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

Abstract

Interpreting the phenotypes of bla _SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla _SHV alleles or additional plasmid-borne bla _SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla _SHV-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 mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla _SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla _SHV alleles but no other acquired β-lactamases to assess genotype-phenotype relationships for bla _SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla _SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was 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 the understanding of resistance mechanisms.

Keywords: AMR; BLI resistance; K. pneumoniae; SHV; extended-spectrum β-lactamase (ESBL); genotype; prediction; β-lactamase.

Fig. 1.. Amino acid substitution profiles associated with n = 181 bla_SHV alleles. (a) Positions studied in the article by Neubauer et al. and tracked in Kleborate v2 are shown as columns; these include sites where substitutions have a clear association with ESBL activity [148, 179, 238 and 240, omega-loop (positions 164–178)] or BLI resistance activity (69, 234 and 235), plus some sites (25, 35, 146 and 156) that are associated with increased MIC to ceftaroline but not ceftriaxone or inhibitor resistance. Position 179 is also a part of the omega-loop and is specifically separated to show its association with ESBL profiles. Position 130 is not included, as it is found only in SHV-10 (BLI resistant) for which there is no nt sequence available. Each row indicates a unique combination of aas across these variable sites. The aas present in SHV-1 are indicated in grey at the top of the panel, where the omega-loop sequence (Ω) is RWETELNEALPGDARD. (b) The number of unique nt alleles associated with each aa profile (row) is shown as a barplot. Colours indicate the functional class assigned to these alleles, on the basis of the mutations shown here and supporting literature (cited in the text and in Table S1).

Fig. 2.. Cladogram for n = 181 bla_SHV alleles. The cladogram was inferred from a pairwise genetic distance matrix calculated from nt sequences using BioNJ, rooted on SHV-1. Tips are labelled with the SHV allele name and coloured to indicate the mutation profile (black, WT; red, orange and pink, ESBL profiles; and blue and purple, BLI-resistant profiles). For alleles classed as non-WT, the class-modifying mutation is included in the label (e.g. 238S indicates substitution of serine at Ambler site 238 in the encoded protein; ‘omegaINS’ refers to a 6-aa insertion in the omega-loop between Ambler codons 167 and 168). Shading indicates clusters of alleles referred to in the text, which may share class-modifying mutations via vertical inheritance.

Fig. 3.. A comparison of the genomic context of SHV allele clusters. Upstream (10 kbp) and downstream (10 kbp) sequences of each bla_SHV were extracted and aligned. The 7585 bp chromosomal SHV collinear block is highlighted in yellow. Bla_SHV is coloured in blue, while mobile genetic elements, such as insertion sequences and transposons, are illustrated in pink and green, respectively. Percent identity between the genes is shown by the gradient scale bar.

Fig. 4.. AST value distributions for ceftazidime. The size of each circle represents the number of isolates with an SHV allele and no other acquired β-lactamase. (a) MIC and (b) disc diffusion measurements show the distribution of phenotypes for each SHV allele. Average MIC and disc diffusion measurements per SHV allele are indicated by a pink vertical line. Grey circles indicate 1 SHV copy, whilst pink circles indicate >1 SHV copy. SHV alleles are grouped based on ESBL, ESBL- and BLI resistant (EI), BLI resistant (inhibR) and WT phenotype classifications. EUCAST (v13.0) or CLSI (M100 33rd edition) S/I breakpoints are indicated using orange and blue lines, respectively. SHV-187* +69L is SHV-132 in Kleborate v2.4.1. For MIC values, larger values indicate increased resistance; for disc diffusion results, larger zone sizes indicate increased susceptibility.

Fig. 5.. AST value distributions for piperacillin-tazobactam. The size of each circle represents the number of isolates with an SHV allele and no other acquired β-lactamase. (a) MIC and (b) disc diffusion measurements show the distribution of phenotypes for each SHV allele. The average MIC and disc diffusion measurements per SHV allele are indicated by a pink vertical line. Grey circles indicate 1 SHV copy, whilst pink circles indicate >1 SHV copy. SHV alleles are grouped based on ESBL, ESBL and BLI resistant (EI), BLI resistant (inhibR),and WT phenotype classifications. EUCAST (v13.0) or CLSI (M100 33rd edition) S/I breakpoints are indicated using orange and blue lines, respectively. SHV-187* +69L is SHV-132 in Kleborate v2.4.1. For MIC values, larger values indicate increased resistance; for disc diffusion results, larger zone sizes indicate increased susceptibility.

Fig. 6.. Presence of porin defects and copy number effects amongst isolates with WT-assigned alleles with genomes tested against 3GCs. Violin plots show the distribution of susceptibility testing measures, coloured by copy number, for WT SHV alleles (n = 1659 isolates tested against ceftazidime and n = 1937 isolates tested against ceftriaxone). EUCAST (v13.0) or CLSI (M100 33rd edition) I/R breakpoints are indicated using orange and blue lines, respectively.

Fig. 7.. Presence of porin defects and copy number effects amongst isolates with WT-assigned alleles with genomes tested against piperacillin-tazobactam. Violin plots show the distribution of susceptibility testing measures, coloured by copy number, for n = 2268 isolates with WT SHV alleles. EUCAST (v13.0) or CLSI (M100 33rd edition) I/R breakpoints are indicated using orange and blue lines, respectively.