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

Diversity and function of OXA-48-like β-lactamase variants in environmental Shewanella isolates from Stockholm, Sweden | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diversity and function of OXA-48-like β-lactamase variants in environmental Shewanella isolates from Stockholm, Sweden Víctor Fernández-Juárez, Marija Petrovic, Indiwari Mihindukulasooriya, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8584107/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The genus Shewanella is a recognized reservoir of antibiotic resistance genes (ARGs), including chromosomally encoded bla OXA alleles that have given rise to clinically relevant OXA-48-like β-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, primarily affiliated with Shewanella baltica and related genospecies, revealed that each carried a chromosomal OXA-48-like β-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 Δ bla OXA mutants of S . baltica strains carrying divergent variants revealed variable contributions to β-lactam resistance. In contrast, heterologous expression of these enzymes in Escherichia coli conferred high resistance to ampicillin and β-lactamase activity comparable to the reference OXA-551, as demonstrated by nitrocefin hydrolysis kinetics. Comparative analysis of S . baltica -like bla OXA−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 β-lactamases and highlight environmental Shewanella as reservoirs of OXA-48-like diversity. IMPORTANCE Antimicrobial resistance (AMR) is a major global health challenge, with β-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 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. General Microbiology beta-lactamase Shewanella baltica nitrocefin hydrolysis antimicrobial resistance environmental reservoirs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The genus Shewanella is widespread in aquatic and sedimentary environments worldwide, with one species, Shewanella algae , emerging as a human pathogen of increasing clinical relevance [ 1 ]. Shewanella spp. are progenitors of several subclasses of Ambler class D β-lactamases, [ 2 – 4 ] as well as other antibiotic resistance genes (ARGs) of clinical concern, such as qnrA , found in S . algae and closely related species [ 5 ], conferring resistance to fluoroquinolones, and mcr-4.3 , thought to have originated from Shewanella frigidimarina [ 6 ], which mediates resistance towards colistin, a last resort antibiotic against infections caused by multidrug-resistant Gram-negative bacteria. All these chromosomally encoded Shewanella genes have been identified in an array of mobile genetic elements (MGEs), including plasmids, that are currently widespread among clinically important human pathogens [ 7 – 10 ]. Diverse ARGs occur naturally in environmental bacteria and can be mobilized into conjugative plasmids through recombination and subsequent dissemination via horizontal gene transfer [ 11 ]. This process requires either the coexistence of native (primarily environmental) and non-native (primarily human-associated) bacterial hosts for sufficient periods of time, or efficient transmission routes across distant hosts, that is, from environmental bacterial communities to human bacterial pathogens, which are still poorly delineated. Not infrequently, ARG carriage is not associated with phenotypic resistance in the native bacterial host, which is typically attributed to low expression levels of the gene in question, less functional gene products, or various genetic factors [ 12 , 13 ]. These so-called ‘silent’ genes may nonetheless effectively contribute to resistance when expressed in non-native hosts [ 12 , 13 ]. Class D β-lactamases, also known as oxacillinases (OXAs) due to their substrate preference, are of particular concern due to their widespread occurrence in Enterobacterale s and their recent spread to high-risk, multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumanii [ 14 ]. Among them, OXA-48-like enzymes, the most widespread in Shewanella genomes, are known to contribute to carbapenem resistance and represent the preponderant carbapenemases across large areas of Europe, the Middle East, and Northern Africa [ 15 ]. Within Europe, OXA-48 carbapenemases are so widespread in Enterobacterales that they are currently considered endemic in Belgium, France, and Spain [ 16 ]. Currently, there are nearly 1,400 class D beta-lactamases registered in the Beta-Lactamase DataBase (BLDB) [ 17 ], including 68 OXA-48-like variants, which showcases the extensive sequence variability across this family of enzymes. Some Shewanella -native, chromosome-encoded OXA-48-like oxacillinases such as OXA-54 in Shewanella oneidensis [ 18 ], OXA-55 in S . algae [ 19 ], or OXA-181 in S . xiamenensis [ 20 ] have been characterized biochemically and genetically. However, the evolutionary selective pressures shaping the diversity of OXA proteins and the impact of such diversity on enzyme function are still not well defined. Our team has devoted efforts to the surveillance of antimicrobial resistance (AMR) and ARG dissemination in waterborne bacteria, including Shewanella populations closely related to Shewanella baltica collectively referred to as the S . baltica complex [ 21 ]. Hence, this study was designed to i) assess the diversity of class D β-lactamases in environmental Shewanella retrieved from Baltic Sea environments in the surroundings of Stockholm, Sweden; ii) investigate the distribution of mutations in these enzymes in similar Shewanella hosts worldwide; iii) evaluate the contribution of bla OXA to AMR in its native Shewanella hosts, and iv) functionally characterize novel variants in a heterologous enterobacterial host, mimicking horizontal acquisition. MATERIALS AND METHODS Bacterial isolation Water and sediment samples were collected from Nynäshamn (58° 53' 57.29" N, 17° 57' 2.23" E; May 14, 2022), Notholmen, Tyresö (59°13'58"N 18°18'40"E; May 14, 2022), and Hagaparken (59°21'21.0"N 18°02'37.0"E; April 11, 2022) representing brackish environments in the Baltic Sea (Nynäshamn and Notholmen) or Lake Brunnsviken (Hagaparken), connected to the Baltic Sea through the Ålkistan canal. Shewanella spp. were isolated from water samples upon filtration through 0.45 µm mixed cellulose ester membrane filters (Millipore) and plating on Lyngby’s Iron Agar (LIA) containing 0.04% (w/v) L-cysteine and either colistin (8 mg/L, due to the intrinsic resistance of some Shewanella sp.), or no antibiotic. For isolation from sediments, scooped shoreline sediment samples in 50 ml sterile centrifuge tubes were overlaid with sterile PBS, vigorously shaken, and serially ten-fold diluted before plating on colistin-supplemented or plain LIA. Shewanella -like colonies were identified as H 2 S producers in this medium after incubation at 28 ºC for 24 h, as previously described [ 21 , 22 ], and purified by re-streaking on the same medium. Genus-level identification was achieved by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis (MALDI Biotyper Sirius System, Bruker). Whole-genome sequencing and extraction of bla OXA genes Shewanella strains were grown overnight, and genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) and sequenced, as previously described [ 5 ]. Briefly, 50 ng of genomic DNA was used for library preparation with the MGIEasy FS Library Prep Set (MGI Tech). Equimolar pooled libraries were then circularized using the MGIEasy Circularization Kit (MGI Tech) and sequenced as 2 × 100 bp paired-end reads on an NBSEQ-G400 platform (MGI Tech). Following genome assembly and annotation with BACTPipe v3.1.0, annotated bla OXA alleles were extracted and deposited in the NCBI GenBank under accessions PX283710-PX283731 and PX713573-PX713575. Phylogenetic analyses and bioinformatics Sequence alignments were generated with ESPript 3.0 [ 23 ] using the sequence and structural data of OXA-48, available from the Protein Data Bank (PDB, entry 3HBR), as a reference. A maximum-likelihood phylogenetic analysis of bla OXA amino acid sequences was performed with MEGA X [ 24 ], using a Jones-Taylor-Thornton (JTT) matrix-based model with Gamma (G) distribution and 1000 bootstrap iterations. DnaSP v6 [ 25 ] was used for the calculation of Tajima’s and Fu & Li’s statistics from DNA sequences. A total of 106 sequences from Shewanella baltica (combining the 25 from this study and those publicly available from the NCBI GenBank), 219 sequences from Shewanella algae , 32 sequences from S. oncorhynchi , and 98 sequences from S. xiamenensis ( File S1 ) were aligned using the Clustal Omega tool (EMBL-EBI), in four different batches, respectively. The resulting multiple sequence alignments were used to generate a sequence logo via the WebLogo server ( https://weblogo.berkeley.edu ) and to analyze the distribution of amino acid residues across the alignment, as well as the degree of conservation at variable positions, according the Gonnet substitution matrix. Protein structures and structure-based functional annotations of the OXA enzymes from strains N1WShe5-IV (OXA-1408), N1WShe6 (OXA-1410), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417) were predicted using I-TASSER (Iterative Threading ASSEmbly Refinement) [ 26 ] and the resulting models were visualized and aligned with the Klebsiella pneumoniae OXA-48 reference structure (3HBR) in Pymol v3.0 (Schrödinger, LLC). Putative promoters and transcription factor binding sites were predicted with BPROM ( www.softberry.com ) and visualized using a custom Python script. Synteny plots were generated with the Clinker pipeline [ 27 ]. Strains and growth conditions Escherichia coli TOP10 was grown in Miller’s LB medium at 37°C, supplemented with tetracycline (Tc, 15 µg/ml) when required. Shewanella sp. N1WShe5-IV, N1WShe6, T1SShe5-III, and T1WShe4, along with their isogenic Δ bla OXA mutant derivatives, were routinely cultured in Miller’s LB medium at 28°C. E . coli MFDpir was grown on LB agar supplemented with diaminopimelic acid (0.3 mM) and, when required, Tc, at 37 ºC, except during conjugal mating with Shewanella , that was conducted at 28 ºC. The strains employed in this study are presented in Table S1 . Genetic manipulations To express distinct OXA variants from a promoterless vector, we generated a custom pGEN-MCS derivative [ 28 ], named pGEN-MCS-Tc, in which the bla selection cassette and its promoter had been replaced by a tet cassette and its promoter (obtained from plasmid pSIM27 [ 29 ]) using the XbaI and SpeI restriction sites ( Table S2 ). Divergent bla OXA−551 -like alleles of strains N1WShe5-IV, N1WShe6, T1SShe5-III, and T1WShe4 were cloned into plasmid pGEN-MCS-Tc using the primers indicated in Table S2 , which spanned the gene coding sequence and the 333 bp upstream region containing the putative promoter. The reference bla OXA−551 sequence in the Comprehensive Antibiotic Resistance Database (CARD) (Accession ARO:3005775) and its native promoter, obtained from Shewanella sp. VAX-SP0-4CM-5 (V. Fernández-Juárez, A. J. Martín-Rodríguez, unpublished), were cloned into the same sites. Upon sequence verification by Sanger sequencing, the recombinant plasmids and the empty vector were mobilized into E . coli TOP10 by electroporation. A bla OXA in-frame deletion mutant of Shewanella sp. N1WShe5-IV, T1SShe5-III, and T1WShe4 were generated by allelic replacement following described procedures [ 30 ]. In brief, the upstream (585 bp) and downstream (600 bp) regions flanking the gene coding sequence were sequentially cloned into plasmid pKNG101 using the primers listed in Table S2 . The suicide vector was mobilized into calcium-competent E . coli MFDpir and transferred to Shewanella sp. N1-W-She5-IV by biparental mating. Merodiploids were resolved by plating on LB agar supplemented with 10% sucrose (w/v). Gene deletion was confirmed by gel electrophoresis with PCR primers flanking the recombination sites ( Table S2 ). Antibiotic susceptibility testing The susceptibility of environmental Shewanella isolates and E . coli TOP10 strains expressing the reference or divergent bla OXA−551 variants, or carrying the empty cloning plasmid, towards ampicillin (AMP, 10 µg), ceftazidime (CAZ, 10 µg), cefotaxime (CTX, 30 µg), imipenem (IMP, 10 µg), and meropenem (MEM, 10 µg) was determined by the disc diffusion method. For Shewanella , the medium was supplemented with 0.5% NaCl (w/v), due to the comparatively poor growth observed for these strains on plain Mueller Hinton medium, and the incubation temperature was 28 ºC, instead of 35 ºC, as the latter was found to be nearly non-permissive for the growth of many of the retrieved strains. The minimal inhibitory concentration (MIC) of the same antibiotics to E . coli TOP10 strains was additionally tested by