Incidence of diverse integrons and beta-lactamase genes in environmental Enterobacteriaceae isolates from Jiaozhou Bay, China

Environmental microbiology investigation was performed to determine the molecular diversity of beta-lactamase genes among ampicillin-resistant bacteria from Jiaozhou Bay. beta-lactamase genes were detected in 93.8% of the bacterial isolates identified as Enterobacteriaceae. The most frequently detected gene was bla(TEM), followed by bla(SHV), bla(OAX-1), bla(MOX) and bla(CMY). Most of the isolates (68.8%) were positive for the intI1 integrase gene, and two isolates were also found for the intI2 gene. The dfr and aadA gene cassettes were predominant. Anthropogenic contamination from onshore sewage processing plants might contribute predominantly to the beta-lactamase gene reservoir in the studied coastal waters. Environmental antibiotic-resistant bacteria and resistance genes may serve as bioindicators of coastal environmental quality or biotracers of the potential contamination sources. This is the first report of the prevalence and characterization of beta-lactamase genes and integrons in coastal Enterobacteriaceae from China.

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus

After demonstrating the lack of effectiveness of standard antibiotics against the acquired antibiotic resistance of Bacillus cereus (NCTC 10989), Escherichia coli (NCTC 1186), and Staphylococcus aureus (ATCC 12715), we showed that the following natural substances were antibacterial against these resistant pathogens: cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (beta-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine). Exposure of the three pathogens to a dilution series of the test compounds showed that oregano oil was the most active substance. The oils and pure compounds exhibited exceptional activity against B. cereus vegetative cells, with oregano oil being active at nanogram, per milliliter levels. In contrast, activities against B. cereus spores were very low. Activities of the test compounds were in the following approximate order: oregano oil > thyme oil approximate to carvacrol > cinnamon oil > perillaldehyde > dopamine > beta-resorcylic acid. The order of susceptibilities of the pathogens to inactivation was as follows: B. cereus (vegetative) much greater than S. aureus approximate to E. coli much greater than B. cereus (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds.

Burkholderia cenocepacia O polysaccharide chain contributes to caspase-1-dependent IL-1 beta production in macrophages

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1 beta secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1 beta in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1 beta production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1 beta production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation. J. Leukoc. Biol. 89: 481-488; 2011.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Evaluation of the VITEK 2 AST N-054 test card for the detection of extended-spectrum beta-lactamase production in Escherichia coli with CTX-M phenotypes

A new VITEK 2 antibiotic susceptibility testing (AST) card, AST N-054, was introduced for aerobic gram-negative bacilli in 2007 and has been widely adopted for routine use in the UK. We evaluated its performance for detecting extended-spectrum beta-lactamase (ESBL) production in Escherichia coli.

Antibiotic resistance in enterobacteriaceae isolated from Portuguese deli meats

This study aimed to identify the presence of β-lactam-resistant bacteria in different types of Portuguese deli meats. The numbers of ampicillin resistant bacteria varied from negative in 25 g to 1.0 × 108colony-forming units/g. Within 78 randomly selected β-lactam-resistant bacteria, 24 different resistant phenotypes were found and 35.9% were multidrug resistant (MDR). The majority (87.2%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the blaTEM gene was detected in 23 out of 67 isolates (34.3%) and 16 of them presented MDR phenotypes. Four Klebsiella oxytoca isolates (6%) harbored a gene for the CTX-M/OXY-type enzyme. The direct sequencing of their purified amplicons confirmed the presence of three types of blaOXYgenes (blaOXY-1, blaOXY-2 and blaOXY-5). These results suggest that without good hygienic practices, deli meats may act as a vehicle of transfer of β-lactam-resistant bacteria to the gastrointestinal tract of consumers.

