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Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics.

ABSTRACT: Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.

SUBMITTER: Fleurbaaij F

PROVIDER: S-EPMC5554287 | biostudies-other | 2017 Apr

REPOSITORIES: biostudies-other

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Epidemiology and genetics of CTX-M extended-spectrum ?-lactamases in Gram-negative bacteria.

Project description:CTX-M enzymes, the plasmid-mediated cefotaximases, constitute a rapidly growing family of extended-spectrum ?-lactamases (ESBLs) with significant clinical impact. CTX-Ms are found in at least 26 bacterial species, particularly in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. At least 109 members in CTX-M family are identified and can be divided into seven clusters based on their phylogeny. CTX-M-15 and CTX-M-14 are the most dominant variants. Chromosome-encoded intrinsic cefotaximases in Kluyvera spp. are proposed to be the progenitors of CTX-Ms, while ISEcp1, ISCR1 and plasmid are closely associated with their mobilization and dissemination.

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Project description:Extended-spectrum ?-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M ?-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL ?-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 ?-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

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Project description:The CTX-M-type extended-spectrum ?-lactamases (ESBLs) present a serious public health threat as they have become nearly ubiquitous among clinical gram-negative pathogens, particularly the enterobacteria. To aid in the understanding and eventual control of the spread of such resistance genes, we sought to determine the diversity of CTX-M ESBLs not among clinical isolates, but in the environment, where weaker and more diverse selective pressures may allow greater enzyme diversification. This was done by examining the CTX-M diversity in municipal wastewater and urban coastal wetlands in southern California, United States, by Sanger sequencing of polymerase chain reaction amplicons. Of the five known CTX-M phylogroups (1, 2, 8, 9, and 25), only genes from groups 1 and 2 were detected in both wastewater treatment plants (WWTPs), and group 1 genes were also detected in one of the two wetlands after a winter rain. The highest relative abundance of blaCTX-M group 1 genes was in the sludge of one WWTP (2.1?×?10(-4) blaCTX-M copies/16S rRNA gene copy). Gene libraries revealed surprisingly high nucleotide sequence diversity, with 157 new variants not found in GenBank, representing 99 novel amino acid sequences. Our results indicate that the resistomes of WWTPs and urban wetlands contain diverse blaCTX-M ESBLs, which may constitute a mobile reservoir of clinically relevant resistance genes.

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