the broth microdilution method, and interpreted following EUCAST guidelines [ 31 ]. Kinetic assays of nitrocefin hydrolysis E . coli TOP10 strains carrying the recombinant bla OXA expression plasmids or the empty vector control were pre-grown in LB broth with Tc overnight at 37 ºC. Overnight cultures were diluted 1:100 in 20 ml of fresh LB medium without antibiotic and grown to an OD 600 of 0.58 ± 0.02 Cell pellets were then obtained by centrifugation at 3273 x g , 4 ºC, and stored frozen at -80 ºC for 24 h. Cell lysis was completed by addition of 2 ml PBS (0.01 M, pH 7.4) to the thawed pellets and sonication on ice (8 x 10 s, with 30 s resting periods between sonication steps, amplitude 50%). Cell debris was removed from the whole-cell lysates by centrifugation at 16000 x g , 4 ºC, 20 min. Upon quantification of total protein content by the BCA method [ 32 ], equal amounts of protein (10 µl) were mixed, in duplicates, with 90 µl of nitrocefin solution in PBS (200 µM, final concentration) inside the wells of a 96-well plate. Nitrocefin hydrolysis was simultaneously monitored by recording the OD 490 (proportional to the generation of its hydrolytic product) and the OD 380 (decay proportional to the loss of intact nitrocefin) every 2 min for 2 h. Data representation and statistical analysis GraphPad Prism v10.0.2 was employed for data visualization and statistics, unless otherwise indicated. RESULTS Baltic Sea Shewanella isolates carry bla OXA−48 -like variants with substantial N-terminal diversity To investigate the diversity of OXA proteins in the local Shewanella populations, we extracted the bla OXA coding sequences from 25 arbitrarily selected and whole genome sequenced Shewanella isolates retrieved from water and sediment samples collected in Nynäshamn, Tyresö, and Hagaparken, as representative members of the natural Shewanella communities in the Baltic Sea region surrounding Stockholm. The species affiliation of the isolates was Shewanella baltica or closely related genospecies. Each isolate harbored a single, chromosomal bla OXA gene, encoding a class D OXA-48-like β-lactamase closely related to the OXA-551 subtype, which altogether represented 20 novel OXA-48-like variants, termed OXA-1401 to OXA-1418, OXA-1428, and OXA-1429. To delineate the evolutionary relationships of these Shewanella OXA β-lactamases within the broader Shewanella OXA-48-like family, a phylogenetic reconstruction was generated using the best-fit model (JTT + G) as implemented in MEGA X (Fig. 1 A). This analysis evidenced well-supported associations that correlate with the taxonomic position of the harboring strains, with the OXA variants of S . baltica and closely related species, such as Shewanella scandinavica , Shewanella vaxholmensis , Shewanella septentrionalis or Shewanella hafniensis clustering apart from those of other more distant species inhabiting similar environments, such as Shewanella oncorhynchi or Shewanella oneidensis , the latter closely related to the archetypal OXA-48 reference of Klebsiella pneumoniae used as an outgroup for the phylogenetic reconstruction (Fig. 1 A). This evidence suggests evolution of OXA enzymes in Shewanella parallel to species diversification. To evaluate whether bla OXA genes were under non-neutral selection in this Shewanella population, we carried out the Fu and Li test (D* = -0.56827, p > 0.10; F* = -0.85327, p > 0.10), which indicated non-significant deviation from neutrality. This was further corroborated by the Tajima test (D = -1.06065, p > 0.10), with this analysis evidencing codons with multiple evolutionary paths. An amino acid sequence alignment, using the sequence and structure of OXA-48 (PDB: 3HBR) and the OXA-551 sequence (CARD entry ARO:3005775) as references, revealed diverse polymorphic sites across the S. baltica OXA proteins, preponderantly located in the N-terminal region of the mature enzyme ( Figure S1 ). This is exemplified in Fig. 1 B with the OXA sequences of isolates N1WShe5-IV (OXA-1408), N1WShe6 (OXA-1410), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417). To understand the structural impact of these mutations, we generated three-dimensional reconstructions of the representative divergent variants using deep-learning algorithms [ 26 ]. Thus, compared to OXA-551, OXA-1408 and OXA-1417 exhibit deletions of five and eight amino acids, respectively (Fig. 1 B), which affect the conformation of the first α-helix or results in its absence (Fig. 2 A, B). In contrast, OXA-1410 and OXA-1413 carry insertions of five amino acids each (Fig. 1 B), which, in the case of OXA-1410, leads to the formation of a second α-helix in the N-terminal region (Fig. 2 C, D). N-terminal mutations and non-conservative substitutions are widespread in Shewanella baltica OXA-48-like enzymes To investigate whether the observed sequence variability was endemic to local S . baltica populations or broadly distributed in this species instead, we retrieved all S . baltica genomes from NCBI GenBank (n = 81, accessed 6 September 2025), extracted their chromosomal bla OXA genes, and, together with the 25 sequences of this study, constructed a sequence logo based on aligned amino acid sequences (Fig. 3 A). The frequency of amino acid residues at each position is shown in Fig. 3 B. These reconstructions evidenced two regions that concentrated most amino acid substitutions, insertions, or deletions, located at positions 27–30 and 61–79 in the alignment. Key amino acids involved in catalysis by serine beta-lactamases such as OXA-48, namely the catalytic serine (position 114 in the sequence logo), carboxylated lysine (position 117), and the universal KTG motif (positions 252–254) [ 33 ] were highly conserved, presumptive of activity preservation despite sequence variation. Most amino acid deviations from the consensus sequence across the 106 analyzed OXA-48-like proteins (Fig. 3 C) were non-conservative, consistent with the frequent insertions and deletions at the N-terminus. For comparative purposes, we performed the same analysis on OXA sequences retrieved from well-represented Shewanella species in terms of genome sequence availability, with variable degrees of taxonomic relatedness with respect to S . baltica , namely S. oncorhynchi (n = 32; OXA-48-like), S. xiamenensis (n = 98; OXA-48-like), and S. algae (n = 219, OXA-55-like), which, in contrast to S. baltica , exhibited a much higher degree of homogeneity, reflected in fewer mutations and the absence of insertions or deletions ( Fig. S2–S4 ). This pattern could indicate higher intrinsic mutation rates in S. baltica compared to other Shewanella species, or alternatively, reflect the influence of selective pressures. Baltic Sea Shewanella exhibit discrete phenotypic resistance towards β-lactam antibiotics Next, to investigate whether bla OXA carriage was associated with phenotypic resistance in S . baltica isolates, the susceptibility profiles of the 25 strains were determined towards a panel of β-lactam antibiotics comprising ampicillin (AMP, 10 µg), meropenem (MEM, 10 µg), imipenem (IMP, 10 µg), ceftazidime (CAZ, 10 µg), and cefotaxime (CTX, 30 µg). The results of disc diffusion tests are presented in Fig. 4 A. Growth inhibition diameters were in the range 15.5–27.1 mm for AMP, 32.1–40.2 mm for MEM, 31.3–40.5 mm for IMP, 30.6–36.8 mm for CAZ and 35.8–44.4 mm for CTX, which, in relative terms, given the absence of specific guidelines for the interpretation of disc diffusion tests with this group of microorganisms, evidenced a high degree of susceptibility to carbapenems (IMP, MEM) and third generation cephalosporins (CAZ, CTX), whereas susceptibility was comparatively lower towards the aminopenicillin ampicillin (AMP). Altogether, these results implied a rather discrete role of bla OXA in S . baltica in mediating substantial resistance towards β-lactam antibiotics under our test conditions. To further dissect the functional role of bla OXA in S. baltica , we generated Δ bla OXA in-frame deletion mutants from three representative strains, N1WShe5-IV (OXA-1408), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417), which captures the structural diversity of the OXA variants within the studied strains (Fig. 1 B, and Fig. S1 ). Attempts to generate the same mutant in the N1WShe6 background failed because of the intrinsic resistance of this strain to sucrose-mediated counter-selection, which is widespread in Shewanella . Mutants were screened, in triplicate, against the same panel of antibiotics, using their respective wild-type strains as references. Our results revealed a statistically significant contribution of bla OXA to AMP resistance in all strains (Fig. 4 B-D), as well as to IMP and MEM in strains N1WShe5-IV and T1SShe5-III (Fig. 4 C-D). Despite a seemingly modest biological impact, these findings confirm the expression and functional activity of the gene product under our experimental conditions. Heterologous expression of Shewanella OXA-551-like enzymes in Escherichia coli stimulates β-lactamase activity There is evidence that OXA β-lactamases originating from Shewanella spp. are currently widely distributed in enterobacterial multidrug resistance plasmids (Fig. 5 A). To investigate the potential impact of the N-terminal mutations found in the OXA-551-like enzymes of our isolates on β-lactamase activity, we cloned the bla OXA gene of the same four representative strains along with its native promoter sequences into a promoter-less plasmid. The same approach was taken for the cloning of OXA-551, used as a reference. In both disk diffusion and broth microdilution assays, E. coli TOP10 expressing either OXA variant showed similar susceptibility to β-lactam antibiotics to the empty vector control (Fig. 5 B, C). A clear exception was observed for ampicillin, where OXA expression enhanced the resistance phenotype, elevating MICs to > 200 µg/ml. The results from antibiotic susceptibility tests suggested a similar performance of the novel OXA variants with respect to OXA-551. To further characterize this, we studied the kinetics of the hydrolytic degradation of nitrocefin, a chromogenic cephalosporin substrate, using cell lysates of E . coli TOP10 expressing either OXA-551 or the four exemplary variants OXA-1408, OXA-1410, OXA-1413 or OXA-1417. The results of this assay (Fig. 5 D) demonstrated similar performance of all the enzymes, with the two variants carrying N-terminal insertions, OXA-1410 and OXA-1413, exhibiting somewhat higher activity than OXA-551 or their counterparts carrying N-terminal deletions. As bla OXA genes are not native to E . coli , we next interrogated factors regulating the expression of in trans -acquired bla OXA−48 alleles. Thus, we compared the promoter region of S . baltica bla OXA with those of a set of bla OXA alleles found in E . coli plasmids mediating antibiotic resistance (Fig. 6 ) BPROM analysis identified canonical σ 70 -type promoters upstream of all analyzed genes, although the predicted regulatory complexity varied markedly between sequences. While several promoters harbor binding sites for multiple global regulators including e.g. CRP, IHF, or RpoH, others show a more restricted regulatory architecture, with only binding sites for redox-associated regulators such as ArcA and FNR predicted in the promoter. Another signature of a putative distinct metabolic regulation of bla OXA expression is the presence of predicted ArgR binding sites in certain promoters, suggesting regulation of gene expression by arginine metabolism. The S . baltica promoter displays a distinct organization, including different RpoD binding motifs and predicted binding sites for CRP and CarP (Fig. 6 ). The predicted binding of CarP putatively links bla OXA expression with arginine and pyrimidine metabolism [ 34 , 35 ]. Noteworthily, two of the exemplary promoters from E . coli plasmids (found in sequences CP194965.1 and CP048327.1, Fig. 6 ) are predicted to have unusually long 5’ untranslated regions (5’ UTRs), which might affect mRNA stability and OXA expression. DISCUSSION AMR represents one of the greatest public health concerns of our times, with an estimate of over 10 million annual deaths by the year 2050 directly attributable to or associated with infections caused by drug-resistant pathogens [ 36 ]. With broad spectrum penicillins and cephalosporins being the most prescribed antibiotics worldwide [ 37 ], the dissemination of β-lactamase resistance across bacterial pathogens is a growing concern. OXA-48-like β-lactamases are particularly problematic because of the low levels of phenotypic resistance associated with their carriage in vitro , despite evidence linking bla OXA carriage with failure of carbapenem-based antimicrobial chemotherapy [ 38 ]. Our study showed, indeed, that the phenotypic resistance