nitrocefin and Escherichia-coli-Infections

Silver nanoparticles (AgNPs) are used as an antimicrobial agent since the ages. However, it is unknown whether AgNPs exert inhibitory effects over the bacterial cells carrying metallo-beta-lactamases (MBLs). Here, using bio-surfactin stabilised AgNPs having a size range from 5 to 25 nm we established its antimicrobial effects against NDMs harbouring cells. Antimicrobial effectiveness of AgNPs is assessed on the E. coli cells carrying New Delhi MBL (NDM) genes, which shows that the cells expressing NDM becomes susceptible to AgNPs and when combined with various groups of beta-lactam a synergistic increase in sensitivity is observed. Purified NDMs are also inhibited by AgNPs as revealed by the hydrolysis of nitrocefin (a chromogenic cephalosporin), though the expression NDM genes remain unchanged. Further, the results obtained from biochemical analysis attribute that the Ag+ ions possibly bind to sulfhydryl (SH) group of cystine in NDMs to inactivate these enzymes. Nonetheless, these AgNPs has the ability to exert antimicrobial activity without affecting the host cell viability when used at a moderate concentration. Overall, we conclude that bio-surfactin-stabilised AgNP is a good candidate to serve as an inhibitor of NDMs, either individually or in combination with beta-lactams.

Topics: Anti-Bacterial Agents; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactams; Biofilms; Cephalosporins; Escherichia coli; Escherichia coli Infections; Metal Nanoparticles; Microbial Sensitivity Tests; Silver; Surface-Active Agents

We explored and evaluated for the first time colorimetric nitrocefin assay in conjunction with the double disc test and PCR assay. We suggested the use of nitrocefin assay for rapid screening of ESBL-production by Enteroaggregative Escherichia coli.

Topics: Animals; Anti-Bacterial Agents; beta-Lactamases; Cephalosporins; DNA, Bacterial; Escherichia coli; Escherichia coli Infections; Humans; Microbial Sensitivity Tests; Polymerase Chain Reaction

β-Lactamase-mediated resistance to β-lactam antibiotics has been significantly threatening the efficacy of these clinically important antibacterial drugs. Although some β-lactamase inhibitors are prescribed in combination with β-lactam antibiotics to overcome this resistance, the emergence of enzymes resistant to current inhibitors necessitates the development of novel β-lactamase inhibitors. In this study, we evaluated the inhibitory effect of dinucleotides on an extended-spectrum class C β-lactamase, AmpC BER. Of the dinucleotides tested, NADPH, a cellular metabolite, decreased the nitrocefin-hydrolyzing activity of the enzyme with a

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Ceftazidime; Cephalosporins; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Escherichia coli; Escherichia coli Infections; Hydrolysis; Indicators and Reagents; Mice; Microbial Sensitivity Tests; NADP; Survival Analysis; Treatment Outcome

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.

Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; beta-Lactamases; Cell Membrane Permeability; Cephalosporins; Drug Resistance, Multiple, Bacterial; Enzyme Activation; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Humans; Hydrolysis; Meropenem; Microbial Sensitivity Tests; Periplasm; Plasmids; Protein Binding; Thienamycins; Young Adult

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed. Hydrolysis rates were obtained and residual enzymatic activity was determined. Cefepime and ertapenem were more active than piperacillin/tazobactam. In contrast, increasing the relative proportion of tazobactam improved susceptibility testing. Twenty micromolar tazobactam inhibited total beta-lactamase activity (as measured by nitrocefin hydrolysis rates) by greater than 75% against all isolates tested: in 11 of 13 E. coli isolates, total beta-lactamase activity was inhibited by 90%. The observed differences between MIC determinations and susceptibility to enzymatic inactivation by tazobactam against E. coli containing AmpC and other -lactamases may be due to the final tazobactam concentration achieved in the periplasmic space. Factors determining this are critical considerations in assessing beta-lactamase inhibitor potency.

Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactams; Cefepime; Ceftazidime; Cephalosporins; Drug Resistance, Bacterial; Drug Therapy, Combination; Enzyme Inhibitors; Ertapenem; Escherichia coli; Escherichia coli Infections; Lactams; Microbial Sensitivity Tests; Penicillanic Acid; Tazobactam