination disc methods. Out of the 457 E. Coli and 226 Klebsiella pneumoniae isolated in Assam Medical College, 29.76% and 53.1% were resistant to two cephalosporins of which 29.41% and 29.16% strains showed production of ESBL. However, 6 (4.41%) and 16 (13.34%) strains additionally showed production of ESBL when tested with combination disc method. Though the Jarlier technique is popular, for detection of ESBL, yet false negative results warrants for alternative method. In the absence of molecular detection methods in routine clinical microbiology laboratory, combination disc method appears to be a better option. PMID:17183880

  13. Multidrug-resistant gram-negative bacteria colonization of healthy US military personnel in the US and Afghanistan

    PubMed Central

    2013-01-01

    Background The US military has seen steady increases in multidrug-resistant (MDR) gram-negative bacteria (GNB) infections in casualties from Iraq and Afghanistan. This study evaluates the prevalence of MDR GNB colonization in US military personnel. Methods GNB colonization surveillance of healthy, asymptomatic military personnel (101 in the US and 100 in Afghanistan) was performed by swabbing 7 anatomical sites. US-based personnel had received no antibiotics within 30 days of specimen collection, and Afghanistan-based personnel were receiving doxycycline for malaria chemoprophylaxis at time of specimen collection. Isolates underwent genotypic and phenotypic characterization. Results The only colonizing MDR GNB recovered in both populations was Escherichia coli (p=0.01), which was seen in 2% of US-based personnel (all perirectal) and 11% of Afghanistan-based personnel (10 perirectal, 1 foot+groin). Individuals with higher off-base exposures in Afghanistan did not show a difference in overall GNB colonization or MDR E. coli colonization, compared with those with limited off-base exposures. Conclusion Healthy US- and Afghanistan-based military personnel have community onset-MDR E. coli colonization, with Afghanistan-based personnel showing a 5.5-fold higher prevalence. The association of doxycycline prophylaxis or other exposures with antimicrobial resistance and increased rates of MDR E. coli colonization needs further evaluation. PMID:23384348

  14. A large family of antivirulence regulators modulates the effects of transcriptional activators in Gram-negative pathogenic bacteria.

    PubMed

    Santiago, Araceli E; Ruiz-Perez, Fernando; Jo, Noah Y; Vijayakumar, Vidhya; Gong, Mei Q; Nataro, James P

    2014-05-01

    We have reported that transcription of a hypothetical small open reading frame (orf60) in enteroaggregative E. coli (EAEC) strain 042 is impaired after mutation of aggR, which encodes a global virulence activator. We have also reported that the cryptic orf60 locus was linked to protection against EAEC diarrhea in two epidemiologic studies. Here, we report that the orf60 product acts as a negative regulator of aggR itself. The orf60 protein product lacks homology to known repressors, but displays 44-100% similarity to at least fifty previously undescribed small (<10 kDa) hypothetical proteins found in many gram negative pathogen genomes. Expression of orf60 homologs from enterotoxigenic E. coli (ETEC) repressed the expression of the AraC-transcriptional ETEC regulator CfaD/Rns and its regulon in ETEC strain H10407. Complementation in trans of EAEC 042orf60 by orf60 homologs from ETEC and the mouse pathogen Citrobacter rodentium resulted in dramatic suppression of aggR. A C. rodentium orf60 homolog mutant showed increased levels of activator RegA and increased colonization of the adult mouse. We propose the name Aar (AggR-activated regulator) for the clinically and epidemiologically important orf60 product in EAEC, and postulate the existence of a large family of homologs among pathogenic Enterobacteriaceae and Pasteurellaceae. We propose the name ANR (AraC Negative Regulators) for this family. PMID:24875828

  15. Activity of the colistin-rifampicin combination against colistin-resistant, carbapenemase-producing Gram-negative bacteria.

    PubMed

    Nastro, Marcela; Rodríguez, Carlos Hernan; Monge, Renata; Zintgraff, Jonathan; Neira, Liliana; Rebollo, Mirta; Vay, Carlos; Famiglietti, Angela

    2014-08-01

    Colistin (COL) is one of the few antimicrobials that retain activity against carbapenemase-producing Gram-negative bacteria (GNB). However, the emergence of COL resistance has renewed the use of combination therapy. The aim of this study was to determine the activity of COL plus rifampicin (RIF) against clinical isolates of COL-resistant, carbapenemase-producing GNB. We employed 36 COL-resistant carbapenemase-producing isolates (27 Klebsiella pneumoniae, 5 Serratia marcescens, and 4 Acinetobacter baumannii) belonging to 36 patients. E-test/agar dilution of all strains was performed with E-test strips of COL placed on agar plates with and without RIF. In 11 patients, the synergy was confirmed by time-kill studies. Synergy was detected in 34 isolates, whereas indifference was detected in two S. marcescens. The E-test/agar dilution method showed comparable results to the time-kill studies. Seven patients infected with these isolates (two meningitis, four sepsis, and one urinary tract infection) were treated with the combination successfully. PMID:24070502

  16. Structure-Activity Relationships of a Novel Pyranopyridine Series of Gram-negative Bacterial Efflux Pump Inhibitors

    PubMed Central

    Nguyen, Son T.; Kwasny, Steven M.; Ding, Xiaoyuan; Cardinale, Steven C.; McCarthy, Courtney T.; Kim, Hong-Suk; Nikaido, Hiroshi; Peet, Norton P.; Williams, John D.; Bowlin, Terry L.; Opperman, Timothy J.

    2015-01-01

    Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d–f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli. PMID:25818767

  17. Silver-doped manganese dioxide and trioxide nanoparticles inhibit both gram positive and gram negative pathogenic bacteria.