of S. baltica -like strains naturally carrying a chromosomally encoded bla OXA gene (which is a widespread genomic characteristic in the genus Shewanella [ 3 ]) was weak towards a panel of diverse β-lactam antibiotics including ampicillin, which is typically proficiently hydrolyzed by OXA-48-like enzymes [ 39 , 40 ]. This is not uncommon and could be related to variability in OXA expression under the experimental conditions. For example, a recent study involving diverse carbapenem-susceptible and a carbapenem-resistant S . algae isolate demonstrated higher transcript levels of bla OXA mRNA in the resistant isolate as compared to the susceptible strains, despite all strains carrying minimally different bla OXA alleles [ 4 ]. This is also consistent with our mutational analyses, with Δ bla OXA deletion mutants displaying, overall, relatively discrete susceptibility defects towards β-lactam antibiotics as compared to the corresponding WT parental strains. While there is wide agreement on Shewanella spp. as progenitors of OXA-48-like β-lactamases, it is less clear whether these enzymes have mobilized from the chromosome of the Shewanella donors into enterobacterial plasmids directly or through intermediate hosts [ 41 ]. OXA-48-like enzymes preponderantly disseminate across clinically relevant Enterobacterales within broad-host conjugative IncL/M-type conjugative plasmids associated with Tn 1999 insertion [ 9 , 42 ]. Other OXA-48-like variants originating from Shewanella have been found in plasmids with other replicon types, including ColE2, IncX2, IncN1, IncT and IncA/C [ 9 ]. Novel allelic variants of Shewanella -derived OXA-48-like enzymes are emerging, in part due to increasing surveillance and sequencing efforts, raising clinical concerns [ 43 ]. A heterologous analysis of Shewanella oneidensis OXA-54 has previously been performed through in trans expression under a lac promoter on a high-copy-number plasmid [ 18 ]. In our study, we employed a nature-mimicking approach to simulate the horizontal acquisition of S . baltica bla OXA by an enterobacterial host. To that end, instead of expressing the gene from an inducible promoter, we cloned native genes and their promoter regions into a low copy-number, promoter-less plasmid that is stable in E . coli in the absence of antibiotic selective pressure [ 44 ]. This approach circumvents the bias introduced by protein overexpression from E . coli -adapted promoters or high copy number expression shuttles, allowing a more realistic assessment of phenotypic resistance acquisition. We showed that, compared to the phenotypic resistance exhibited by the native hosts, expression of S . baltica OXA-48-like in E . coli stimulates phenotypic resistance against AMP while displaying discrete phenotypic resistance to carbapenems or third generation cephalosporins, consistent with phenotypic evidence of OXA-48-like carriage by clinical strain subsets [ 45 ] or heterologous functional studies with other Shewanella OXA-48-like enzymes [ 43 ]. OXA enzymes show remarkable sequence diversity. In Shewanella , the phylogenetic distribution of OXA-48-like carbapenemases resembles the taxonomy of the genus, suggesting parallel evolution across different taxa [ 2 ]. We have shown here that S . baltica and closely related species harbor an array of OXA-48-like variants that differ primarily in their N-termini, in some cases presenting in-frame insertions or deletions. In fact, of the 25 strains of this study, 20 carried a novel OXA variant, which showcases the still unknown environmental diversity pool of ARGs and the evolutionary forces that drive their diversification. Through a comparative analysis of two populated whole genome-sequenced clades, S . baltica and S . algae , we have shown that S . baltica OXA-48-like enzymes are substantially more variable and accumulate more non-conservative mutations than those encoded in the genomes of other Shewanella species. From a genomic standpoint, S . baltica is known to be a highly heterogeneous and recombination-prone clade [ 21 , 46 – 48 ], in contrast to S . algae , which is substantially more uniform [ 47 , 48 ]. The higher degree of sequence variability in S . baltica OXA-48-like enzymes could indicate higher intrinsic mutation frequency in this species as compared to S . algae , which is currently unknown, although it could also be the result of evolutionary forces favoring the propagation of N-terminal mutations. In OXA-48-like enzymes, residues in or around the β5-β6 loop and Ω loop are important determinants of enzymatic activity and substrate specificity [ 49 – 52 ]. While none of these regions are in the N-terminus of the protein, the high degree of N-terminal sequence and structural divergence has been previously highlighted in novel OXA-48 variants [ 53 ]. Given the prima facie marginal contribution to phenotypic resistance in the native host, we reasoned that mutations affecting enzyme function might have minimal fitness consequences. We therefore studied whether rearrangements in the N-terminal region of S . baltica OXA proteins could potentially affect enzyme performance. Computational analyses using four representative OXA enzymes carrying five amino acid insertions or deletions with respect to the closest OXA-48-like subtype, OXA-551, predicted structural alterations affecting N-terminal α-helices. Our experimental analyses of OXA-1408, OXA-1410, OXA-1413, and OXA-1417 demonstrated that in-frame alterations in this region do not substantially affect β-lactamase activity in vivo when expressed in a heterologous host. Similarly, in vitro antibiotic hydrolysis assays showed that the enzymes harboring insertions exhibit a modestly increased hydrolytic capacity than OXA-551 or enzymes carrying N-terminal deletions. The lack of non-deleterious mutations also implies that, despite their overall low contribution to phenotypic resistance, OXA enzymes may play a significant role in the eco-physiology of Shewanella and support the propagation of mutant variants without functional consequences across natural populations. Promoter architecture and regulatory variability have emerged as important determinants of ARG expression [ 54 ]. Promoter sequence variants of clinically relevant ARGs, including bla OXA−48 , are associated with differences in expression levels under various environmental conditions, and specific transcription factors such as FNR, ArcA or ArgR can influence expression in response to distinct metabolic or stress signals [ 54 ]. Experimental work in other systems has also demonstrated that point mutations within core promoter elements, such as the − 10 box, can modulate β-lactamase expression and resistance levels, as seen for the bla OXA−61 promoter in Campylobacter jejuni [ 55 ] and in chromosomal β-lactamase promoters of Klebsiella oxytoca where alterations in -10 and − 35 motifs significantly changed promoter strength [ 56 ]. Horizontal mobilization of ARGs can include adjacent upstream sequences, such as parts of the native promoter region. In these cases, binding sites for transcription factors present in the original chromosomal context would also be co-transferred, making them potentially available to influence gene expression in the new host. In this study, we have shown that the S . baltica bla OXA promoter is predicted to accommodate the binding of global regulators such as RpoD and CRP, as well as CarP. In E . coli , CarP acts as a pyrimidine-mediated repressor of carAB transcription [ 34 , 35 ], which encodes carbamoyl phosphate synthase, a key enzyme in arginine and pyrimidine biosynthesis, thereby potentially extending previously proposed links between bla OXA expression and arginine metabolism [ 54 ]. Of note, CarP is also necessary for the resolution of ColE1 plasmid multimers [ 34 ]. Besides, in our comparative analysis of the S . baltica bla OXA promoter with respect to E . coli plasmid-borne counterparts, we identified some unusually long 5’ UTRs (> 200–300 nt), as predicted by BPROM. Such lengths are atypical, considering that the length of most 5’ UTRs in E . coli genes is 25–35 nt, although 5’ UTRs as long as 700 bp have also been reported [ 57 ]. The length of 5’ UTRs have profound effects on mRNA stability and translation [ 58 – 60 ], which could potentially relate to the known variability in the levels of phenotypic resistance associated with bla OXA carriage in different bacterial hosts and cellular physiological contexts [ 12 ]. While in silico predictions require empirical validation, these initial observations warrant further investigation. Taken together, this work reaffirms Shewanella communities as a substantial reservoir of OXA diversity in aquatic environments, prone to potential horizontal dissemination. Declarations Acknowledgements This work was funded by grants from the Swedish Research Council for Sustainable Development (FORMAS, Ref. AC-2023/0032), the Längmanska Kulturfonden (Ref. BA25-0527), and the Hans Dahlbergs Stiftelse för Miljö och Hälsa to AJMR. EJ, ÅS, and AJMR would like to thank the EU and Swedish Research Council for funding in the frame of the collaborative international consortium PARRTAE (Reference Number: ID 351), financed under the ERA-NET Aquatic Pollutants Joint Transnational Call (GA nº869178). This ERA-NET is an integral part of the activities developed by the Water, Oceans and AMR Joint Programming activities. MP is grateful to the AMGEN Foundation for a scholarship. References Martin-Rodriguez AJ (2025) Shewanella algae. Trends Microbiol 33(8):920–921 Tacão M, Araújo S, Vendas M, Alves A, Henriques I (2018) Shewanella species as the origin of blaOXA-48 genes: insights into gene diversity, associated phenotypes and possible transfer mechanisms. Int J Antimicrob Agents 51(3):340–348 Dong N, Zhang Y, Wu Y, Ju X, Yan Z et al (2025) Genetic insights into Shewanella spp., progenitor of the bla (OXA-48)-like genes: a large-scale study. 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Sci Rep 15(1):26632 Zeng X, Brown S, Gillespie B, Lin J (2014) A single nucleotide in the promoter region modulates the expression of the beta-lactamase OXA-61 in Campylobacter jejuni. J Antimicrob Chemother 69(5):1215–1223 Fournier B, Gravel A, Hooper DC, Roy PH (1999) Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca. Antimicrob Agents Chemother 43(4):850–855 Kim D, Hong JS, Qiu Y, Nagarajan H, Seo JH et al (2012) Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling. PLoS Genet 8(8):e1002867 Adams PP, Baniulyte G, Esnault C, Chegireddy K, Singh N et al (2021) Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping. Elife ;10 Chen F, Cocaign-Bousquet M, Girbal L, Nouaille S (2022) 5'UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability. Front Microbiol 13:1088941 Evfratov SA, Osterman IA, Komarova ES, Pogorelskaya AM, Rubtsova MP et al (2017) Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli. Nucleic Acids Res 45(6):3487–3502 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementalR.pdf Supplemental information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8584107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573462063,"identity":"6525e8ed-fcd4-48a6-866e-c66697dcd9a9","order_by":0,"name":"Víctor Fernández-Juárez","email":"","orcid":"https://orcid.org/0000-0002-8090-5154","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Víctor","middleName":"","lastName":"Fernández-Juárez","suffix":""},{"id":573462110,"identity":"184c10dc-b215-4037-b9d9-1b82b9c307be","order_by":1,"name":"Marija Petrovic","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Marija","middleName":"","lastName":"Petrovic","suffix":""},{"id":573468427,"identity":"3f313d9d-1109-4ef1-82f7-61650ff7cfcf","order_by":2,"name":"Indiwari Mihindukulasooriya","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Indiwari","middleName":"","lastName":"Mihindukulasooriya","suffix":""},{"id":573468428,"identity":"62868722-fe82-4bdc-a8c2-afadba55e2e9","order_by":3,"name":"Enrique Joffré","email":"","orcid":"https://orcid.org/0000-0003-0328-518X","institution":"Karolinska Institute and Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"","lastName":"Joffré","suffix":""},{"id":573468429,"identity":"d280e7e0-e1ad-498a-8796-b0f880e37478","order_by":4,"name":"Åsa Sjöling","email":"","orcid":"https://orcid.org/0000-0003-1529-1720","institution":"Karolinska Institute and University of Gothenburg","correspondingAuthor":false,"prefix":"","firstName":"Åsa","middleName":"","lastName":"Sjöling","suffix":""},{"id":573468430,"identity":"d00951d4-369d-4a1d-848c-78362b71c37e","order_by":5,"name":"Alberto