Diversity, functional classification and genotyping of SHV β-lactamases inKlebsiella pneumoniae

Abstract

AbstractInterpreting phenotypes ofblaSHValleles inKlebsiella pneumoniaegenomes is complex. While all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomalblaSHValleles or additional plasmid-borneblaSHValleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/amino acid changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases (e.g., NCBI’s Reference Gene Catalog and the β-lactamase database (BLDB)) over the specific functionality of individual SHV protein variants. Therefore, identification of ESBL-producing strains fromK. pneumoniaegenome data is complicated.Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific amino-acid substitutions. We then systematically assigned SHV alleles to functional classes (wildtype, ESBL, BLI-resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with current assignments in NCBI’s Reference Gene Catalog and/or BLDB (21 to wildtype, 12 to ESBL, 4 to BLI-resistant). Phylogenetic and comparative genomic analyses support that; i) SHV-1 (encoded byblaSHV-1) is the ancestral chromosomal variant; ii) ESBL and BLI-resistant variants have evolved multiple times through parallel substitution mutations; iii) ESBL variants are mostly mobilised to plasmids; iv) BLI-resistant variants mostly result from mutations in chromosomalblaSHV. We used matched genome-phenotype data from the KlebNET-GSP Genotype-Phenotype Group to identify 3,999K. pneumoniaeisolates carrying one or moreblaSHValleles but no other acquired β-lactamases, with which we assessed genotype-phenotype relationships forblaSHV. This collection includes human, animal, and environmental isolates collected between 2001 to 2021 from 24 countries across six continents. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, while most omega-loop substitutions do not. Our data also provide direct support for wildtype assignment of 67 protein variants, including eight that were noted in public databases as ESBL. We reclassified these eight variants as wildtype, because they lack ESBL-associated mutations, and our phenotype data support susceptibility to 3GCs (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164, SHV-187).The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotypingK. pneumoniaefrom genome assemblies), whereby known and novelblaSHValleles are classified based on causative mutations. Kleborate v2.4.1 was also updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve understanding of resistance mechanisms.Impact statementSince everyK. pneumoniaegenome has an intrinsic SHV β-lactamase and may also carry additional mobile forms, the correct interpretation ofblaSHVgenes detected in genome data can be challenging and can lead toK. pneumoniaebeing misclassified as ESBL-producing. Here, we use matchedK. pneumoniaegenome and drug susceptibility data contributed from dozens of studies, together with systematic literature review of experimental evidence, to improve our understanding ofblaSHVallele variation and mapping of genotype to phenotype. This study shows the value of coordinated data sharing, in this case via the KlebNET-GSP Genotype-Phenotype Group, to improve our understanding of the evolutionary history and functionality ofblaSHVgenes. The results are captured in an open-source AMR dictionary utilised by the Kleborate genotyping tool, that could easily be incorporated into or used to update other tools and AMR gene databases. This work is part of the wider efforts of the KlebNET-GSP group to develop and support a unified platform tailored for the analysis and interpretation ofK. pneumoniaegenomes by a wide range of stakeholders.Data summaryBlaSHVallele sequences and class assignments are distributed with Kleborate, v2.4.1, DOI:10.5281/zenodo.10469001.Table S1provides a summary ofblaSHValleles, including primary accessions, class-modifying mutations, and supporting evidence for class assignments that differ from NCBI’s Reference Gene Catalog or BLDB. Whole genome sequence data are publicly available as reads and/or assemblies, individual accessions are given inTable S2; corresponding genotypes and antibiotic susceptibility phenotypes and measurements are available inTables S3andS4, respectively.