    PubMed

    Kunkalekar, R K; Prabhu, M S; Naik, M M; Salker, A V

    2014-01-01

    Palladium, ruthenium and silver-doped MnO2 and silver doped Mn2O3 nanoparticles were synthesized by simple co-precipitation technique. SEM-TEM analysis revealed the nano-size of these synthesized samples. XPS data illustrates that Mn is present in 4+ and 3+ oxidation states in MnO2 and Mn2O3 respectively. Thermal analysis gave significant evidence for the phase changes with increasing temperature. Antibacterial activity of these synthesized nanoparticles on three Gram positive bacterial cultures (Staphylococcus aureus ATCC 6538, Streptococcus epidermis ATCC 12228, Bacillus subtilis ATCC 6633) and three Gram negative cultures (Escherichia coli ATCC 8739, Salmonella abony NCTC 6017 and Klebsiella pneumoniae ATCC 1003) was investigated using a disc diffusion method and live/dead assay. Only Ag-doped MnO2 and Ag-doped Mn2O3 nanoparticles showed antibacterial property against all six-test bacteria but Ag-doped MnO2 was found to be more effective than Ag-doped Mn2O3. PMID:24140741

  18. Neisseria gonorrhoeae filamentous phage NgoΦ6 is capable of infecting a variety of Gram-negative bacteria.

    PubMed

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Szczêsna, Ewa; Piechucki, Marcin; Kwiatek, Agnieszka; Maugel, Timothy K; Stein, Daniel C

    2014-01-01

    We constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage NgoΦ6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::Φ6). Escherichia coli cells harboring pBS::Φ6 were able to produce a biologically active phagemid, NgoΦ6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli, Haemophilus influenzae, Neisseria sicca, Pseudomonas sp., and Paracoccus methylutens. A derivative of pBS::Φ6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli, Haemophilus influenzae, N. sicca, Pseudomonas sp., and Paracoccus methylutens, indicating that NgoΦ6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that NgoΦ6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that NgoΦ6 is the first broad-host-range filamentous bacteriophage described. PMID:24198404

  19. Synergistic interactions between doxycycline and terpenic components of essential oils encapsulated within lipid nanocapsules against gram negative bacteria.

    PubMed

    Valcourt, C; Saulnier, P; Umerska, A; Zanelli, M P; Montagu, A; Rossines, E; Joly-Guillou, M L

    2016-02-10

    The combination of essential oils (EOs) with antibiotics provides a promising strategy towards combating resistant bacteria. We have selected a mixture of 3 major components extracted from EOs: carvacrol (oregano oil), eugenol (clove oil) and cinnamaldehyde (cinnamon oil). These compounds were successfully encapsulated within lipid nanocapsules (LNCs). The EOs-loaded LNCs were characterised by a noticeably high drug loading of 20% and a very small particle diameter of 114nm. The in vitro interactions between EOs-loaded LNCs and doxycycline were examined via checkerboard titration and time-kill assay against 5 Gram-negative strains: Acinetobacter baumannii SAN, A. baumannii RCH, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. No growth inhibition interactions were found between EOs-loaded LNCs and doxycycline (FIC index between 0.7 and 1.30). However, when bactericidal effects were considered, a synergistic interaction was observed (FBC index equal to 0.5) against all tested strains. A synergistic effect was also observed in time-kill assay (a difference of at least 3 log between the combination and the most active agent alone). Scanning electron microscopy (SEM) was used to visualise the changes in the bacterial membrane. The holes in bacterial envelope and leakage of cellular contents were observed in SE micrographs after exposure to the EOs-LNCs and the doxycycline combination. PMID:26631640

  20. A Large Family of Antivirulence Regulators Modulates the Effects of Transcriptional Activators in Gram-negative Pathogenic Bacteria

    PubMed Central

    Santiago, Araceli E.; Ruiz-Perez, Fernando; Jo, Noah Y.; Vijayakumar, Vidhya; Gong, Mei Q.; Nataro, James P.

    2014-01-01

    We have reported that transcription of a hypothetical small open reading frame (orf60) in enteroaggregative E. coli (EAEC) strain 042 is impaired after mutation of aggR, which encodes a global virulence activator. We have also reported that the cryptic orf60 locus was linked to protection against EAEC diarrhea in two epidemiologic studies. Here, we report that the orf60 product acts as a negative regulator of aggR itself. The orf60 protein product lacks homology to known repressors, but displays 44–100% similarity to at least fifty previously undescribed small (<10 kDa) hypothetical proteins found in many gram negative pathogen genomes. Expression of orf60 homologs from enterotoxigenic E. coli (ETEC) repressed the expression of the AraC-transcriptional ETEC regulator CfaD/Rns and its regulon in ETEC strain H10407. Complementation in trans of EAEC 042orf60 by orf60 homologs from ETEC and the mouse pathogen Citrobacter rodentium resulted in dramatic suppression of aggR. A C. rodentium orf60 homolog mutant showed increased levels of activator RegA and increased colonization of the adult mouse. We propose the name Aar (AggR-activated regulator) for the clinically and epidemiologically important orf60 product in EAEC, and postulate the existence of a large family of homologs among pathogenic Enterobacteriaceae and Pasteurellaceae. We propose the name ANR (AraC Negative Regulators) for this family. PMID:24875828

  1. Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria

    PubMed Central

    Rahman, Mokhlasur; Kühn, Inger; Rahman, Motiur; Olsson-Liljequist, Barbro; Möllby, Roland

    2004-01-01

    We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log2 dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria. PMID:15066837

  2. Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods.

    PubMed

    Osei Sekyere, J; Govinden, U; Bester, L A; Essack, S Y

    2016-09-01

    A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools. PMID:27153928

  3. In vitro activities of quinolones, beta-lactams, tobramycin, and trimethoprim-sulfamethoxazole against nonfermentative gram-negative bacilli.

