Diversity and function of OXA-48-like β-lactamase variants in environmental <i>Shewanella</i> isolates from Stockholm, Sweden

Abstract

The genus Shewanella is a recognized reservoir of antibiotic resistance genes (ARGs), including chromosomally encoded blaOXA alleles that have given rise to clinically relevant OXA-48-like beta-lactamases in Enterobacterales. However, our understanding of these enzymes in environmental Shewanella populations remains limited. Here, we investigated their distribution, evolution, and function in Shewanella spp. isolated from Baltic Sea environments near Stockholm, Sweden. Whole-genome sequencing of 25 isolates affiliated with Shewanella baltica and related genospecies revealed that each carried a chromosomal OXA-48-like beta-lactamase closely related to OXA-551. These enzymes exhibited substantial N-terminal polymorphisms, including indels, defining 20 novel variants. Phylogenetic analyses showed that the diversification of OXA variants closely mirrored the host species taxonomy, suggesting parallel evolution. Phenotypic susceptibility testing demonstrated that native Shewanella hosts remained largely susceptible to carbapenems, third-generation cephalosporins, and, to a lesser extent, ampicillin, suggesting limited expression or activity under native regulatory control. Functional assays using Delta blaOXA mutants of S. baltica strains carrying divergent variants revealed variable contributions to beta-lactam resistance. In contrast, heterologous expression of these enzymes in Escherichia coli conferred high resistance to ampicillin and beta-lactamase activity comparable to the reference OXA-551, as demonstrated by nitrocefin hydrolysis kinetics. Comparative analysis of S. baltica-like blaOXA-48 promoters and those associated with plasmid-borne OXA-48 variants in E. coli revealed conserved sigma70-dependent regulation, besides additional predicted transcription factor binding sites clustered near the -10 box, suggestive of a fine-tuned regulation in Shewanella. Our findings expand functional insights into OXA-48-like beta-lactamases and highlight environmental Shewanella as reservoirs of OXA-48-like diversity.IMPORTANCEAntimicrobial resistance (AMR) is a major global health challenge, with beta-lactamase-mediated resistance undermining the efficacy of last-line antibiotics, such as carbapenems. OXA-48-like carbapenemases, now endemic across various parts of the world, trace their origin to Shewanella species. Understanding how these enzymes diversify, function, and transition from chromosomal genes to transmittable, clinically concerning resistance determinants is critical for AMR surveillance and risk assessment. This study demonstrates that Baltic Sea Shewanella baltica populations harbor diverse OXA-48-like enzymes with relatively limited phenotypic impact in their native hosts, but more active when expressed in E. coli mimicking real-life acquisition. By linking natural sequence variation to enzyme activity, we show that polymorphisms in the N-terminal region of these enzymes do not have substantial functional consequences, indicating that many naturally occurring variants reflect evolutionary mutations that have not affected enzyme performance. These findings reinforce the importance of aquatic environments as reservoirs of AMR determinants poised for mobilization.Antimicrobial resistance (AMR) is a major global health challenge, with beta-lactamase-mediated resistance undermining the efficacy of last-line antibiotics, such as carbapenems. OXA-48-like carbapenemases, now endemic across various parts of the world, trace their origin to Shewanella species. Understanding how these enzymes diversify, function, and transition from chromosomal genes to transmittable, clinically concerning resistance determinants is critical for AMR surveillance and risk assessment. This study demonstrates that Baltic Sea Shewanella baltica populations harbor diverse OXA-48-like enzymes with relatively limited phenotypic impact in their native hosts, but more active when expressed in E. coli mimicking real-life acquisition. By linking natural sequence variation to enzyme activity, we show that polymorphisms in the N-terminal region of these enzymes do not have substantial functional consequences, indicating that many naturally occurring variants reflect evolutionary mutations that have not affected enzyme performance. These findings reinforce the importance of aquatic environments as reservoirs of AMR determinants poised for mobilization.