OXA-830, a Novel Chromosomally Encoded Extended-Spectrum Class D β-Lactamase in Aeromonas simiae

The diversity of class D β-lactamases mediating resistance to β-lactams has been increasingly reported recently. In this study, a novel class D oxacillinase named OXA-830 was identified in a fully sequenced Aeromonas simiae strain, which was isolated from sewage discharged from a farm in southern China. OXA-830 shares the highest amino acid identity of 79.3% with an OXA-12-like variant named OXA-725. When expressed in E. coli DH5α, OXA-830 conferred resistance to penicillins and selected β-lactamase inhibitors but not to cephalosporins and carbapenems. Kinetic analysis of OXA-830 revealed a broad substrate profile including penicillins, cefazolin, cefoxitin, and ceftazidime but not carbapenems. The hydrolytic activity was significantly inhibited by sulbactam, followed by tazobactam, but was less effectively inhibited by clavulanic acid. The blaOXA–830 gene was located on the A. simiae A6 chromosome and the blaOXA–830-related region was bracketed by a pair of perfect inverted repeats.


INTRODUCTION
The genus Aeromonas is a distinct group of oxidase-positive, facultatively anaerobic, Gram-negative bacilli of the family Aeromonadaceae (Colwell et al., 1986). Members of Aeromonas can be isolated from every environmental niche where bacterial ecosystems exist, including aquatic habitats and fish as well as food products, and are implicated in human and animal infections (Janda and Abbott, 2010). The species Aeromonas simiae was first described in two strains (CIP 107798 and CIP 107797) isolated from feces of healthy monkeys from Mauritius (Harf-Monteil et al., 2004). Further study revealed that these two strains may originate from the same clone because they share entirely identical 16S rRNA, gyrB, and rpoD genes with each other (Saavedra et al., 2006). To date, there have been a large number of reports about the presence of β-lactamase genes among Aeromonas strains (Carvalho et al., 2012;Chen et al., 2012).
Oxacillin-hydrolyzing (OXA)-type β-lactamases (OXAs) constitute most of the members of Ambler class D active-serine-site β-lactamases (Bush et al., 1995) and are widely identified among clinically relevant Gram-negative bacteria, such as Pseudomonas spp., Acinetobacter spp., Aeromonas spp. and Enterobacteriaceae (Poirel et al., 2010). Most of the OXAs possess the ability to hydrolyze cloxacillin or oxacillin at a rate of >50% that for benzylpenicillin and are typically not inhibited by commercially available β-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam (Payne et al., 1994;Bush et al., 1995). According to the Bush functional classification scheme for β-lactamases (Bush et al., 1995), OXAs are classified into group 2d. Although most of the early identified OXAs exhibited a substrate profile strictly restricted to penicillin, oxacillin, cloxacillin and nitrocefin, several OXA members have been demonstrated to be active against extended-spectrum cephalosporins which is typically due to a small number of point mutations occurring in parental narrow-spectrum class D β-lactamases (DBLs), such as the derivatives of OXA-10 (Poirel et al., 2010;Leonard et al., 2013). For instance, compared to OXA-10,  exhibits the ability to hydrolyze ceftazidime (Hall et al., 1993); OXA-17 (N76S) has an increased hydrolytic ability for cefotaxime as well as a decreased capacity for ceftazidime (Danel et al., 1999); and OXA-19, which differed from OXA-10 by nine amino acids, can hydrolyze ceftazidime with a low activity (Mugnier et al., 1998). Some OXAs have evolved to exhibit hydrolytic activity toward β-lactams of "last resort, " i.e., carbapenems (El Garch et al., 2011;Antonelli et al., 2015). Similar to other antimicrobial resistance genes, many OXA β-lactamase genes have been identified on both plasmids and chromosomes with diverse mobile genetic elements (MGEs), such as integrons, insertion sequences and transposons (Poirel et al., 2010). For example, the bla OXA−1 -like, bla OXA−2 -like, and bla OXA−10 -like genes were commonly captured as gene cassettes by integrons in plasmids (Naas and Nordmann, 1999); the bla OXA−23 gene from the chromosome of Acinetobacter radioresistens may be transferred onto plasmids diffusing into Acinetobacter baumannii through the ISAba1-based composite transposon Tn2006 or transposon-like structure named Tn2008, or a single copy of ISAba4 upstream of the gene (Corvec et al., 2007;Adams-Haduch et al., 2008); and the bla OXA−58 gene-encoding regions in A. baumannii isolated from different countries (France, Spain, Romania, and Turkey) were bracketed by ISAba3 on one side and ISAba3, ISAba1 or IS18 on the other side (Poirel and Nordmann, 2006). However, according to the literature, there are intrinsic chromosomally encoded OXAs in many bacterial species (Poirel et al., 2010). The first such gene, identified in 1994, originated from the chromosome of Aeromonas jandaei (formerly Aeromonas sobria), which was not associated with an integron or transposon and was named bla OXA−12 (Rasmussen et al., 1994).
In this study, for the first time, we determined the complete genome sequence of A. simiae, i.e., a sewage-derived A. simiae strain A6. Based on sequence analysis, we identified and characterized a novel chromosomally encoded DBL named OXA-830, which is quite divergent from the other OXA β-lactamases.