Chicken Caecal Microbiome Modifications Induced by Campylobacter jejuni Colonization and by a Non-Antibiotic Feed Additive

Campylobacter jejuni is an important zoonotic foodborne pathogen causing acute gastroenteritis in humans. Chickens are often colonized at very high numbers by C. jejuni, up to 109 CFU per gram of caecal content, with no detrimental effects on their health. Farm control strategies are being developed to lower the C. jejuni contamination of chicken food products in an effort to reduce human campylobacteriosis incidence. It is believed that intestinal microbiome composition may affect gut colonization by such undesirable bacteria but, although the chicken microbiome is being increasingly characterized, information is lacking on the factors affecting its modulation, especially by foodborne pathogens. This study monitored the effects of C. jejuni chicken caecal colonization on the chicken microbiome in healthy chickens. It also evaluated the capacity of a feed additive to affect caecal bacterial populations and to lower C. jejuni colonization. From day-0, chickens received or not a microencapsulated feed additive and were inoculated or not with C. jejuni at 14 days of age. Fresh caecal content was harvested at 35 days of age. The caecal microbiome was characterized by real time quantitative PCR and Ion Torrent sequencing. We observed that the feed additive lowered C. jejuni caecal count by 0.7 log (p<0.05). Alpha-diversity of the caecal microbiome was not affected by C. jejuni colonization or by the feed additive. C. jejuni colonization modified the caecal beta-diversity while the feed additive did not. We observed that C. jejuni colonization was associated with an increase of Bifidobacterium and affected Clostridia and Mollicutes relative abundances. The feed additive was associated with a lower Streptococcus relative abundance. The caecal microbiome remained relatively unchanged despite high C. jejuni colonization. The feed additive was efficient in lowering C. jejuni colonization while not disturbing the caecal microbiome.

Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the bla(CARB-2) (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition bla(TEM) I primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for bla(TEM), aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. bla(TEM)-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.

The multiple antibiotic resistance (mar) locus and its significance

Chromosomally encoded systems involved in low level resistance of bacteria to different classes of antibiotics (mainly beta-lactams, chloramphenicol, quinolones and tetracycline), disinfectants and in resistance to organic solvents have been the focus of considerable interest in recent years. The multiple antibiotic resistance (mar) locus of Escherichia coli and Salmonella is perhaps the best described system involved in this type of resistance which is induced by MarA, the activator protein encoded by the marRAB locus. The mar-locus is reported to mediate resistance primarily by up-regulating efflux of some antibiotics, disinfectants and organic solvents via the AcrAB-TolC efflux pump and down regulating influx through Outer Membrane Protein F (OmpF). Whilst the level of antibiotic resistance conferred by marRAB is only low level, there are increasing data to suggest that marRAB and related systems are important in clinical antibiotic resistance, possibly as a 'stepping stone' to higher levels of resistance. Other related systems include up-regulation of RobA, SoxS and AcrAB which give rise to a similar resistance phenotype to that conferred by up-regulation of MarA. The aim of this paper is to review the function and significance of the mar-locus and related systems with a particular focus on its implications in veterinary medicine. (C) 2002 Published by Elsevier Science Ltd.

Bottled mineral water as a potential source of antibiotic resistant bacteria

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Degradation of the antibiotic amoxicillin by photo-Fenton process - Chemical and toxicological assessment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

β-lactam antibiotics epitope mapping with STD NMR spectroscopy: A study of drug-human serum albumin interaction

Molecular recognition events are key issues in many biological processes. STD NMR (saturation transfer difference nuclear magnetic resonance spectroscopy) is one of the techniques used to understand such biological interactions. Herein, we have investigated the interactions of four β-lactam antibiotics belonging to two classes (cephalosporins and penicillins) with human serum albumin (HSA) by 1H STD NMR revealing that the interaction between the aromatic moiety and HSA is responsible for the binding efficiency. Thus, the structural differences from the five to six-membered thio ring in penicillins and cephalosporins do not seem to influence antibiotic-albumin interactions. © 2012 Sociedade Brasileira de Química.

Estudo da composição de meios de cultura para a produção de cefamicina C por Streptomuces clavuligerus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção e identificação dos genes de beta-lactamases blaSHV, blaTEM e blaCTX-M em Klebsiella penumoniae isoladas em um Hospital Terciário do Estado de São Paulo

Pós-graduação em Microbiologia - IBILCE