    PubMed

    Fass, R J; Barnishan, J; Solomon, M C; Ayers, L W

    1996-06-01

    From 1991 to 1995, 8,975 nonfermentative gram-negative bacilli were isolated from patients at The Ohio State University Medical Center: 71% Pseudomonas aeruginosa, 14% Stenotrophomonas maltophilia, 7.6% Acinetobacter baumannii, and < 2% each of 25 other species. The MICs of trovafloxacin (CP-99,219), ciprofloxacin, ofloxacin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, ceftazidime, cefoperazone, ceftriaxone, imipenem, tobramycin, and trimethoprim-sulfamethoxazole (TMP-SMZ) were determined for 308 isolates, representing 13 species, by a standardized broth microdilution method. The activities of all drugs were species dependent. The fluoroquinolones had inconsistent activity against most species, although several relatively uncommon nonfermenters were consistently susceptible or resistant. Trovafloxacin was considerably more active than ciprofloxacin and ofloxacin against S. maltophilia, A. baumannii, and several less common species. Among the beta-lactams, relative activities varied considerably; overall, imipenem had the broadest spectrum of activity but was inactive against S. maltophilia and Burkholderia cepacia isolates. Tobramycin and TMP-SMZ had stereotypic spectra of activity. Tobramycin was active against most species except S. maltophilia, Alcaligenes xylosoxidans subsp. xylosoxidans, Burkholderia spp., and Weeksella virosa. TMP-SMZ was active against most species except P. aeruginosa and Pseudomonas fluorescens-putida. A review of laboratory records indicated few changes in susceptibility patterns from 1991 to 1995; the only clear trend was toward increasing P. aeruginosa resistance to all classes of drugs. PMID:8726011

  4. Propionispora vibrioides, nov. gen., nov. sp., a new gram-negative, spore-forming anaerobe that ferments sugar alcohols.

    PubMed

    Biebl, H; Schwab-Hanisch, H; Spröer, C; Lünsdorf, H

    2000-10-01

    Anaerobic enrichment cultures, with erythritol as substrate, resulted in the isolation of a strain with properties not yet found in an existing genus in this combination. The strain, FKBS1, was strictly anaerobic, stained gram-negative and formed spores. Cells were small motile vibrios with flagella inserted at the concave side of the cell. Spores were located terminally and caused only slight swelling of the cells if compared to related spore-forming genera. FKBS1 fermented fructose, mannitol, sorbitol, xylitol and erythritol to propionic acid, acetic acid, CO2 and small amounts of H2 to balance the difference in the oxidation-reduction value between substrate and cell mass. The 16S rDNA sequence revealed relationship to the Sporomusa-Pectinatus-Selenomonas group. However, the phylogenetic distance to any of its members was too great to allow it to be placed in one of the existing genera. Morphologically the strain resembled Sporomusa, which, however, performs an acetogenic type of fermentation. The propionic-acid-forming genera of the group are either not spore-formers or, in the case of Dendrosporobacter quercicolus (syn. Clostridium quercicolum), morphologically different. It is therefore proposed to classify strain FKBS1 as a new genus and species, Propionispora vibrioides. PMID:11081792

  5. Scrutinizing the Scaffolds of Marine Biosynthetics from Different Source Organisms: Gram-Negative Cultured Bacterial Products Enter Center Stage

    PubMed Central

    Still, Patrick C.; Johnson, Tyler A.; Theodore, Christine M.; Loveridge, Steven T.; Crews, Phillip

    2014-01-01

    Compounds from macro marine organisms are presumed to owe their biosynthetic origins to associated microbial symbionts, although few definitive examples exist. An upsurge in the recent literature from 2012 to 2013 has shown that four compounds previously reported from macro marine organisms are in fact biosynthesized by non-photosynthetic Gram-negative bacteria (NPGNB). Structural parallels between compounds isolated from macro marine organisms and NPGNB producers form the basis of this review. Although less attention has been given to investigating the chemistry of NPGNB sources, there exists a significant list of structural parallels between NPGNB and macro marine organism-derived compounds. Alternatively, of the thousands of compounds isolated from Gram-positive actinomycetes, few structural parallels with macro marine organisms are known. A summary of small molecules isolated from marine NPGNB sources is presented, including compounds isolated from marine myxobacteria. From this assemblage of structural parallels and diverse chemical structures, it is hypothesized that the potential for the discovery of inspirational molecules from NPGNB sources is vast and that the recent spike in the literature of macro marine compounds owing their biosynthetic origin to NPGNB producers represents a turning point in the field. PMID:24571234

  6. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  7. Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

    PubMed Central

    Tomasek, P H; Karns, J S

    1989-01-01

    A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase. Images PMID:2661544

  8. Biosynthetic Products from a Nearshore-Derived Gram-Negative Bacterium Enable Reassessment of the Kailuin Depsipeptides

    PubMed Central

    Theodore, Christine M.; Lorig-Roach, Nicholas; Still, Patrick C.; Johnson, Tyler A.; Drašković, Marija; Schwochert, Joshua A.; Naphen, Cassandra N.; Crews, Mitchell S.; Barker, Simone A.; Valeriote, Frederick A.; Lokey, R. Scott; Crews, Phillip

    2015-01-01

    Sampling of California nearshore sediments resulted in the isolation of a Gram-negative bacterium, Photobacterium halotolerans, capable of producing unusual biosynthetic products. Liquid culture in artificial seawater-based media provided cyclic depsipeptides including four known compounds, kailuins B–E (2–5), and two new analogues, kailuins G and H (7 and 8). The structures of the new and known compounds were confirmed through extensive spectroscopic and Marfey's analyses. During the course of these studies, a correction was made to the previously reported double-bond geometry of kailuin D (4). Additionally, through the application of a combination of derivatization with Mosher's reagent and extensive 13C NMR shift analysis, the previously unassigned chiral center at position C-3 of the β-acyloxy group of all compounds was determined. To evaluate bioactivity and structure–activity relationships, the kailuin core (13) and kailuin lactam (14) were prepared by chiral synthesis using an Fmoc solid-phase peptide strategy followed by solution-phase cyclization. All isolated compounds and synthetic cores were assayed for solid tumor cell cytotoxicity and showed only minimal activity, contrary to other published reports. Additional phenotypic screenings were done on 4 and 5, with little evidence of activity. PMID:25699470

  9. A zebrafish intelectin ortholog agglutinates both Gram-negative and Gram-positive bacteria with binding capacity to bacterial polysaccharide.