J. Martín-Rodríguez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABMklEQVRIie2QMUvDQBTH3xFol5Ssl0H9BMKVwKFQ7FcxFNJFcZMOHa7LZTl1rd/E8cJBp6RZMziYJV0NYkmXatRUEpIPIHi/4e7eu/vxfxyARvMHQaxeXVar/Noss3nZUOTh3FBs0a1886vUs4CE3a8N/y54e50/w6kfbV7SJ7ix+tdp8M5HYydUDBWz9mBiPcFylQENp0PmhnD+KDZEHXHPoNGCGWY7Ci2vCJY9BVR6iLkcCEk8UDZXPRojZiDepTiF3JdKnDWUD9N5QAzt9l0KxQEvlaSWEuRcYjJYMBi0Pw2JkJ5F98qkSYaWLsfEFhkoWE8IDgOmzFVLGfrCSWZbdUxjD/IdHxGr7xl5cXsxtsQ0TYt5W6mCzarGP19/KGVLADjp6JXjFp1tjUaj+ad8AlkEdDnjzqksAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2422-129X","institution":"Karolinska Institute and University of Las Palmas de Gran Canaria","correspondingAuthor":true,"prefix":"","firstName":"Alberto","middleName":"J.","lastName":"Martín-Rodríguez","suffix":""}],"badges":[],"createdAt":"2026-01-12 17:05:38","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8584107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8584107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100410681,"identity":"aaf9864c-7eed-4f23-9970-171d236917c8","added_by":"auto","created_at":"2026-01-16 13:08:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179087,"visible":true,"origin":"","legend":"","description":"","filename":"MsblaOXAclean.docx","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/587bdc767474628f5e487eff.docx"},{"id":100410791,"identity":"3556525e-832a-46c8-ac0c-6da322f49ce5","added_by":"auto","created_at":"2026-01-16 13:09:07","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs8584107.json","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/a2997bdd71c6a95e1ef0237c.json"},{"id":100409507,"identity":"edec1ffa-5e48-4c6b-b5c6-75c454e1f0b0","added_by":"auto","created_at":"2026-01-16 13:07:16","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146329,"visible":true,"origin":"","legend":"","description":"","filename":"rs85841071enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/6042441befb2e9a0eda9ec1d.xml"},{"id":100411141,"identity":"4ca6c9b2-a86f-4146-b4a4-6e6807497881","added_by":"auto","created_at":"2026-01-16 13:09:55","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144206,"visible":true,"origin":"","legend":"","description":"","filename":"rs85841071structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/4b93863f3c9ae0f9571f0d9e.xml"},{"id":100410769,"identity":"0d52ae00-c888-40bb-9578-c19d839a74f2","added_by":"auto","created_at":"2026-01-16 13:08:54","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161956,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/db34b5923f682f535fd18f9c.html"},{"id":100411137,"identity":"e36612ef-d0ce-4a9c-978e-7b58607a2f27","added_by":"auto","created_at":"2026-01-16 13:09:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":985538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiversity and phylogeny of OXA-48-like variants of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. baltica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e complex strains. A.\u003c/strong\u003e Phylogenetic reconstruction of precursor sequences of OXA enzymes extracted from the whole genome sequences of the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e strains of this study and representative sequences from closely related and more distant \u003cem\u003eShewanella\u003c/em\u003especies, as well as the reference OXA-48 sequence. GenBank accessions of full-length protein sequences are given in parentheses. The maximum-likelihood evolutionary relationships were reconstructed using the JTT+G model after 1000 bootstrap iterations. \u003cstrong\u003eB. \u003c/strong\u003eSequence alignment of OXA-48, OXA-551 (CARD accession \u003ca href=\"https://card.mcmaster.ca/ontology/44237\"\u003eARO:3005775\u003c/a\u003e), and a novel variant carried by strain N1WShe5-IV (OXA-1408) presenting diverse polymorphisms with respect to OXA-551. Secondary structural elements are based on the crystal structure of OXA-48 (PDB entry \u003ca href=\"https://www.rcsb.org/structure/3HBR\"\u003e3HBR\u003c/a\u003e). Red and green shadows in the plot indicate deletions and insertions, respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/46a0687a8e58aca430ab4a05.png"},{"id":100411133,"identity":"2e47c295-ffdd-4389-b5ba-3ffa2390bd77","added_by":"auto","created_at":"2026-01-16 13:09:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5506119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein structure prediction of OXA-48-like variants of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. baltica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains. \u003c/strong\u003ePredicted tridimensional structures of OXA variants for (\u003cstrong\u003eA\u003c/strong\u003e) N1WShe5-IV (OXA-1408) and (\u003cstrong\u003eB\u003c/strong\u003e) T1WShe4 (OXA-1417), shown as examples of N-terminal deletions, and (\u003cstrong\u003eC\u003c/strong\u003e) N1WShe6 (OXA-1410) and (\u003cstrong\u003eD\u003c/strong\u003e) T1SShe5-III (OXA-1413), shown as examples of N-terminal insertions, and all aligned with the reference crystal structure of OXA-48 from \u003cem\u003eK. pneumoniae\u003c/em\u003e (PDB: 3HBR), shown in light pink. The signal peptide in each structure is shown in grey, and alpha-helix variations in the N-terminal part of the protein are depicted in different colors.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/ee96ce26a0077254050c64b5.png"},{"id":100422476,"identity":"a45ddc6b-bd6b-41df-bf74-3df3896b0fe7","added_by":"auto","created_at":"2026-01-16 14:07:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVariability of OXA-48-like sequences in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. baltica \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecomplex strains. A. \u003c/strong\u003eMultiple sequence alignment of the amino acid sequence obtained in this study, along with publicly available sequences from NCBI (81 genomes downloaded on September 6, 2025). Each sequence logo displays stacks of symbols representing the different amino acids, one for each position in the alignment, and the relative height of each letter indicates the frequency of the corresponding amino acid.\u003cstrong\u003e B. \u003c/strong\u003eStacked bar chart of amino acid residue frequencies per alignment position across the N = 106 sequences. Each bar shows the counts of residues observed at that site, color-coded by residue identity. \u003cstrong\u003eC. \u003c/strong\u003eDistribution of amino acid substitutions relative to the consensus sequence (MRSFAISTVLVMSSLLASSIIAAPTFAPTFASTAAKTEWQETRSWDAIFTQHQVEPQQAKQQQAKQQQAKQQQAKPQKTKSQQASGVVVLWNENKQQGYTNNLKRANQGFLPASTFKIPNSLIALELGVVKDEHQVFKWDGKSRDIATWNRDHNLITGMKYSVVPVYQEFARQIGEARMSKMIASFDYGNEDISGNLDSFWLDGGIRISATEQIDFLRRLYHNKIHASERSQRIVKQAMLTEANSDYIIRAKTGYAVRAEPSIGWWVGWVELDDNVWFFAMNMDIPDAAGLPLRQAITKEVLKLEHVIP) across the 106 aligned sequences. At each alignment position, non-consensus residues are shown as stacked bars and are color-coded by residue ID. The horizontal bar above the plot represents the degree of conservation at each position, i.e., green indicates positions with no substitutions, blue denotes conservative substitutions, orange indicates semi-conservative substitutions, and red marks non-conservative substitutions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/1b73811eeb232b00c1cece2c.png"},{"id":100422236,"identity":"2c927033-cb6b-4519-87bf-bf5bb03d416d","added_by":"auto","created_at":"2026-01-16 14:07:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":715428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSusceptibility profiles of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. baltica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e complex isolates to β-lactam antibiotics and effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eOXA\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e deletion on antibiotic resistance. A. \u003c/strong\u003eInhibition zones, determined by the disc diffusion method, of 25 \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e strains using discs loaded with ampicillin (AMP, 10 µg), ceftazidime (CAZ, 10 µg), cefotaxime (CTX, 30 µg), imipenem (IMP, 10 µg), and meropenem (MEM, 10 µg). \u003cstrong\u003eB. \u003c/strong\u003eComparative susceptibility data of N1WShe5-IV (OXA-1408), N1WShe6 (OXA-1410), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417) wild-type (WT) and Δ\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA \u003c/sub\u003emutants on susceptibility towards the same β-lactam antibiotics. The statistical significance of the differences were determined with unpaired t-tests, using \u003cem\u003eP\u003c/em\u003e = 0.05 as threshold (* \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; ns = not significant).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/c6cd7b5646bea10120a9431b.png"},{"id":100410524,"identity":"2b2c7585-e879-45c7-ab5f-8611ed43f2bc","added_by":"auto","created_at":"2026-01-16 13:08:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1288833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePerformance of reference and novel OXA variants in a heterologous host. A. \u003c/strong\u003eSynteny plot showcasing the integration of \u003cem\u003eShewanella\u003c/em\u003e chromosomal \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e and an adjacent LysR-type transcriptional regulation into an enterobacterial plasmid, illustrating horizontal transmission to \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. \u003cstrong\u003eB.\u003c/strong\u003e Susceptibility of \u003cem\u003eEscherichia coli \u003c/em\u003eTOP10 expressing OXA-551, OXA-1408, OXA-1410, OXA-1413, and OXA-1417, or carrying the empty cloning plasmid (VC, vector control) towards β-lactam antibiotics, expressed as inhibition zones from disc diffusion assays. \u003cstrong\u003eC.\u003c/strong\u003e Minimal inhibitory concentrations of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 strains towards β-lactam antibiotics, expressed as µg/ml (S = susceptible; R = resistant). \u003cstrong\u003eD. \u003c/strong\u003eKinetics of nitrocefin hydrolysis by whole-cell lysates of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli \u003c/em\u003eTOP10 strains expressing either OXA variant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/1d8ca2679173a97a69930625.png"},{"id":100410400,"identity":"73183449-b69c-43fa-a270-d950eac94884","added_by":"auto","created_at":"2026-01-16 13:08:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromoter architecture of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eOXA \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003egenes. \u003c/strong\u003ePredictions of σ⁷⁰-type promoters and transcription factor binding sites for selected DNA regions upstream \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e alleles are shown. Each horizontal black line represents the intergenic region upstream of a given \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e open reading frame, oriented 5′-3′, with the start codon (ATG) indicated at the right end. Vertical lines mark key motifs: blue lines indicate -10 boxes, red lines indicate -35 boxes, and color-coded vertical ticks represent transcription factor binding sites. The scale is approximate.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/d12ae53e025ffacca5cdf76e.png"},{"id":100424109,"identity":"a85e5576-8db0-4248-a89b-8940eab89043","added_by":"auto","created_at":"2026-01-16 14:15:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18662625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/fe29ecfb-c111-4a1e-a78d-4cb23db59016.pdf"},{"id":100411160,"identity":"f12f82de-42e6-49ef-a1a8-9bb425d6dbd7","added_by":"auto","created_at":"2026-01-16 13:10:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1495438,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental information\u003c/p\u003e","description":"","filename":"SupplementalR.