Characterization and identification of a novel chromosomal class C β-lactamase, LAQ-1, and comparative genomic analysis of a multidrug resistance plasmid in Lelliottia amnigena P13

Abstract

IntroductionLelliottia amnigena, a bacterium usually isolated from natural environments, may cause human infections and has been suggested to be naturally resistant to second- and third-generation cephalosporins.MethodsIn this study, we determined the whole-genome sequence of an isolate, L. Amnigena P13, isolated from animal farm sewage. On the basis of genome sequence analysis, susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis, we identified a novel chromosome-encoded AmpC β-lactamase, LAQ-1.Results and DiscussionblaLAQ-1 is resistant to penicillin G, ampicillin, and several first- to fourth-generation cephalosporins, such as cefazolin, cefoxitin and cefepime. The MIC levels of some β-lactams, such as cefoxitin, cefepime, aztreonam and cefazolin, for the recombinant clone (pUCP24-blaLAQ-1/DH5α) increased by approximately 4- to 64-fold compared with those of the control strain (pUCP24/DH5α). The kinetic properties of LAQ-1, with the highest catalytic activity observed toward piperacillin, were basically the same as those of typical class C β-lactamases, and avibactam had a strong inhibitory effect on its hydrolytic activity. The genetic background of blaLAQ-1 was relatively conserved, and no mobile genetic element (MGE) was found around it. The plasmid pP13-67 of L. amnigena P13 harbored 12 resistance genes [qnrS1, aph(6)-Id, aadA2, sul1, sul2,blaTEM-1, qacEΔ1, dfrA12, tetA and floR] related to different mobile genetic elements within an ~22 kb multidrug resistance region. The multidrug resistance region shared the highest nucleotide sequence similarities with those of the chromosomes or plasmids of different bacterial species, indicating the possibility of horizontal transfer of these resistance genes among different bacterial species.

Lelliottia amnigena recovered from the lung of a harbour porpoise, and comparative analyses with Lelliottia spp.

“…S2), sharing 99.3 % nucleotide and 99.5 % amino acid pairwise identity with the reference sequence (accession MZ497396). In agreement with [25], we found that bla LAQ-1 encoded by GFKo1 had the obligatory serine active site of the β-lactamase catalytic motif S–V–S–K (serine–valine–serine–lysine) at positions 83–86, the typical class C β-lactamase motif Y–A–N (tryptophan–alanine–asparagine) at positions 169–171, D/E (a peptide segment containing two dicarboxylic amino acids) at positions 236–238 and the conserved triad K–T–G (lysine–threonine–glycine) at positions 334–336 (Fig. S3).…”

“…The Bla LAQ-1 sequence of L. amnigena P13 represents the reference for this chromosomally encoded AmpC β-lactamase [25]. Twelve other AmpC β-lactamases (ACT-12, ACT-22, BIL-1, CMY-2, CMY-20, LAT-1, CFE-1, YRC-1, MIR-1, MIR-23, ACT-6, ACT-10 [25]) were included in the analysis for comparative purposes; the accessions for the amino acid sequences of these proteins are given in parentheses.…”

“…Despite bla LAQ being a core gene of all sequenced L. amnigena isolates, it is evident that broad-spectrum resistance to β-lactam antibiotics is not a uniform feature of the species. Resistance to penicillins is reported frequently, but resistance to specific cephalosporins is highly variable [25,[45][46][47]. Genome sequence data are rarely available for the strains characterized in these studies, making it difficult to determine the genotypic factors that contribute to the observed resistant phenotypes.…”

“…Bakta-annotated protein sequences for all genomes (Table 1) were searched against this sequence, with hits >70 % coverage and >70 % identity retained. The 'hit' protein sequences were extracted from the .faa Bakta-annotated files using Biostrings v2.64.0 (R v4.3.1, RStudio v2023.06.1) and used to create a multiplesequence alignment (Clustal Omega v1.2.2; Geneious Prime v2023.0.1) with the protein sequences of the 12 AmpC β-lactamases (ACT-12, ACT-22, BIL-1, CMY-2, CMY-20, LAT-1, CFE-1, YRC-1, MIR-1, MIR-23, ACT-6, ACT-10) included in the study in which the functionality of the bla LAQ-1 protein was demonstrated [25]. A phylogenetic tree was created from the sequence alignment using PhyML v3.3.20180621 (Blosum62 matrix) [38], with bootstrap values determined based on 100 replications.…”

“…Only rarely have L. amnigena and L. nimipressuralis been associated with opportunistic disease in humans [21][22][23][24]. There are few reports in the literature of the carriage of antimicrobial resistance (AMR) genes by Lelliottia spp., though a new chromosomally encoded AmpC β-lactamase, bla LAQ-1 , conferring resistance to ampicillin and several cephalosporins was recently described for an L. amnigena strain isolated from animal farm sewage in PR China [25,26].…”