    PubMed

    Chen, Lei; Yan, Jie; Sun, Weiping; Zhang, Yan; Sui, Chao; Qi, Jing; Du, Yijun; Feng, Lijun

    2016-08-01

    Intelectins are glycan-binding lectins found in various species including cephalochordates, urochordates, fish, amphibians and mammals. But their detailed functions are not well studied in zebrafish which is a good model to study native immunity. In this study, we cloned a zebrafish intelectin ortholog, zebrafish intelectin 2 (zITLN2), which contains a conserved fibrinogen-related domain (FReD) in the N-terminus and the unique intelectin domain in the C-terminus. We examined the tissue distribution of zITLN2 in adult zebrafish and found that zITLN2 was expressed in various organs with the highest level in intestine. Like amphioxus intelectins, zITLN2 expression was upregulated in adult zebrafish infected with Staphylococcus aureus with the highest expression level at 12 h after challenge. Recombinant zITLN2 protein expressed in E. coli was able to agglutinate both Gram-negative and Gram-positive bacteria to similar degrees in a calcium-dependent manner. Furthermore, recombinant zITLN2 bound lipopolysaccharide (LPS) and peptidoglycan (PGN) comparably. Our work on zITLN2 provided further information to understand functions of this new family of lectins and the innate immunity in vertebrates. PMID:27329687

  10. Screening of antibiotic susceptibility to β-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis.

    PubMed

    Chung, Cheng-Che; Cheng, I-Fang; Chen, Hung-Mo; Kan, Heng-Chuan; Yang, Wen-Horng; Chang, Hsien-Chang

    2012-04-01

    We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria. PMID:22404714

  11. Colistin combination therapy improves microbiologic cure in critically ill patients with multi-drug resistant gram-negative pneumonia.

    PubMed

    Parchem, N L; Bauer, K A; Cook, C H; Mangino, J E; Jones, C D; Porter, K; Murphy, C V

    2016-09-01

    Currently, in vitro synergy with colistin has not translated into improved clinical outcomes. This study aimed to compare colistin combination therapy to colistin monotherapy in critically ill patients with multi-drug resistant gram-negative (MDR-GN) pneumonia. This was a retrospective analysis of critically ill adult patients receiving intravenous colistin for MDR-GN pneumonia comparing colistin combination therapy to colistin monotherapy with a primary endpoint of clinical cure. Combination therapy was defined by administration of another antibiotic to which the MDR-GN pathogen was reported as susceptible or intermediate. Ninety patients were included for evaluation (41 combination therapy and 49 monotherapy). Baseline characteristics were similar between groups. No difference in clinical cure was observed between combination therapy and monotherapy in univariate analysis, nor when adjusted for APACHE II score and time to appropriate antibiotic therapy (57.1 vs. 63.4 %, adjusted OR 1.15, p = 0.78). Microbiological cure was significantly higher for combination therapy (87 vs. 35.5 %, p < 0.001). Colistin combination therapy was associated with a significant improvement in microbiological cure, without improvement in clinical cure. Based on the in vitro synergy and improvement in microbiological clearance, colistin combination therapy should be prescribed for MDR-GN pneumonia. Further research is warranted to determine if in vitro synergy with colistin translates into improved clinical outcomes. PMID:27230510

  12. Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid

    SciTech Connect

    Wunschel, David S.; Webb-Robertson, Bobbie-Jo M.; Frevert, Charles W.; Skerret, Shawn J.; Beagley, Nathaniel; Willse, Alan R.; Colburn, Heather A.; Antolick, Kathryn C.

    2009-09-16

    The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a of mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas auriginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame.

  13. Multidrug-resistant and extensively drug-resistant Gram-negative pathogens: current and emerging therapeutic approaches

    PubMed Central

    Karaiskos, Ilias; Giamarellou, Helen

    2014-01-01

    Introduction: In the era of multidrug-resistant, extensively drug-resistant (XDR) and even pandrug-resistant Gram-negative microorganisms, the medical community is facing the threat of untreatable infections particularly those caused by carbapenemase-producing bacteria, that is, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Therefore, all the presently available antibiotics, as well as for the near future compounds, are presented and discussed. Areas covered: Current knowledge concerning mechanisms of action, in vitro activity and interactions, pharmacokinetic/pharmacodynamics, clinical efficacy and toxicity issues for revived and novel antimicrobial agents overcoming current resistance mechanisms, including colistin, tigecycline, fosfomycin, temocillin, carbapenems, and antibiotics still under development for the near future such as plazomicin, eravacycline and carbapenemase inhibitors is discussed. Expert opinion: Colistin is active in vitro and effective in vivo against XDR carbapenemase-producing microorganisms in the critically ill host, whereas tigecycline, with the exception of P. aeruginosa, has a similar spectrum of activity. The efficacy of combination therapy in bacteremias and ventilator-associated pneumonia caused by K. pneumoniae carbapenemase producers seems to be obligatory, whereas in cases of P. aeruginosa and A. baumannii its efficacy is questionable. Fosfomycin, which is active against P. aeruginosa and K. pneumoniae, although promising, shares poor experience in XDR infections. The in vivo validity of the newer potent compounds still necessitates the evaluation of Phase III clinical trials particularly in XDR infections. PMID:24766095

  14. Outcome of Transplantation Using Organs From Donors Infected or Colonized With Carbapenem-Resistant Gram-Negative Bacteria.