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8584107/v1/bdf399beaacbe4fdd370172c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDiversity and function of OXA-48-like β-lactamase variants in environmental \u003cem\u003eShewanella\u003c/em\u003e isolates from Stockholm, Sweden\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe genus \u003cem\u003eShewanella\u003c/em\u003e is widespread in aquatic and sedimentary environments worldwide, with one species, \u003cem\u003eShewanella algae\u003c/em\u003e, emerging as a human pathogen of increasing clinical relevance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eShewanella\u003c/em\u003e spp. are progenitors of several subclasses of Ambler class D β-lactamases, [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] as well as other antibiotic resistance genes (ARGs) of clinical concern, such as \u003cem\u003eqnrA\u003c/em\u003e, found in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e and closely related species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], conferring resistance to fluoroquinolones, and \u003cem\u003emcr-4.3\u003c/em\u003e, thought to have originated from \u003cem\u003eShewanella frigidimarina\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which mediates resistance towards colistin, a last resort antibiotic against infections caused by multidrug-resistant Gram-negative bacteria. All these chromosomally encoded \u003cem\u003eShewanella\u003c/em\u003e genes have been identified in an array of mobile genetic elements (MGEs), including plasmids, that are currently widespread among clinically important human pathogens [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDiverse ARGs occur naturally in environmental bacteria and can be mobilized into conjugative plasmids through recombination and subsequent dissemination via horizontal gene transfer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This process requires either the coexistence of native (primarily environmental) and non-native (primarily human-associated) bacterial hosts for sufficient periods of time, or efficient transmission routes across distant hosts, that is, from environmental bacterial communities to human bacterial pathogens, which are still poorly delineated. Not infrequently, ARG carriage is not associated with phenotypic resistance in the native bacterial host, which is typically attributed to low expression levels of the gene in question, less functional gene products, or various genetic factors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These so-called \u0026lsquo;silent\u0026rsquo; genes may nonetheless effectively contribute to resistance when expressed in non-native hosts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClass D β-lactamases, also known as oxacillinases (OXAs) due to their substrate preference, are of particular concern due to their widespread occurrence in \u003cem\u003eEnterobacterale\u003c/em\u003es and their recent spread to high-risk, multidrug-resistant strains of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eAcinetobacter baumanii\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among them, OXA-48-like enzymes, the most widespread in \u003cem\u003eShewanella\u003c/em\u003e genomes, are known to contribute to carbapenem resistance and represent the preponderant carbapenemases across large areas of Europe, the Middle East, and Northern Africa [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Within Europe, OXA-48 carbapenemases are so widespread in \u003cem\u003eEnterobacterales\u003c/em\u003e that they are currently considered endemic in Belgium, France, and Spain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Currently, there are nearly 1,400 class D beta-lactamases registered in the Beta-Lactamase DataBase (BLDB) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], including 68 OXA-48-like variants, which showcases the extensive sequence variability across this family of enzymes. Some \u003cem\u003eShewanella\u003c/em\u003e-native, chromosome-encoded OXA-48-like oxacillinases such as OXA-54 in \u003cem\u003eShewanella oneidensis\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], OXA-55 in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], or OXA-181 in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003exiamenensis\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] have been characterized biochemically and genetically. However, the evolutionary selective pressures shaping the diversity of OXA proteins and the impact of such diversity on enzyme function are still not well defined.\u003c/p\u003e \u003cp\u003eOur team has devoted efforts to the surveillance of antimicrobial resistance (AMR) and ARG dissemination in waterborne bacteria, including \u003cem\u003eShewanella\u003c/em\u003e populations closely related to \u003cem\u003eShewanella baltica\u003c/em\u003e collectively referred to as the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e complex [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Hence, this study was designed to i) assess the diversity of class D β-lactamases in environmental \u003cem\u003eShewanella\u003c/em\u003e retrieved from Baltic Sea environments in the surroundings of Stockholm, Sweden; ii) investigate the distribution of mutations in these enzymes in similar \u003cem\u003eShewanella\u003c/em\u003e hosts worldwide; iii) evaluate the contribution of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e to AMR in its native \u003cem\u003eShewanella\u003c/em\u003e hosts, and iv) functionally characterize novel variants in a heterologous enterobacterial host, mimicking horizontal acquisition.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolation\u003c/h2\u003e \u003cp\u003eWater and sediment samples were collected from Nyn\u0026auml;shamn (58\u0026deg; 53' 57.29\" N, 17\u0026deg; 57' 2.23\" E; May 14, 2022), Notholmen, Tyres\u0026ouml; (59\u0026deg;13'58\"N 18\u0026deg;18'40\"E; May 14, 2022), and Hagaparken (59\u0026deg;21'21.0\"N 18\u0026deg;02'37.0\"E; April 11, 2022) representing brackish environments in the Baltic Sea (Nyn\u0026auml;shamn and Notholmen) or Lake Brunnsviken (Hagaparken), connected to the Baltic Sea through the \u0026Aring;lkistan canal. \u003cem\u003eShewanella\u003c/em\u003e spp. were isolated from water samples upon filtration through 0.45 \u0026micro;m mixed cellulose ester membrane filters (Millipore) and plating on Lyngby\u0026rsquo;s Iron Agar (LIA) containing 0.04% (w/v) L-cysteine and either colistin (8 mg/L, due to the intrinsic resistance of some \u003cem\u003eShewanella\u003c/em\u003e sp.), or no antibiotic. For isolation from sediments, scooped shoreline sediment samples in 50 ml sterile centrifuge tubes were overlaid with sterile PBS, vigorously shaken, and serially ten-fold diluted before plating on colistin-supplemented or plain LIA. \u003cem\u003eShewanella\u003c/em\u003e-like colonies were identified as H\u003csub\u003e2\u003c/sub\u003eS producers in this medium after incubation at 28 \u0026ordm;C for 24 h, as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and purified by re-streaking on the same medium. Genus-level identification was achieved by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis (MALDI Biotyper Sirius System, Bruker).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole-genome sequencing and extraction of\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eOXA\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eShewanella\u003c/em\u003e strains were grown overnight, and genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) and sequenced, as previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, 50 ng of genomic DNA was used for library preparation with the MGIEasy FS Library Prep Set (MGI Tech). Equimolar pooled libraries were then circularized using the MGIEasy Circularization Kit (MGI Tech) and sequenced as 2 \u0026times; 100 bp paired-end reads on an NBSEQ-G400 platform (MGI Tech). Following genome assembly and annotation with BACTPipe v3.1.0, annotated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e alleles were extracted and deposited in the NCBI GenBank under accessions PX283710-PX283731 and PX713573-PX713575.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic analyses and bioinformatics\u003c/h3\u003e\n\u003cp\u003eSequence alignments were generated with ESPript 3.0 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] using the sequence and structural data of OXA-48, available from the Protein Data Bank (PDB, entry 3HBR), as a reference. A maximum-likelihood phylogenetic analysis of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e amino acid sequences was performed with MEGA X [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], using a Jones-Taylor-Thornton (JTT) matrix-based model with Gamma (G) distribution and 1000 bootstrap iterations. DnaSP v6 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] was used for the calculation of Tajima\u0026rsquo;s and Fu \u0026amp; Li\u0026rsquo;s statistics from DNA sequences.\u003c/p\u003e \u003cp\u003eA total of 106 sequences from \u003cem\u003eShewanella baltica\u003c/em\u003e (combining the 25 from this study and those publicly available from the NCBI GenBank), 219 sequences from \u003cem\u003eShewanella algae\u003c/em\u003e, 32 sequences from \u003cem\u003eS. oncorhynchi\u003c/em\u003e, and 98 sequences from \u003cem\u003eS. xiamenensis\u003c/em\u003e (\u003cb\u003eFile S1\u003c/b\u003e) were aligned using the Clustal Omega tool (EMBL-EBI), in four different batches, respectively. The resulting multiple sequence alignments were used to generate a sequence logo via the WebLogo server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://weblogo.berkeley.edu\u003c/span\u003e\u003cspan address=\"https://weblogo.berkeley.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and to analyze the distribution of amino acid residues across the alignment, as well as the degree of conservation at variable positions, according the Gonnet substitution matrix.\u003c/p\u003e \u003cp\u003eProtein structures and structure-based functional annotations of the OXA enzymes from strains N1WShe5-IV (OXA-1408), N1WShe6 (OXA-1410), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417) were predicted using I-TASSER (Iterative Threading ASSEmbly Refinement) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and the resulting models were visualized and aligned with the \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e OXA-48 reference structure (3HBR) in Pymol v3.0 (Schr\u0026ouml;dinger, LLC).\u003c/p\u003e \u003cp\u003ePutative promoters and transcription factor binding sites were predicted with BPROM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://weblogo.berkeley.edu\" target=\"_blank\"\u003ewww.softberry.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.softberry.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized using a custom Python script. Synteny plots were generated with the Clinker pipeline [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStrains and growth conditions\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e TOP10 was grown in Miller\u0026rsquo;s LB medium at 37\u0026deg;C, supplemented with tetracycline (Tc, 15 \u0026micro;g/ml) when required. \u003cem\u003eShewanella\u003c/em\u003e sp. N1WShe5-IV, N1WShe6, T1SShe5-III, and T1WShe4, along with their isogenic Δ\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e mutant derivatives, were routinely cultured in Miller\u0026rsquo;s LB medium at 28\u0026deg;C. \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e MFDpir was grown on LB agar supplemented with diaminopimelic acid (0.3 mM) and, when required, Tc, at 37 \u0026ordm;C, except during conjugal mating with \u003cem\u003eShewanella\u003c/em\u003e, that was conducted at 28 \u0026ordm;C. The strains employed in this study are presented in \u003cb\u003eTable S1\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eGenetic manipulations\u003c/h3\u003e\n\u003cp\u003eTo express distinct OXA variants from a promoterless vector, we generated a custom pGEN-MCS derivative [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], named pGEN-MCS-Tc, in which the \u003cem\u003ebla\u003c/em\u003e selection cassette and its promoter had been replaced by a \u003cem\u003etet\u003c/em\u003e cassette and its promoter (obtained from plasmid pSIM27 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]) using the XbaI and SpeI restriction sites (\u003cb\u003eTable S2\u003c/b\u003e). Divergent \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;551\u003c/sub\u003e-like alleles of strains N1WShe5-IV, N1WShe6, T1SShe5-III, and T1WShe4 were cloned into plasmid pGEN-MCS-Tc using the primers indicated in \u003cb\u003eTable S2\u003c/b\u003e, which spanned the gene coding sequence and the 333 bp upstream region containing the putative promoter. The reference \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;551\u003c/sub\u003e sequence in the Comprehensive Antibiotic Resistance Database (CARD) (Accession ARO:3005775) and its native promoter, obtained from \u003cem\u003eShewanella\u003c/em\u003e sp. VAX-SP0-4CM-5 (V. Fern\u0026aacute;ndez-Ju\u0026aacute;rez, A. J. Mart\u0026iacute;n-Rodr\u0026iacute;guez, unpublished), were cloned into the same sites. Upon sequence verification by Sanger sequencing, the recombinant plasmids and the empty vector were mobilized into \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 by electroporation.