Bacterial Strains
The host strain A. simiae A6 carrying the bla OXA−830 gene was isolated in November 2017 from sewage discharged from a farm in Wenzhou, China. Species identification was conducted using the Vitek-60 microorganism autoanalysis system (BioMérieux Corporate, Craponne, France). Further verification was performed based on the 16S rRNA sequencing method. Moreover, considering limited resolution the 16S rRNA gene provides to discriminate among closely related species of the genus Aeromonas (Alperi et al., 2008), a multilocus phylogenetic analysis (MLPA) of the concatenated sequences of 6 housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA, and atpD) as previously reported (Beaz-Hidalgo et al., 2015) as well as whole-genome sequence-based phylogenetic analysis using kSNP3.0 (Gardner et al., 2015) were conducted to determine the evolutionary relationship of A. simiae A6 with 32 other Aeromonas sp. strains of different species. Two neighbor-joining phylogenetic trees were generated by using MEGA7 with 1,000 bootstrap replications (Kumar et al., 2016). The bacteria and plasmids used in this work are listed in Supplementary Table S1.

Antimicrobial Susceptibility Testing
The minimum inhibitory concentrations (MICs) were determined using the agar dilution method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Susceptibility patterns were interpreted according to the CLSI breakpoint criteria (CLSI, 2019)

Whole-Genome Sequencing and Sequence Analysis
Whole-cell DNA of A.simiae A6 was extracted using the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen Scientific, Union City, CA, United States) and sequenced on a PacBio RS II instrument (Pacific Biosciences). The PacBio long reads were initially assembled by Canu v1.6 (Koren et al., 2017), and then two FASTQ sequence files generated using the Illumina HiSeq 2500 platform were mapped onto the primary assembly to control assembly quality and to correct possible misidentified bases by using Bwa and the Genome Analysis Toolkit (McKenna et al., 2010). The consensus sequence was obtained by a customderived script written in Python 1 . Potential open reading frames (ORFs) were predicted using Glimmer software (Delcher et al., 2007) and annotated against the UniProt/Swiss-Prot and nonredundant protein databases using the BLASTX program with an e-value threshold of 1e-5. GView was used to construct basic genomic features (Petkau et al., 2010). Annotation of MGEs and resistance genes was performed using ISfinder (Siguier et al., 2006), INTEGRALL (Moura et al., 2009) and ResFinder (Zankari et al., 2012) with default parameters. The molecular weight and pI value of OXA-830 was predicted using ProtParam 2 . The putative signal peptide cleavage site of OXA-830 was identified by SignalP 5.0 (Almagro Armenteros et al., 2019).
Amino acid alignment and the neighbor-joining phylogenetic tree construction of OXA-830 with other DBLs were performed using the MAFFT program and MEGA7 with a bootstrap of 1,000 replicates, respectively (Katoh and Standley, 2013;Kumar et al., 2016). Comparisons of the nucleotide sequences were performed using BLASTN. Other bioinformatics tools were written using Python (see text footnote 1) and Biopython (Cock et al., 2009).
Cloning of the bla OXA−830 Gene and Expression and Purification of OXA-830 The gene encoding OXA-830 along with its promoter was amplified by PCR using forward (5 -CGGAATTCAGACACAGATTGGCACAGCA-3 ) and reverse (5 -CCCAAGCTTGCCCGGTGAAGAAGAAGTGA-3 ) primers with a pair of flanking restriction endonuclease adapters. Then, the PCR product was eluted from an agarose gel, digested with the EcoRI and HindIII, and ligated into the pUCP24 vector with a T4 DNA ligase cloning kit (Takara Bio, Inc., Dalian, China). The recombinant plasmids were transformed into competent E. coli DH5α by the calcium chloride method, and bacterial colonies were cultured on Luria-Bertani agar plates supplemented with 20 µg/mL gentamicin. The recombinant plasmids were verified by both restriction enzyme digestion and Sanger sequencing (Shanghai Sunny Biotechnology Co., Ltd., Shanghai, China). The similar procedure was also applied to cloned complete ORF of bla OXA−830 into pET-28b. The recombinant plasmid (pET-OXA-830) was transformed into competent E. coli BL21 cells by the calcium chloride method, and transformants were selected on Luria-Bertani agar plates supplemented with 50 µg/mL kanamycin. The authenticity of cloned fragments was confirmed by Sanger sequencing (Shanghai Sunny Biotechnology Co., Ltd., Shanghai, China). The above-mentioned E. coli BL21 transformant was grown in Luria-Bertani medium with 50 µg/mL kanamycin at 37 • C. Overnight cultures were diluted 100-fold in 200 mL of Luria-Bertani medium and incubated for hours at 37 • C with orbital shaking. Isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma Chemicals Co., St. Louis, MO, United States) was added to a final concentration of 1 mM until the cultures reached an OD 600 between 0.6 and 0.8, and incubation was continued for an additional 4 h. OXA-830 was isolated from the periplasm and purified by affinity chromatography based on the instructions of the His-tag Protein Purification Kit (P2226, Beyotime, China).

