ABSTRACT
A novel metallo-ß-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.
Subject(s)
Proteus mirabilis/drug effects , Proteus mirabilis/genetics , beta-Lactamases/genetics , Aztreonam/pharmacology , Ceftazidime/pharmacokinetics , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Ertapenem , Hydrolysis , Integrons , Microbial Sensitivity Tests , Penicillanic Acid/analogs & derivatives , Penicillanic Acid/pharmacology , Piperacillin/pharmacology , Piperacillin, Tazobactam Drug Combination , Proteus Infections/microbiology , Proteus mirabilis/isolation & purification , United States , beta-Lactamases/metabolism , beta-Lactams/pharmacologyABSTRACT
OBJECTIVES: High levels of ß-lactamase production can impact treatment with a ß-lactam/ß-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. METHODS: Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. RESULTS: mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm. CONCLUSIONS: CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.
Subject(s)
Escherichia coli/enzymology , RNA, Messenger/analysis , beta-Lactamases/analysis , Anti-Bacterial Agents/pharmacology , Cephalosporins/pharmacology , Disk Diffusion Antimicrobial Tests , Escherichia coli/classification , Escherichia coli/genetics , Genotype , Humans , Immunoblotting , Penicillanic Acid/analogs & derivatives , Penicillanic Acid/pharmacology , Piperacillin/pharmacology , Piperacillin, Tazobactam Drug Combination , RNA Stability , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tazobactam , beta-Lactamases/geneticsABSTRACT
High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively.
Subject(s)
Bacterial Proteins/analysis , Bacterial Proteins/genetics , Enterobacteriaceae/enzymology , Multiplex Polymerase Chain Reaction/methods , Plasmids , Real-Time Polymerase Chain Reaction/methods , beta-Lactamases/analysis , beta-Lactamases/genetics , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/microbiology , Humans , Sensitivity and Specificity , Transition TemperatureABSTRACT
A multiplex, real-time TaqMan assay was designed to identify clinical isolates carrying plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 100% when testing characterized AmpC/non-AmpC-producing isolates and randomly selected clinical isolates. This is a rapid assay that can be performed in a clinical microbiology laboratory.
Subject(s)
Bacterial Proteins/genetics , Bacteriological Techniques/methods , Enterobacteriaceae/enzymology , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , beta-Lactamases/genetics , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Enterobacteriaceae Infections/microbiology , Humans , Plasmids , Sensitivity and SpecificityABSTRACT
A previously designed end-point multiplex PCR assay and singleplex assays used to detect ß-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.