\u003c/p\u003e \u003cp\u003eA \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e in-frame deletion mutant of \u003cem\u003eShewanella\u003c/em\u003e sp. N1WShe5-IV, T1SShe5-III, and T1WShe4 were generated by allelic replacement following described procedures [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In brief, the upstream (585 bp) and downstream (600 bp) regions flanking the gene coding sequence were sequentially cloned into plasmid pKNG101 using the primers listed in \u003cb\u003eTable S2\u003c/b\u003e. The suicide vector was mobilized into calcium-competent \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e MFDpir and transferred to \u003cem\u003eShewanella\u003c/em\u003e sp. N1-W-She5-IV by biparental mating. Merodiploids were resolved by plating on LB agar supplemented with 10% sucrose (w/v). Gene deletion was confirmed by gel electrophoresis with PCR primers flanking the recombination sites (\u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eAntibiotic susceptibility testing\u003c/h3\u003e\n\u003cp\u003eThe susceptibility of environmental \u003cem\u003eShewanella\u003c/em\u003e isolates and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 strains expressing the reference or divergent \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;551\u003c/sub\u003e variants, or carrying the empty cloning plasmid, towards ampicillin (AMP, 10 \u0026micro;g), ceftazidime (CAZ, 10 \u0026micro;g), cefotaxime (CTX, 30 \u0026micro;g), imipenem (IMP, 10 \u0026micro;g), and meropenem (MEM, 10 \u0026micro;g) was determined by the disc diffusion method. For \u003cem\u003eShewanella\u003c/em\u003e, the medium was supplemented with 0.5% NaCl (w/v), due to the comparatively poor growth observed for these strains on plain Mueller Hinton medium, and the incubation temperature was 28 \u0026ordm;C, instead of 35 \u0026ordm;C, as the latter was found to be nearly non-permissive for the growth of many of the retrieved strains. The minimal inhibitory concentration (MIC) of the same antibiotics to \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 strains was additionally tested by the broth microdilution method, and interpreted following EUCAST guidelines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKinetic assays of nitrocefin hydrolysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 strains carrying the recombinant \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e expression plasmids or the empty vector control were pre-grown in LB broth with Tc overnight at 37 \u0026ordm;C. Overnight cultures were diluted 1:100 in 20 ml of fresh LB medium without antibiotic and grown to an OD\u003csub\u003e600\u003c/sub\u003e of 0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 Cell pellets were then obtained by centrifugation at 3273 x \u003cem\u003eg\u003c/em\u003e, 4 \u0026ordm;C, and stored frozen at -80 \u0026ordm;C for 24 h. Cell lysis was completed by addition of 2 ml PBS (0.01 M, pH 7.4) to the thawed pellets and sonication on ice (8 x 10 s, with 30 s resting periods between sonication steps, amplitude 50%). Cell debris was removed from the whole-cell lysates by centrifugation at 16000 x \u003cem\u003eg\u003c/em\u003e, 4 \u0026ordm;C, 20 min. Upon quantification of total protein content by the BCA method [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], equal amounts of protein (10 \u0026micro;l) were mixed, in duplicates, with 90 \u0026micro;l of nitrocefin solution in PBS (200 \u0026micro;M, final concentration) inside the wells of a 96-well plate. Nitrocefin hydrolysis was simultaneously monitored by recording the OD\u003csub\u003e490\u003c/sub\u003e (proportional to the generation of its hydrolytic product) and the OD\u003csub\u003e380\u003c/sub\u003e (decay proportional to the loss of intact nitrocefin) every 2 min for 2 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData representation and statistical analysis\u003c/h3\u003e\n\u003cp\u003eGraphPad Prism v10.0.2 was employed for data visualization and statistics, unless otherwise indicated.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eBaltic Sea\u003c/b\u003e \u003cb\u003eShewanella\u003c/b\u003e \u003cb\u003eisolates carry\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eOXA\u0026minus;48\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-like variants with substantial N-terminal diversity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the diversity of OXA proteins in the local \u003cem\u003eShewanella\u003c/em\u003e populations, we extracted the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e coding sequences from 25 arbitrarily selected and whole genome sequenced \u003cem\u003eShewanella\u003c/em\u003e isolates retrieved from water and sediment samples collected in Nyn\u0026auml;shamn, Tyres\u0026ouml;, and Hagaparken, as representative members of the natural \u003cem\u003eShewanella\u003c/em\u003e communities in the Baltic Sea region surrounding Stockholm. The species affiliation of the isolates was \u003cem\u003eShewanella baltica\u003c/em\u003e or closely related genospecies. Each isolate harbored a single, chromosomal \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e gene, encoding a class D OXA-48-like β-lactamase closely related to the OXA-551 subtype, which altogether represented 20 novel OXA-48-like variants, termed OXA-1401 to OXA-1418, OXA-1428, and OXA-1429.\u003c/p\u003e \u003cp\u003eTo delineate the evolutionary relationships of these \u003cem\u003eShewanella\u003c/em\u003e OXA β-lactamases within the broader \u003cem\u003eShewanella\u003c/em\u003e OXA-48-like family, a phylogenetic reconstruction was generated using the best-fit model (JTT\u0026thinsp;+\u0026thinsp;G) as implemented in MEGA X (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This analysis evidenced well-supported associations that correlate with the taxonomic position of the harboring strains, with the OXA variants of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e and closely related species, such as \u003cem\u003eShewanella scandinavica\u003c/em\u003e, \u003cem\u003eShewanella vaxholmensis\u003c/em\u003e, \u003cem\u003eShewanella septentrionalis\u003c/em\u003e or \u003cem\u003eShewanella hafniensis\u003c/em\u003e clustering apart from those of other more distant species inhabiting similar environments, such as \u003cem\u003eShewanella oncorhynchi\u003c/em\u003e or \u003cem\u003eShewanella oneidensis\u003c/em\u003e, the latter closely related to the archetypal OXA-48 reference of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e used as an outgroup for the phylogenetic reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This evidence suggests evolution of OXA enzymes in \u003cem\u003eShewanella\u003c/em\u003e parallel to species diversification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e genes were under non-neutral selection in this \u003cem\u003eShewanella\u003c/em\u003e population, we carried out the Fu and Li test (D* = -0.56827, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10; F* = -0.85327, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10), which indicated non-significant deviation from neutrality. This was further corroborated by the Tajima test (D = -1.06065, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10), with this analysis evidencing codons with multiple evolutionary paths. An amino acid sequence alignment, using the sequence and structure of OXA-48 (PDB: 3HBR) and the OXA-551 sequence (CARD entry ARO:3005775) as references, revealed diverse polymorphic sites across the \u003cem\u003eS. baltica\u003c/em\u003e OXA proteins, preponderantly located in the N-terminal region of the mature enzyme (\u003cb\u003eFigure S1\u003c/b\u003e). This is exemplified in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB with the OXA sequences of isolates N1WShe5-IV (OXA-1408), N1WShe6 (OXA-1410), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417). To understand the structural impact of these mutations, we generated three-dimensional reconstructions of the representative divergent variants using deep-learning algorithms [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, compared to OXA-551, OXA-1408 and OXA-1417 exhibit deletions of five and eight amino acids, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which affect the conformation of the first α-helix or results in its absence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). In contrast, OXA-1410 and OXA-1413 carry insertions of five amino acids each (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which, in the case of OXA-1410, leads to the formation of a second α-helix in the N-terminal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eN-terminal mutations and non-conservative substitutions are widespread in\u003c/b\u003e \u003cb\u003eShewanella baltica\u003c/b\u003e \u003cb\u003eOXA-48-like enzymes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether the observed sequence variability was endemic to local \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e populations or broadly distributed in this species instead, we retrieved all \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e genomes from NCBI GenBank (n\u0026thinsp;=\u0026thinsp;81, accessed 6 September 2025), extracted their chromosomal \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e genes, and, together with the 25 sequences of this study, constructed a sequence logo based on aligned amino acid sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The frequency of amino acid residues at each position is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. These reconstructions evidenced two regions that concentrated most amino acid substitutions, insertions, or deletions, located at positions 27\u0026ndash;30 and 61\u0026ndash;79 in the alignment. Key amino acids involved in catalysis by serine beta-lactamases such as OXA-48, namely the catalytic serine (position 114 in the sequence logo), carboxylated lysine (position 117), and the universal KTG motif (positions 252\u0026ndash;254) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] were highly conserved, presumptive of activity preservation despite sequence variation. Most amino acid deviations from the consensus sequence across the 106 analyzed OXA-48-like proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) were non-conservative, consistent with the frequent insertions and deletions at the N-terminus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor comparative purposes, we performed the same analysis on OXA sequences retrieved from well-represented \u003cem\u003eShewanella\u003c/em\u003e species in terms of genome sequence availability, with variable degrees of taxonomic relatedness with respect to \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e, namely \u003cem\u003eS. oncorhynchi\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;32; OXA-48-like), \u003cem\u003eS. xiamenensis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;98; OXA-48-like), and \u003cem\u003eS. algae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;219, OXA-55-like), which, in contrast to \u003cem\u003eS. baltica\u003c/em\u003e, exhibited a much higher degree of homogeneity, reflected in fewer mutations and the absence of insertions or deletions (\u003cb\u003eFig. S2\u0026ndash;S4\u003c/b\u003e). This pattern could indicate higher intrinsic mutation rates in \u003cem\u003eS. baltica\u003c/em\u003e compared to other \u003cem\u003eShewanella\u003c/em\u003e species, or alternatively, reflect the influence of selective pressures.