Characterization of Chromosome-Mediated BlaOXA-894 in Shewanella xiamenensis Isolated from Pig Wastewater

A new variant of the blaOXA-546 gene, namely blaOXA-894, was identified on the chromosome of Shewanella xiamenensis isolated from pig wastewater in rural China. OXA-894 differs from OXA-546 (A46V, I219del) and OXA-48 (T167I, I219del) with two amino acid substitutions, respectively. The isolate was resistant to ampicillin, aztreonam, imipenem, meropenem and fosfomycin. Carba NP test confirmed S. xiamenensis strain sx20 as a carbapenemase-producer. The blaOXA-894 gene was located between the gene encoding a LysR family transcriptional regulator and the C15 gene. Its gene environment was similar to other S. xiamenensis with chromosome-located blaOXA-48-like genes. The T24H and T94V amino acid substitutions of LuxS protein were predicted to be deleterious, which may affect the virulence phenotype. The occurrence and potential health risk of carbapenem-resistant S. xiamenensis in a water environment is of concern.


Introduction
The bla OXA-48 gene, encoding class D beta-lactamases, was first reported in Klebsiella pneumoniae from a patient with urinary tract and skin burns in Turkey in 2001 [1]. The enzyme usually hydrolyzed penicillins at high levels, but hydrolyzed carbapenems at a low level [2]. Bla OXA-48 -like genes were widely reported among K. pneumoniae and other Enterobacteriaceae [3]. To date, 91 bla OXA-48 -like variants have been identified, with classical bla OXA-48 being the most widespread [4][5][6][7]. Bla OXA-48 -like gene carriers caused outbreaks of nosocomial and community infections in many countries, including China [3,8]. The number of reservoirs for these organisms was increasing among humans, animals and in the environment [3]. Thus, the rapid dissemination of carbapenem-resistant species harboring bla OXA-48 -like genes in different ecosystems has posed a severe threat to human health.
Shewanella xiamenensis, Gram-negative rods, 0.7-0.8 × 2.5-4.0 µm, was isolated from coastal sediments for the first time in China [9]. S. xiamenensis was usually detected in the marine and freshwater environment, and rarely isolated from an animal source [10]. It has become an emerging pathogen contributing to intestinal colonization and abdominal cavity infection [11]. S. xiamenensis has been regarded as a progenitor of OXA-48 family class D beta-lactamase [12]. The chromosome-mediated bla OXA-48 -like genes may be mobilized onto plasmids by insertion sequences, then plasmids could be transferred to clinically relevant pathogens (such as Escherichia coli). Increasing numbers of bla OXA-48 -variants were reported in S. xiamenensis from human and environmental sources, such as bla OXA-181 , bla OXA-199 , bla OXA-204 , and bla OXA-538 [13][14][15][16]. Thus, the purpose of this study was to characterize a gene belonging to the bla OXA-48 family, and investigate its genetic environment in S. xiamenensis.

Antibiotic Susceptibility Testing
The minimum inhibitory concentrations (MICs) of S. xiamenensis strain sx20 were determined using the broth microdilution method with the following agents: amikacin, ampicillin, ampicillin-sulbactam, aztreonam, cefazolin, ceftazidime, cefotetan, ceftriaxone, cefepime, ciprofloxacin, ertapenem, fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, nitrofurantoin, piperacillin-tazobactam, tobramycin (Meilun, Dalian, China). The control strain was E. coli ATCC 25922. The results were interpreted according to EUCAST guidelines. Phenotypic detection of carbapenemase was performed using the Carba-direct NP test [18]. Bacteria were cultured on Mueller-Hinton agar overnight (Oxoid, Basingstoke, UK). The bacterial mass was scraped off with a 1-µL loop and suspended in a 1.5-mL Eppendorf tube containing 100 µL of 20 mM Tris-HCl lysis buffer. This lysate was mixed with 100 µL of an aqueous indicator solution which contained 0.05% phenol red with 0.1 mmol/L ZnSO 4 and 6 mg/mL imipenem, and the phenol red solution without antibiotic as a control tube.