    PubMed

    Mularoni, A; Bertani, A; Vizzini, G; Gona, F; Campanella, M; Spada, M; Gruttadauria, S; Vitulo, P; Conaldi, P; Luca, A; Gridelli, B; Grossi, P

    2015-10-01

    Donor-derived infections due to multidrug-resistant bacteria are a growing problem in solid organ transplantation, and optimal management options are not clear. In a 2-year period, 30/214 (14%) recipients received an organ from 18/170 (10.5%) deceased donors with infection or colonization caused by a carbapenem-resistant gram-negative bacteria that was unknown at the time of transplantation. Among them, 14/30 recipients (47%) received a transplant from a donor with bacteremia or with infection/colonization of the transplanted organ and were considered at high risk of donor-derived infection transmission. The remaining 16/30 (53%) recipients received an organ from a nonbacteremic donor with colonization of a nontransplanted organ and were considered at low risk of infection transmission. Proven transmission occurred in 4 of the 14 high-risk recipients because donor infection was either not recognized, underestimated, or not communicated. These recipients received late, short or inappropriate posttransplant antibiotic therapy. Transmission did not occur in high-risk recipients who received appropriate and prompt antibiotic therapy for at least 7 days. The safe use of organs from donors with multidrug-resistant bacteria requires intra- and inter-institutional communication to allow appropriate management and prompt treatment of recipients in order to avoid transmission of infection. PMID:25981339

  15. Co-selection of antibiotic and metal(loid) resistance in gram-negative epiphytic bacteria from contaminated salt marshes.

    PubMed

    Henriques, Isabel; Tacão, Marta; Leite, Laura; Fidalgo, Cátia; Araújo, Susana; Oliveira, Cláudia; Alves, Artur

    2016-08-15

    The goal of this study was to investigate co-selection of antibiotic resistance in gram-negative epiphytic bacteria. Halimione portulacoides samples were collected from metal(loid)-contaminated and non-contaminated salt marshes. Bacterial isolates (n=137) affiliated with Vibrio, Pseudomonas, Shewanella, Comamonas, Aeromonas and with Enterobacteriaceae. Vibrio isolates were more frequent in control site while Pseudomonas was common in contaminated sites. Metal(loid) and antibiotic resistance phenotypes varied significantly according to site contamination, and multiresistance was more frequent in contaminated sites. However, differences among sites were not observed in terms of prevalence or diversity of acquired antibiotic resistance genes, integrons and plasmids. Gene merA, encoding mercury resistance, was only detected in isolates from contaminated sites, most of which were multiresistant to antibiotics. Results indicate that metal(loid) contamination selects for antibiotic resistance in plant surfaces. In salt marshes, antibiotic resistance may be subsequently transferred to other environmental compartments, such as estuarine water or animals, with potential human health risks. PMID:27210560

  16. Synthesis of the cell surface during the division cycle of rod-shaped, gram-negative bacteria.

    PubMed Central

    Cooper, S

    1991-01-01

    When the growth of the gram-negative bacterial cell wall is considered in relation to the synthesis of the other components of the cell, a new understanding of the pattern of wall synthesis emerges. Rather than a switch in synthesis between the side wall and pole, there is a partitioning of synthesis such that the volume of the cell increases exponentially and thus perfectly encloses the exponentially increasing cytoplasm. This allows the density of the cell to remain constant during the division cycle. This model is explored at both the cellular and molecular levels to give a unified description of wall synthesis which has the following components: (i) there is no demonstrable turnover of peptidoglycan during cell growth, (ii) the side wall grows by diffuse intercalation, (iii) pole synthesis starts by some mechanism and is preferentially synthesized compared with side wall, and (iv) the combined side wall and pole syntheses enclose the newly synthesized cytoplasm at a constant cell density. The central role of the surface stress model in wall growth is distinguished from, and preferred to, models that propose cell-cycle-specific signals as triggers of changes in the rate of wall synthesis. The actual rate of wall synthesis during the division cycle is neither exponential nor linear, but is close to exponential when compared with protein synthesis during the division cycle. PMID:1779930

  17. Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils.

    PubMed

    Bach, Evelise; Daroit, Daniel Joner; Corrêa, Ana Paula Folmer; Brandelli, Adriano

    2011-11-01

    The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45-55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts. PMID:21526391

  18. Lipopolysaccharide-binding protein of Bombyx mori participates in a hemocyte-mediated defense reaction against gram-negative bacteria.

    PubMed

    Koizumi, N; Imai, Y; Morozumi, A; Imamura, M; Kadotani, T; Yaoi, K; Iwahana, H; Sato, R

    1999-09-01

    BmLBP is a lipopolysaccharide-binding protein in B. mori and participates in bacterial clearance in vivo. Here, we investigated the function of BmLBP more specifically. More than 90% of injected gram-negative rough strains to which BmLBP binds were removed from the plasma within 30 min post-injection, whereas it required 8h for the clearance of smooth strains to which BmLBP does not bind. Observation of the hemocoel after the injection of Escherichia coli rough strain showed that melanized nodules were formed at 30 min post-injection when the clearance of injected E. coli cells had occurred. Fluorescence microscope observation revealed that E. coli cells were actually trapped in the nodules formed in vivo. Furthermore, plasma pre-treated E. coli rough cells (BmLBP bound) added to hemocytes isolated in vitro caused vigorous hemocyte aggregations with the bacteria, while plasma pre-treated smooth cells did not. The formation of aggregates was inhibited by anti-BmLBP serum pre-treatment, suggesting that BmLBP causes the clearance of bacteria by promoting hemocyte nodule formation. PMID:12770298

  19. A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria

    PubMed Central

    Chu, Ya-Yun; Nega, Mulugeta; Wölfle, Martina; Plener, Laure; Grond, Stephanie; Jung, Kirsten; Götz, Friedrich

    2013-01-01

    The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community. PMID:24098134

  20. Photocatalytic degradation of methylene blue and inactivation of gram-negative bacteria by TiO2 nanoparticles in aqueous suspension

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The photocatalytic degradation of methylene blue (MB) and inactivation of Gram-negative bacteria E. coli K12 and P. aeruginosa by TiO2 nanoparticles in aqueous suspension were studied. TiO2 resulted in significant reduction in MB absorption and a shift of MB absorption peak from 664 nm to 658 nm aft...