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBaltic Sea\u003c/b\u003e \u003cb\u003eShewanella\u003c/b\u003e \u003cb\u003eexhibit discrete phenotypic resistance towards β-lactam antibiotics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, to investigate whether \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e carriage was associated with phenotypic resistance in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e isolates, the susceptibility profiles of the 25 strains were determined towards a panel of β-lactam antibiotics comprising ampicillin (AMP, 10 \u0026micro;g), meropenem (MEM, 10 \u0026micro;g), imipenem (IMP, 10 \u0026micro;g), ceftazidime (CAZ, 10 \u0026micro;g), and cefotaxime (CTX, 30 \u0026micro;g). The results of disc diffusion tests are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Growth inhibition diameters were in the range 15.5\u0026ndash;27.1 mm for AMP, 32.1\u0026ndash;40.2 mm for MEM, 31.3\u0026ndash;40.5 mm for IMP, 30.6\u0026ndash;36.8 mm for CAZ and 35.8\u0026ndash;44.4 mm for CTX, which, in relative terms, given the absence of specific guidelines for the interpretation of disc diffusion tests with this group of microorganisms, evidenced a high degree of susceptibility to carbapenems (IMP, MEM) and third generation cephalosporins (CAZ, CTX), whereas susceptibility was comparatively lower towards the aminopenicillin ampicillin (AMP). Altogether, these results implied a rather discrete role of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e in mediating substantial resistance towards β-lactam antibiotics under our test conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further dissect the functional role of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e in \u003cem\u003eS. baltica\u003c/em\u003e, we generated Δ\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e in-frame deletion mutants from three representative strains, N1WShe5-IV (OXA-1408), T1SShe5-III (OXA-1413), and T1WShe4 (OXA-1417), which captures the structural diversity of the OXA variants within the studied strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, and \u003cb\u003eFig. S1\u003c/b\u003e). Attempts to generate the same mutant in the N1WShe6 background failed because of the intrinsic resistance of this strain to sucrose-mediated counter-selection, which is widespread in \u003cem\u003eShewanella\u003c/em\u003e. Mutants were screened, in triplicate, against the same panel of antibiotics, using their respective wild-type strains as references. Our results revealed a statistically significant contribution of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e to AMP resistance in all strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D), as well as to IMP and MEM in strains N1WShe5-IV and T1SShe5-III (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Despite a seemingly modest biological impact, these findings confirm the expression and functional activity of the gene product under our experimental conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous expression of\u003c/b\u003e \u003cb\u003eShewanella\u003c/b\u003e \u003cb\u003eOXA-551-like enzymes in\u003c/b\u003e \u003cb\u003eEscherichia coli\u003c/b\u003e \u003cb\u003estimulates β-lactamase activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThere is evidence that OXA β-lactamases originating from \u003cem\u003eShewanella\u003c/em\u003e spp. are currently widely distributed in enterobacterial multidrug resistance plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To investigate the potential impact of the N-terminal mutations found in the OXA-551-like enzymes of our isolates on β-lactamase activity, we cloned the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e gene of the same four representative strains along with its native promoter sequences into a promoter-less plasmid. The same approach was taken for the cloning of OXA-551, used as a reference. In both disk diffusion and broth microdilution assays, \u003cem\u003eE. coli\u003c/em\u003e TOP10 expressing either OXA variant showed similar susceptibility to β-lactam antibiotics to the empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). A clear exception was observed for ampicillin, where OXA expression enhanced the resistance phenotype, elevating MICs to \u0026gt;\u0026thinsp;200 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results from antibiotic susceptibility tests suggested a similar performance of the novel OXA variants with respect to OXA-551. To further characterize this, we studied the kinetics of the hydrolytic degradation of nitrocefin, a chromogenic cephalosporin substrate, using cell lysates of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10 expressing either OXA-551 or the four exemplary variants OXA-1408, OXA-1410, OXA-1413 or OXA-1417. The results of this assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) demonstrated similar performance of all the enzymes, with the two variants carrying N-terminal insertions, OXA-1410 and OXA-1413, exhibiting somewhat higher activity than OXA-551 or their counterparts carrying N-terminal deletions.\u003c/p\u003e \u003cp\u003eAs \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e genes are not native to \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, we next interrogated factors regulating the expression of \u003cem\u003ein trans\u003c/em\u003e-acquired \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e alleles. Thus, we compared the promoter region of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica bla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e with those of a set of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e alleles found in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e plasmids mediating antibiotic resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) BPROM analysis identified canonical σ\u003csup\u003e70\u003c/sup\u003e-type promoters upstream of all analyzed genes, although the predicted regulatory complexity varied markedly between sequences. While several promoters harbor binding sites for multiple global regulators including e.g. CRP, IHF, or RpoH, others show a more restricted regulatory architecture, with only binding sites for redox-associated regulators such as ArcA and FNR predicted in the promoter. Another signature of a putative distinct metabolic regulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e expression is the presence of predicted ArgR binding sites in certain promoters, suggesting regulation of gene expression by arginine metabolism. The \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e promoter displays a distinct organization, including different RpoD binding motifs and predicted binding sites for CRP and CarP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The predicted binding of CarP putatively links \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e expression with arginine and pyrimidine metabolism [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Noteworthily, two of the exemplary promoters from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e plasmids (found in sequences CP194965.1 and CP048327.1, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) are predicted to have unusually long 5\u0026rsquo; untranslated regions (5\u0026rsquo; UTRs), which might affect mRNA stability and OXA expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAMR represents one of the greatest public health concerns of our times, with an estimate of over 10\u0026nbsp;million annual deaths by the year 2050 directly attributable to or associated with infections caused by drug-resistant pathogens [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. With broad spectrum penicillins and cephalosporins being the most prescribed antibiotics worldwide [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the dissemination of β-lactamase resistance across bacterial pathogens is a growing concern. OXA-48-like β-lactamases are particularly problematic because of the low levels of phenotypic resistance associated with their carriage \u003cem\u003ein vitro\u003c/em\u003e, despite evidence linking \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e carriage with failure of carbapenem-based antimicrobial chemotherapy [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our study showed, indeed, that the phenotypic resistance of \u003cem\u003eS. baltica\u003c/em\u003e-like strains naturally carrying a chromosomally encoded \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e gene (which is a widespread genomic characteristic in the genus \u003cem\u003eShewanella\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]) was weak towards a panel of diverse β-lactam antibiotics including ampicillin, which is typically proficiently hydrolyzed by OXA-48-like enzymes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This is not uncommon and could be related to variability in OXA expression under the experimental conditions. For example, a recent study involving diverse carbapenem-susceptible and a carbapenem-resistant \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e isolate demonstrated higher transcript levels of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e mRNA in the resistant isolate as compared to the susceptible strains, despite all strains carrying minimally different \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e alleles [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This is also consistent with our mutational analyses, with Δ\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e deletion mutants displaying, overall, relatively discrete susceptibility defects towards β-lactam antibiotics as compared to the corresponding WT parental strains.\u003c/p\u003e \u003cp\u003eWhile there is wide agreement on \u003cem\u003eShewanella\u003c/em\u003e spp. as progenitors of OXA-48-like β-lactamases, it is less clear whether these enzymes have mobilized from the chromosome of the \u003cem\u003eShewanella\u003c/em\u003e donors into enterobacterial plasmids directly or through intermediate hosts [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. OXA-48-like enzymes preponderantly disseminate across clinically relevant \u003cem\u003eEnterobacterales\u003c/em\u003e within broad-host conjugative IncL/M-type conjugative plasmids associated with Tn\u003cem\u003e1999\u003c/em\u003e insertion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Other OXA-48-like variants originating from \u003cem\u003eShewanella\u003c/em\u003e have been found in plasmids with other replicon types, including ColE2, IncX2, IncN1, IncT and IncA/C [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Novel allelic variants of \u003cem\u003eShewanella\u003c/em\u003e-derived OXA-48-like enzymes are emerging, in part due to increasing surveillance and sequencing efforts, raising clinical concerns [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A heterologous analysis of \u003cem\u003eShewanella oneidensis\u003c/em\u003e OXA-54 has previously been performed through \u003cem\u003ein trans\u003c/em\u003e expression under a \u003cem\u003elac\u003c/em\u003e promoter on a high-copy-number plasmid [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our study, we employed a nature-mimicking approach to simulate the horizontal acquisition of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica bla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e by an enterobacterial host. To that end, instead of expressing the gene from an inducible promoter, we cloned native genes and their promoter regions into a low copy-number, promoter-less plasmid that is stable in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e in the absence of antibiotic selective pressure [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This approach circumvents the bias introduced by protein overexpression from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e-adapted promoters or high copy number expression shuttles, allowing a more realistic assessment of phenotypic resistance acquisition. We showed that, compared to the phenotypic resistance exhibited by the native hosts, expression of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e OXA-48-like in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e stimulates phenotypic resistance against AMP while displaying discrete phenotypic resistance to carbapenems or third generation cephalosporins, consistent with phenotypic evidence of OXA-48-like carriage by clinical strain subsets [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] or heterologous functional studies with other \u003cem\u003eShewanella\u003c/em\u003e OXA-48-like enzymes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOXA enzymes show remarkable sequence diversity. In \u003cem\u003eShewanella\u003c/em\u003e, the phylogenetic distribution of OXA-48-like carbapenemases resembles the taxonomy of the genus, suggesting parallel evolution across different taxa [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. We have shown here that \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e and closely related species harbor an array of OXA-48-like variants that differ primarily in their N-termini, in some cases presenting in-frame insertions or deletions. In fact, of the 25 strains of this study, 20 carried a novel OXA variant, which showcases the still unknown environmental diversity pool of ARGs and the evolutionary forces that drive their diversification. Through a comparative analysis of two populated whole genome-sequenced clades, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e, we have shown that \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e OXA-48-like enzymes are substantially more variable and accumulate more non-conservative mutations than those encoded in the genomes of other \u003cem\u003eShewanella\u003c/em\u003e species. From a genomic standpoint, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e is known to be a highly heterogeneous and recombination-prone clade [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], in contrast to \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e, which is substantially more uniform [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The higher degree of sequence variability in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e OXA-48-like enzymes could indicate higher intrinsic mutation frequency in this species as compared to \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ealgae\u003c/em\u003e, which is currently unknown, although it could also be the result of evolutionary forces favoring the propagation of N-terminal mutations.