Whole-Genome Sequencing and Analysis
The entire genome of S. xiamenensis strain sx20 was sequenced using whole-genome sequencing (WGS) analysis using an Illumina HiSeq 4000-PE150 platform (Illumina, CA, USA). The sequences were assembled using SPAdes 3.11 and annotated via RAST (http://rast.nmpdr.org/). Antibiotic resistance genes and virulence genes were analyzed by Resfinder (https://cge.cbs.dtu.dk/services/ResFinder/) and the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/). The genetic environment was visualized by Easyfig 2.2.3. Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and Jalview [19] were used to perform the alignment analysis of amino acid sequences. The effect of the biological function of a protein caused by amino acid substitution or indel was predicted by PROVEAN (http://provean.jcvi.org/). The sequencing data of the whole genome and bla OXA-894 gene was deposited under the GenBank accession number SUNE00000000 and MN525568, respectively.

The Conjugation Assay
The conjugation experiment was carried out using the mixed broth method as previously described [20]. Conjugation was performed using E. coli J53 (sodium azide-resistant) as the recipient strain. Transconjugants were selected on LB agar plates (Oxoid, Basingstoke, UK) supplemented with sodium azide (100 mg/L) and meropenem (2 mg/L).

Results and Discussion
In our study, S. xiamenensis strain sx20 was isolated from pig wastewater in rural China. It was resistant to ampicillin, aztreonam, ertapenem, imipenem, meropenem and fosfomycin. The Carba NP test showed it was a carbapenemase producer. Carbapenem MICs of S. xiamenensis strain sx20 were similar to S. xiamenensis IR34 harboring bla OXA-204 gene and S. xiamenensis DDP1 harboring bla OXA-416 (Table 1), but higher than strains of IR24 and IR33 harboring bla OXA-48 gene and S12-harboring bla OXA-181 gene [12,13,22], indicating there may be additional mechanisms for regulating carbapenem resistance in S. xiamenensis. Year means the publication time of the strain.
The results of the phylogenetic analysis ( Figure 2) showed that the bla OXA-894 gene formed a cluster with the bla OXA-546 , bla OXA-48b , bla OXA-547 gene sequences detected in S. xiamenensis. The first such bla OXA-48 -like gene in S. xiamenensis to be reported was from India, in 2011, namely bla OXA-181 gene, which has activity against carbapenems [13]. Until now, at least 10 bla OXA-48 -like variants have been identified in the S. xiamenensis from India, China, Portugal, Italy and Algeria, including bla OXA-48 , bla OXA-48b , bla OXA-181 , bla OXA-199 , bla OXA-204 , bla OXA-416 , bla OXA-538 , bla OXA-546 , bla OXA-894 and bla OXA-547 [10,[12][13][14][15][16]22,23]. It indicated that the bla OXA-48 -like genes are evolving continuously in different regions.  BlaOXA-546 was first reported in the plasmid of S. xiamenensis Sh1 isolated from saltmarsh plants in the USA, 2018 [14]. However, the chromosome-mediated blaOXA-546 gene has not been reported until now. In the current study, the single copy of the blaOXA-894 gene was found in S. xiamenensis sx20. It was located on  The results of the phylogenetic analysis ( Figure 2) showed that the blaOXA-894 gene formed a cluster with the blaOXA-546, blaOXA-48b, blaOXA-547 gene sequences detected in S. xiamenensis. The first such blaOXA-48-like gene in S. xiamenensis to be reported was from India, in 2011, namely blaOXA-181 gene, which has activity against carbapenems [13]. Until now, at least 10 blaOXA-48-like variants have been identified in the S. xiamenensis from India, China, Portugal, Italy and Algeria, including blaOXA-48, blaOXA-48b, blaOXA-181, blaOXA-199, blaOXA-204, blaOXA-416, blaOXA-538, blaOXA-546, blaOXA-894 and blaOXA-547 [10,[12][13][14][15][16]22,23]. It indicated that the blaOXA-48-like genes are evolving continuously in different regions. BlaOXA-546 was first reported in the plasmid of S. xiamenensis Sh1 isolated from saltmarsh plants in the USA, 2018 [14]. However, the chromosome-mediated blaOXA-546 gene has not been reported until now. In the current study, the single copy of the blaOXA-894 gene was found in S. xiamenensis sx20. It was located on Bla OXA-546 was first reported in the plasmid of S. xiamenensis Sh1 isolated from saltmarsh plants in the USA, 2018 [14]. However, the chromosome-mediated bla OXA-546 gene has not been reported until now. In the current study, the single copy of the bla OXA-894 gene was found in S. xiamenensis sx20. It was located on the chromosome and inserted between the LysR family transcriptional regulator and the C15 gene (Figure 3). The genetic context of bla OXA-894 was similar to that previously reported for other bla OXA-48 -like genes in the Shewanella species [14]. The occurrence of bla OXA-894 can increase the diversity of chromosome-mediated carbapenem-hydrolyzing class D β-lactamase genes in Shewanella species. No mobile element was found upstream and downstream of the bla OXA-894 gene in S. xiamenensis strain sx20, indicating a low probability of horizontal gene transfer. But bla OXA-894 gene was detected in an isolate from water, an environment that can be frequently affected by anthropogenic activities (such as discharges of wastewater), which may potentiate the spread of this gene in the environment, in animals and in humans. Therefore, the occurrence and potential health risk of carbapenem-resistant S. xiamenensis in water environment needs to be concerned. the chromosome and inserted between the LysR family transcriptional regulator and the C15 gene ( Figure  3). The genetic context of blaOXA-894 was similar to that previously reported for other blaOXA-48-like genes in the Shewanella species [14]. The occurrence of blaOXA-894 can increase the diversity of chromosome-mediated carbapenem-hydrolyzing class D β-lactamase genes in Shewanella species. No mobile element was found upstream and downstream of the blaOXA-894 gene in S. xiamenensis strain sx20, indicating a low probability of horizontal gene transfer. But blaOXA-894 gene was detected in an isolate from water, an environment that can be frequently affected by anthropogenic activities (such as discharges of wastewater), which may potentiate the spread of this gene in the environment, in animals and in humans. Therefore, the occurrence and potential health risk of carbapenem-resistant S. xiamenensis in water environment needs to be concerned. According to the virulence factor database, only the luxS gene was detected with 82% nucleotide identity with wild type luxS (NC_000913). The deduced LuxS protein differs from wild-type LuxS (NP_417172) by 44 amino acid substitutions, and the T24H and T94V substitutions were predicted to be deleterious by PROVEAN. The luxS gene in the Shewanella encodes an autoinducer-2-like molecule which was the postulated universal bacterial signal. The mutants of the luxS gene could influence the biofilm formation, production of virulence factors and motility of pathogenic bacteria [24]. It indicated that the mutants of luxS gene may affect the virulence phenotype of the S. xiamenensis strain sx20.