  1. Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices.

    PubMed

    Margot, H; Zwietering, M H; Joosten, H; O'Mahony, Emer; Stephan, R

    2015-12-01

    This study evaluated the effects of changing the composition of the pre-enrichment medium buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW variants tested for different food matrices. In products with high levels of Gram-negative background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in any of the BPW variants tested unless the initial level exceeded 10(3) cfu/10 g of sprouts. Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment strategy based on BPW variants tested in this evaluation is not recommended for produce with a high level of Gram-negative background flora due to very high detection limits. PMID:26267889

  2. Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide.

    PubMed

    Kim, Hyun; Jang, Ju Hye; Kim, Sun Chang; Cho, Ju Hyun

    2016-08-01

    Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28-34 of lactoferrin (Lf28-34), amino acids 84-99 of bactericidal/permeability increasing protein (BPI84-99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria. PMID:27242337

  3. 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

  4. Efficacy of a commercial probiotic relative to oxytetracycline as Gram-negative bacterial control agents in a rotifer (Brachionus plicatilis) batch culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two trials were conducted to evaluate two gram-negative bacterial control strategies in batch cultures of the rotifer Brachionus plicatilis. In the first trial, rotifers at an initial density of 47/mL were cultured for 5 d and dosed with a 10-mg/L solution of either oxytetracycline or a commercial p...

  5. [Post-marketing surveillance of antibacterial activities of cefozopran against various clinical isolates--II. Gram-negative bacteria].

    PubMed

    Igari, Jun; Oguri, Toyoko; Hiramatsu, Nobuyoshi; Akiyama, Kazumitsu; Koyama, Tsuneo

    2003-10-01

    As a post-marketing surveillance, the in vitro antibacterial activities of cefozopran (CZOP), an agent of cephems, against various clinical isolates were yearly evaluated and compared with those of other cephems, oxacephems, carbapenems, monobactams, and penicillins. Changes in CZOP susceptibility among bacteria were also evaluated with the bacterial resistance ratio calculated from the breakpoint MIC. Twenty-five species (4,154 strains) of Gram-negative bacteria were isolated from the clinical materials annually collected from 1996 to 2001, and consisted of Moraxella (Branhamella) catarrhalis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Serratia liquefaciens, Citrobacter freundii, Citrobacter koseri, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia spp., Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Acinetobacter baumannii, Acinetobacter Iwoffii, Burkholderia cepacia, Stenotrophomonas maltophilia, Bacteroides fragilis group, and Prevotella/Porphyromonas. CZOP preserved its antibacterial activity against M. (B.) catarrhalis (MIC90: 4 micrograms/mL) and showed comparable activity to carbapenems against H. influenzae (MIC90: 1 microgram/mL). The antibacterial activity of CZOP against E. coli was preferable (MIC90: 0.125 microgram/mL) and comparable to those of cefpirome (CPR), cefepime (CFPM), and imipenem (IPM). The MIC90 of CZOP against K. pneumoniae and K. oxytoca was 1 and 0.25 microgram/mL, respectively. The MIC90 of CZOP against E. cloacae increased during 6 years (32 to 128 micrograms/mL). The antibacterial activity of CZOP against E. aerogenes was preferable (MIC90: 1 microgram/mL). The antibacterial activities of CZOP against S. marcescens and S. liquefaciens were relatively potent (MIC90: 0.5 and 0.25 microgram/mL) and comparable to those of CPR, CFPM, and carumonam. CZOP preserved comparable antibacterial

  6. Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin

    PubMed Central

    Kirchner, Miranda; Mafura, Muriel; Hunt, Theresa; Abu-Oun, Manal; Nunez-Garcia, Javier; Hu, Yanmin; Weile, Jan; Coates, Anthony; Card, Roderick; Anjum, Muna F.

    2014-01-01

    A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the feces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being blaTEM, dfr, strB, tet(A), and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of blaTEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harboring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year. PMID:25566232

  7. Evaluation of marine bacteriocinogenic enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria.

    PubMed

    Ghomrassi, Hamdi; ben Braiek, Olfa; Choiset, Yvan; Haertlé, Thomas; Hani, Khaled; Chobert, Jean-Marc; Ghrairi, Taoufik