\u003c/p\u003e \u003cp\u003eIn OXA-48-like enzymes, residues in or around the β5-β6 loop and Ω loop are important determinants of enzymatic activity and substrate specificity [\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. While none of these regions are in the N-terminus of the protein, the high degree of N-terminal sequence and structural divergence has been previously highlighted in novel OXA-48 variants [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Given the \u003cem\u003eprima facie\u003c/em\u003e marginal contribution to phenotypic resistance in the native host, we reasoned that mutations affecting enzyme function might have minimal fitness consequences. We therefore studied whether rearrangements in the N-terminal region of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e OXA proteins could potentially affect enzyme performance. Computational analyses using four representative OXA enzymes carrying five amino acid insertions or deletions with respect to the closest OXA-48-like subtype, OXA-551, predicted structural alterations affecting N-terminal α-helices. Our experimental analyses of OXA-1408, OXA-1410, OXA-1413, and OXA-1417 demonstrated that in-frame alterations in this region do not substantially affect β-lactamase activity \u003cem\u003ein vivo\u003c/em\u003e when expressed in a heterologous host. Similarly, \u003cem\u003ein vitro\u003c/em\u003e antibiotic hydrolysis assays showed that the enzymes harboring insertions exhibit a modestly increased hydrolytic capacity than OXA-551 or enzymes carrying N-terminal deletions. The lack of non-deleterious mutations also implies that, despite their overall low contribution to phenotypic resistance, OXA enzymes may play a significant role in the eco-physiology of \u003cem\u003eShewanella\u003c/em\u003e and support the propagation of mutant variants without functional consequences across natural populations.\u003c/p\u003e \u003cp\u003ePromoter architecture and regulatory variability have emerged as important determinants of ARG expression [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Promoter sequence variants of clinically relevant ARGs, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXA\u0026minus;48\u003c/em\u003e\u003c/sub\u003e, are associated with differences in expression levels under various environmental conditions, and specific transcription factors such as FNR, ArcA or ArgR can influence expression in response to distinct metabolic or stress signals [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Experimental work in other systems has also demonstrated that point mutations within core promoter elements, such as the \u0026minus;\u0026thinsp;10 box, can modulate β-lactamase expression and resistance levels, as seen for the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;61\u003c/sub\u003e promoter in \u003cem\u003eCampylobacter jejuni\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and in chromosomal β-lactamase promoters of \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e where alterations in -10 and \u0026minus;\u0026thinsp;35 motifs significantly changed promoter strength [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHorizontal mobilization of ARGs can include adjacent upstream sequences, such as parts of the native promoter region. In these cases, binding sites for transcription factors present in the original chromosomal context would also be co-transferred, making them potentially available to influence gene expression in the new host. In this study, we have shown that the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica bla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e promoter is predicted to accommodate the binding of global regulators such as RpoD and CRP, as well as CarP. In \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, CarP acts as a pyrimidine-mediated repressor of \u003cem\u003ecarAB\u003c/em\u003e transcription [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which encodes carbamoyl phosphate synthase, a key enzyme in arginine and pyrimidine biosynthesis, thereby potentially extending previously proposed links between \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e expression and arginine metabolism [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Of note, CarP is also necessary for the resolution of ColE1 plasmid multimers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Besides, in our comparative analysis of the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica bla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e promoter with respect to \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e plasmid-borne counterparts, we identified some unusually long 5\u0026rsquo; UTRs (\u0026gt;\u0026thinsp;200\u0026ndash;300 nt), as predicted by BPROM. Such lengths are atypical, considering that the length of most 5\u0026rsquo; UTRs in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e genes is 25\u0026ndash;35 nt, although 5\u0026rsquo; UTRs as long as 700 bp have also been reported [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The length of 5\u0026rsquo; UTRs have profound effects on mRNA stability and translation [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], which could potentially relate to the known variability in the levels of phenotypic resistance associated with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e carriage in different bacterial hosts and cellular physiological contexts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While \u003cem\u003ein silico\u003c/em\u003e predictions require empirical validation, these initial observations warrant further investigation. Taken together, this work reaffirms \u003cem\u003eShewanella\u003c/em\u003e communities as a substantial reservoir of OXA diversity in aquatic environments, prone to potential horizontal dissemination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants from the Swedish Research Council for Sustainable Development (FORMAS, Ref. AC-2023/0032), the L\u0026auml;ngmanska Kulturfonden (Ref. BA25-0527), and the Hans Dahlbergs Stiftelse f\u0026ouml;r Milj\u0026ouml; och H\u0026auml;lsa to AJMR. EJ, \u0026Aring;S, and AJMR would like to thank the EU and Swedish Research Council for funding in the frame of the collaborative international consortium PARRTAE (Reference Number: ID 351), financed under the ERA-NET Aquatic Pollutants Joint Transnational Call (GA n\u0026ordm;869178). This ERA-NET is an integral part of the activities developed by the Water, Oceans and AMR Joint Programming activities. MP is grateful to the AMGEN Foundation for a scholarship.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartin-Rodriguez AJ (2025) Shewanella algae. Trends Microbiol 33(8):920\u0026ndash;921\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTac\u0026atilde;o M, Ara\u0026uacute;jo S, Vendas M, Alves A, Henriques I (2018) Shewanella species as the origin of blaOXA-48 genes: insights into gene diversity, associated phenotypes and possible transfer mechanisms. Int J Antimicrob Agents 51(3):340\u0026ndash;348\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong N, Zhang Y, Wu Y, Ju X, Yan Z et al (2025) Genetic insights into Shewanella spp., progenitor of the bla (OXA-48)-like genes: a large-scale study. 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Euro Surveill ;22(31)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJamin C, Brouwer MSM, Veldman KT, Beuken E, Witteveen S et al (2023) Mobile colistin resistance mcr-4.3- and mcr-4.6-harbouring plasmids in livestock- and human-retrieved Enterobacterales in the Netherlands. JAC Antimicrob Resist 5(3):dlad053\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartins-Sorenson N, Snesrud E, Xavier DE, Cacci LC, Iavarone AT et al (2020) A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain. J Antimicrob Chemother 75(1):60\u0026ndash;64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePitout JDD, Peirano G, Kock MM, Strydom KA, Matsumura Y (2019) The Global Ascendency of OXA-48-Type Carbapenemases. 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Nucleic Acids Res 45(6):3487\u0026ndash;3502\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"f2b94f8e-b7ab-46ef-8522-534844ee020a","identifier":"10.13039/501100001862","name":"Svenska Forskningsrådet Formas","awardNumber":"AC-2023/0032","order_by":0},{"identity":"04d2cb83-992d-4f17-aae1-fa113d365355","identifier":"10.13039/501100004359","name":"Vetenskapsrådet","awardNumber":"GA nº869178","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Karolinska Institute","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"beta-lactamase, Shewanella baltica, nitrocefin hydrolysis, antimicrobial resistance, environmental reservoirs","lastPublishedDoi":"10.21203/rs.3.rs-8584107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8584107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genus \u003cem\u003eShewanella\u003c/em\u003e is a recognized reservoir of antibiotic resistance genes (ARGs), including chromosomally encoded \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e alleles that have given rise to clinically relevant OXA-48-like β-lactamases in \u003cem\u003eEnterobacterales\u003c/em\u003e. However, our understanding of these enzymes in environmental \u003cem\u003eShewanella\u003c/em\u003e populations remains limited. Here, we investigated their distribution, evolution, and function in \u003cem\u003eShewanella\u003c/em\u003e spp. isolated from Baltic Sea environments near Stockholm, Sweden. Whole-genome sequencing of 25 isolates, primarily affiliated with \u003cem\u003eShewanella baltica\u003c/em\u003e and related genospecies, revealed that each carried a chromosomal OXA-48-like β-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 \u003cem\u003eShewanella\u003c/em\u003e 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 Δ\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e mutants of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e strains carrying divergent variants revealed variable contributions to β-lactam resistance. In contrast, heterologous expression of these enzymes in \u003cem\u003eEscherichia coli\u003c/em\u003e conferred high resistance to ampicillin and β-lactamase activity comparable to the reference OXA-551, as demonstrated by nitrocefin hydrolysis kinetics. Comparative analysis of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebaltica\u003c/em\u003e-like \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e promoters and those associated with plasmid-borne OXA-48 variants in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e revealed conserved sigma70-dependent regulation, besides additional predicted transcription factor binding sites clustered near the \u0026minus;\u0026thinsp;10 box, suggestive of a fine-tuned regulation in \u003cem\u003eShewanella\u003c/em\u003e. Our findings expand functional insights into OXA-48-like β-lactamases and highlight environmental \u003cem\u003eShewanella\u003c/em\u003e as reservoirs of OXA-48-like diversity.\u003c/p\u003e \u003cp\u003eIMPORTANCE\u003c/p\u003e \u003cp\u003eAntimicrobial resistance (AMR) is a major global health challenge, with β-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 \u003cem\u003eShewanella\u003c/em\u003e 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 \u003cem\u003eShewanella baltica\u003c/em\u003e populations harbor diverse OXA-48-like enzymes with relatively limited phenotypic impact in their native hosts, but more active when expressed in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e 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 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.\u003c/p\u003e","manuscriptTitle":"Diversity and function of OXA-48-like β-lactamase variants in environmental Shewanella isolates from Stockholm, Sweden","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 11:12:09","doi":"10.21203/rs.3.rs-8584107/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"666d64fa-70b7-4827-a955-c01eb84cab7f","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61018772,"name":"General Microbiology"}],"tags":[],"updatedAt":"2026-01-16T11:12:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 11:12:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8584107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8584107","identity":"rs-8584107","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}