Conclusions
This is the first report of chromosome-mediated blaOXA-894 gene in S. xiamenensis. The OXA-894 differs from OXA-546 (A46V, I219del), OXA-48 (T167I, I219del) with two amino acid substitutions, respectively. BlaOXA-894 gene was inserted between the LysR family transcriptional regulator and C15 gene. The occurrence of blaOXA-894 can increase the diversity of chromosome-encoded carbapenemhydrolyzing class D β-lactamases identified in Shewanella species. A mutated luxS gene was also identified in this strain, which may affect the virulence phenotype of S. xiamenensis. The occurrence and potential health risk of carbapenem-resistant bacteria in the water environment is of concern. According to the virulence factor database, only the luxS gene was detected with 82% nucleotide identity with wild type luxS (NC_000913). The deduced LuxS protein differs from wild-type LuxS (NP_417172) by 44 amino acid substitutions, and the T24H and T94V substitutions were predicted to be deleterious by PROVEAN. The luxS gene in the Shewanella encodes an autoinducer-2-like molecule which was the postulated universal bacterial signal. The mutants of the luxS gene could influence the biofilm formation, production of virulence factors and motility of pathogenic bacteria [24]. It indicated that the mutants of luxS gene may affect the virulence phenotype of the S. xiamenensis strain sx20.

Conclusions
This is the first report of chromosome-mediated bla OXA-894 gene in S. xiamenensis. The OXA-894 differs from OXA-546 (A46V, I219del), OXA-48 (T167I, I219del) with two amino acid substitutions, respectively. Bla OXA-894 gene was inserted between the LysR family transcriptional regulator and C15 gene. The occurrence of bla OXA-894 can increase the diversity of chromosome-encoded carbapenem-hydrolyzing class D β-lactamases identified in Shewanella species. A mutated luxS gene was also identified in this strain, which may affect the virulence phenotype of S. xiamenensis. The occurrence and potential health risk of carbapenem-resistant bacteria in the water environment is of concern.
Author Contributions: X.L. designed the study; H.X. and Q.Z. contributed to the sampling; H.Z. and Z.Z. performed the experiments; H.Z. analyzed the data and wrote the manuscript. All authors reviewed and revised the manuscript.