    2016-02-11

    Use of lactic acid bacteria (LAB) as probiotics may provide an alternative to the use of antibiotics in aquaculture. LAB strains isolated from wild fish viscera and skin were evaluated for bacteriocin production and safety aspects (lack of antibiotic resistance, production of virulence factors). 16S rRNA gene sequences revealed the presence of Enterococcus faecium (13 isolates) and Lactococcus lactis (3 isolates) from fish samples. Pulsed-field gel electrophoresis analyses of the 13 enterococci isolates showed that they were all clustered, with greater than 95% similarity. However, RAPD analysis revealed significant molecular diversity between enterococci strains. Six enterococci strains were chosen and evaluated for their antibacterial activities. These strains produced a bacteriocin-like substance and exhibited a broad spectrum of inhibition against pathogenic bacteria isolated from diseased fish, including Streptococcus parauberis, Vagococcus spp., and Carnobacterium maltaromaticum, and in particular against the Gram-negative bacteria Flavobacterium frigidarium, Vibrio pectenicida, V. penaeicida, and Photobacterium damselae. The inhibition activity towards bacterial indicator strains was at a maximum when bacteria were grown at 37°C. However, bacteriocin production was observed at 15°C after 12 h of incubation. Only structural genes of enterocins A and B were detected by PCR in the 6 enterococci strains, suggesting the production of these enterocins. In addition, these strains did not harbor any virulence factors or any significant antibiotic resistance, and they tolerated bile. Our results suggest that enterococci are an important part of the bacterial flora of fish and that some strains have the potential to be used as probiotics. PMID:26865233

  8. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons

    SciTech Connect

    Helmann, J.D.; Walsh, C.T. ); Wang, Ying; Mahler, I. )

    1989-01-01

    The authors report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator region in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.

  9. Tracking Colistin-Treated Patients to Monitor the Incidence and Outcome of Carbapenem-Resistant Gram-Negative Infections

    PubMed Central

    Kadri, Sameer S.; Hohmann, Samuel F.; Orav, E. John; Bonne, Stephanie L.; Moffa, Matthew A.; Timpone, Joseph G.; Strich, Jeffrey R.; Palmore, Tara; Christopher, Kenneth B.; Varughese, Christy; Hooper, David C.; Danner, Robert L.

    2015-01-01

    Background. Existing surveillance mechanisms may underestimate the incidence of carbapenem-resistant gram-negative infections (CRGNIs). Although carbapenem resistance increases the risk of death, the trend in mortality over time is unknown. Methods. A retrospective cohort study was conducted at 40 academic medical centers using a discharge database to identify adult hospital admissions without cystic fibrosis in 2006–2012 and received intravenous colistin for >3 consecutive days or died during therapy (termed colistin cases). The primary outcomes were the number of colistin cases per 100 000 admissions per year and change in the hospital mortality rate over time compared with the rate of discharges to home. Secondary outcomes included median overall and intensive care unit lengths of stay. Results. From 2006 to 2012, a total of 5011 unique patients were identified as colistin cases. The number per 100 000 admissions per year increased from 35.56 to 92.98 during the 7-year study (P < .001). The odds of in-hospital death among colistin cases (compared with discharge to home) decreased by a mean of 5.2%/y (P = .04), whereas discharge to an institution (P = .24) or hospice (P = .89) remained steady over time. The median overall and intensive care unit lengths of stay decreased by 7.5 and 6 days, respectively (P < .001). In a 4-hospital chart review, 81.6% of colistin cases were found to have culture-positive CRGNIs. Conversely, 53% of extensively drug-resistant bloodstream CRGNIs at 2 of these hospitals met colistin case criteria. Conclusions. Colistin cases represent a severely ill population with a high probability of having culture-confirmed CRGNIs. Colistin tracking is a novel strategy for monitoring the incidence and mortality of CRGNIs, particularly those caused by extensively drug-resistant bacteria. Although the incidence of colistin cases nearly tripled within 7 years, more of these patients are surviving hospitalization and going home. PMID:25246597

  10. Aerobic Degradation of Mercaptosuccinate by the Gram-Negative Bacterium Variovorax paradoxus Strain B4 ▿ †

    PubMed Central

    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. PMID:21075928

  11. 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

  12. Target affinities of faropenem to and its impact on the morphology of gram-positive and gram-negative bacteria.

    PubMed

    Dalhoff, A; Nasu, T; Okamoto, K

    2003-07-01

    Faropenem is a new oral beta-lactam antibiotic unique from carbapenems and other available beta-lactams. Determinants of the in vitro activity of beta-lactam antibiotics include affinity to penicillin-binding proteins (PBPs) and beta-lactamase stability. In this study, the binding affinity of faropenem to various PBPs and its impact on the morphology of Staphylococcus aureus and Escherichia coli were evaluated. In general, faropenem demonstrated high binding affinity to high-molecular-weight PBPs but low affinity to low-molecular-weight PBPs. In S. aureus and Streptococcus pneumoniae, faropenem exhibited high binding affinity to PBP1, followed by PBP3 and PBP2. In E. coli, faropenem showed the highest affinity for PBP2, followed by PBP1A, PBP1B, PBP3 and PBP4. In Proteus vulgaris, binding was highest to PBP4, followed by PBP1A, PBP2 and PBP3. In Serratia marcescens, faropenem bound preferentially to PBP2 and PBP4. Exposure of S. aureus to faropenem at minimum inhibitory concentrations (MICs) of 1/8 or 1/4 resulted in irregular septum formation. At 1x MIC or higher, a larger number of lysed cells were observed. Exposure of E. coli to 1/8x MIC or 1/4x MIC also induced changes in cellular shape; the normal rod-shaped form changed to a spherical form in a time-dependent manner. After exposure of E. coli to 1x MIC for 2 h, bulging-shaped E. coli cells were observed and after 4 h of exposure cell lysis was demonstrated. In the presence of 4x MIC, spheroplast-like forms and cell lysis were observed. The morphological changes triggered by faropenem are in agreement with the PBP binding affinities reported. Thus, the high binding affinities of faropenem to PBPs from gram-negative and gram-positive bacteria are mirrored by its pronounced and concentration-dependent bactericidal effect. PMID:12886052

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

    PubMed Central

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

    2015-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  14. Antagonistic activities of some Bifidobacterium sp. strains isolated from resident infant gastrointestinal microbiota on Gram-negative enteric pathogens.

    PubMed

    Delcaru, Cristina; Alexandru, Ionela; Podgoreanu, Paulina; Cristea, Violeta Corina; Bleotu, Coralia; Chifiriuc, Mariana Carmen; Bezirtzoglou, Eugenia; Lazar, Veronica

    2016-06-01

    The gastrointestinal microbiota contributes to the consolidation of the anti-infectious barrier against enteric pathogens. The purpose of this study was to investigate the influence of Bifidobacterium sp. strains, recently isolated from infant gastrointestinal microbiota on the in vitro growth and virulence features expression of enteropathogenic bacterial strains. The antibacterial activity of twelve Bifidobacterium sp. strains isolated from human feces was examined in vitro against a wide range of Gram negative pathogenic strains isolated from 30 infant patients (3 days to 5 years old) with diarrhea. Both potential probiotic strains (Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum, Bifidobacterium breve, Bifidobacterium ruminantium) and enteropathogenic strains (EPEC, EIEC, Klebsiella pneumoniae, Salmonella sp., Yersinia enterocolitica, Pseudomonas aeruginosa) were identified by MALDI-TOF and confirmed serologically when needed. The bactericidal activity, growth curve, adherence to the cellular HEp-2 substratum and production of soluble virulence factors have been assessed in the presence of different Bifidobacterium sp. cultures and fractions (whole culture and free-cell supernatants). Among the twelve Bifidobacterium sp. strains, the largest spectrum of antimicrobial activity against 9 of the 18 enteropathogenic strains was revealed for a B. breve strain recently isolated from infant intestinal feces. The whole culture and free-cell supernatant of B. breve culture decreased the multiplication rate, shortened the log phase and the total duration of the growth curve, with an earlier entrance in the decline phase and inhibited the adherence capacity to a cellular substratum and the swimming/swarming motility too. These results indicate the significant probiotic potential of the B. breve strain. PMID:26921694

  15. Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

    PubMed

    Steinbüchel, A

    1986-04-01

    The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains. PMID:2937774

  16. 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

  17. Antibacterial Activity of Sphingoid Bases and Fatty Acids against Gram-Positive and Gram-Negative Bacteria

    PubMed Central

    Fischer, Carol L.; Drake, David R.; Dawson, Deborah V.; Blanchette, Derek R.; Brogden, Kim A.

    2012-01-01

    There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity—the sphingoid bases d-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid—against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. d-Sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection. PMID:22155833

  18. 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

  19. A preliminary study on gram-negative bacteria (GNB) and their endotoxins in a gin house in India.

    PubMed

    Doctor, P B; Bhagia, L J; Derasari, A Y; Vyas, J B; Amin, R J; Ghosh, S K

    2006-12-01

    The presence of byssinosis, an occupational disease found among cotton mill workers, has been well documented in different parts of the world. The disease develops due to exposure to environmental cotton dust. Evidence suggests that the causative agent for the disease is gram-negative bacteria (GNB) and their endotoxins present on the cotton fibers. An investigation was carried out in a gin house in western India. Environmental dust samples were collected by vertical elutriator (VE). Airborne dust concentrations were very high in the working environment: 2.11 mg/m3 in ginning and 0.95 mg/m3 in the press department (p < 0.05), which was higher than the threshold limit value collected by VE (0.2 mg/m3), and higher than the permissible exposure limit for respirable dust (0.5 mg/m3 for nontextile industries using cotton). In the office control site, the dust concentration was 0.31 mg/m3. The Occupational Safety and Health Administration's cotton dust standard permissible exposure limit for respirable dust is 0.2 mg/m3 in yarn manufacturing, 0.75 mg/m3 in slashing and weaving, and 0.5 mg/m3 in nontextile industries using cotton. These samples also showed high concentrations of airborne endotoxin (p < 0.001) in ginning and pressing (2.77 and 1.52 micro g/m3) compared with the office control site (0.009 micro g/m3 measured by Limulus amoebocyte lysate technique). Total enumeration of airborne GNB was carried out qualitatively by the petri plate exposure method and quantitatively by an Andersen 6-stage viable sampler and VE. GNB were recovered in quite high numbers. Among all the GNB, Enterobacter agglomerans were the dominant bacterial flora. Results indicate that gin workers are occupationally exposed to airborne GNB and endotoxins, and require masks. PMID:17133691

  20. Antibacterial activities of the methanol extracts of ten Cameroonian vegetables against Gram-negative multidrug-resistant bacteria

    PubMed Central

    2013-01-01

    Background Many edible plants are used in Cameroon since ancient time to control microbial infections. This study was designed at evaluating the antibacterial activities of the methanol extracts of ten Cameroonian vegetables against a panel of twenty nine Gram negative bacteria including multi-drug resistant (MDR) strains. Methods The broth microdilution method was used to determine the Minimal Inhibitory Concentrations (MIC) and the Minimal Bactericidal Concentrations (MBC) of the studied extracts. When chloramphenicol was used as a reference antibiotic, the MICs were also determined in the presence of Phenylalanine-Arginine β-Naphtylamide (PAβN), an efflux pumps inhibitor (EPI). The phytochemical screening of the extracts was performed using standard methods. Results All tested extracts exhibited antibacterial activities, with the MIC values varying from 128 to 1024 mg/L. The studied extracts showed large spectra of action, those from L. sativa, S. edule, C. pepo and S. nigrum being active on all the 29 bacterial strains tested meanwhile those from Amaranthus hybridus, Vernonia hymenolepsis, Lactuca.carpensis and Manihot esculenta were active on 96.55% of the strains used. The plant extracts were assessed for the presence of large classes of secondary metabolites: alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, steroids, tannins and triterpenes. Each studied plant extract was found to contain compounds belonging to at least two of the above mentioned classes. Conclusion These results confirm the traditional claims and provide promising baseline information for the potential use of the tested vegetables in the fight against bacterial infections involving MDR phenotypes. PMID:23368430