Determination of Kinetic Parameters
Kinetic parameters for hydrolysis of β-lactams by the purified OXA-830 β-lactamase were examined using a UV-VIS spectrophotometer (U-3900, HITACHI, Japan) at 30 • C in 10 mM phosphate buffer (pH 7.0) in a final reaction volume of 300 µL. The steady-state kinetic parameters (k cat and K M ) were determined by non-linear regression of the initial reaction rates with the Michaelis-Menten equation in Prism (version 7) software (GraphPad Software, San Jose, CA, United States). β-lactamase inhibition was studied with benzylpenicillin (500 µM) as the substrate. The β-lactamase inhibitors sulbactam, tazobactam and clavulanic acid at various concentrations were preincubated with the purified OXA-830 β-lactamase for 3 min at 30 • C before addition of substrate. The inhibitor concentration required to reduce the hydrolysis of 500 µM benzylpenicillin by 50% was determined by non-linear regression with the log(inhibitor) vs. response -Variable slope equation in Prism (version 7) software (GraphPad Software, San Jose, CA, United States).

Nucleotide Sequence Accession Number
The complete nucleotide sequences of the chromosome of A. simiae A6 and the bla OXA−830 gene in this work have been submitted to DDBJ/EMBL/GenBank under accession numbers CP040449 and MK926981, respectively.

RESULTS AND DISCUSSION
Identification and Characterization of the OXA-830-Producing Isolate, A. simiae A6 Aeromonas simiae A6 was isolated in 2017 from sewage discharged from a farm in Wenzhou, southern China. A 16S ribosomal RNA gene homology analysis showed that A. simiae  (Figures 1A,B). We finally grouped the strain into the species A. simiae and named it A. simiae A6.
Since the high MICs of the aforementioned antimicrobials and no complete genome sequence of species A. simiae is FIGURE 2 | Genomic comparison of A. simiae A6 with its close relatives. From outside to inside: circles 1 and 2 are homologous regions of A. schubertii WL1483 (accession number CP013067) and A. schubertii LF1708 (CP039611) compared to A. simiae A6, while unmatched regions are left blank; circles 3 and 4 display predicted ORFs encoded in the forward strand and reverse strand, respectively; circles 5 and 6 represent the GC content and GC skew maps, respectively; and circle 7 shows the scale in kb.
currently available in the public database, the complete genome of A. simiae A6 was determined to unveil the potential factors associated with the resistance profiles. The results showed that A. simiae A6 has a circular chromosome (without plasmids) of 3.97 Mb in size that contains 3,633 ORFs with an average GC content of 60.56% (Table 2 and Figure 2). Comparative genomics analysis revealed the genome of A. simiae A6 shared the highest sequence similarities with that of A. schubertii WL1483 (accession number CP013067, at 91.62% identity and 65% coverage) and A. schubertii LF1708 (accession number CP039611, at 91.56% identity and 65% coverage) (Figure 2). Notably, A. simiae A6 encodes two predicted DBL-encoding genes. One is bla OXA−10 located in a truncated class I integron with a gene array of intI1-qnrVC4-cmlA5-bla OXA−10 -aac(6')-Ibcr-aadA1-dfrA14-mobC-IS6100 (ranging from 3261 to 3269 kb), and the other belongs to a novel OXA β-lactamase named Possible Origin of the New Class D β-Lactamase OXA-830 bla OXA−830 gene was 801 bp in length and encoded a 266 amino acid preprotein of ca. 29.4 kDa. A signal peptide cleavage site was predicted to be between an alanine and asparagine at amino acid residues 22 and 23. In addition, the pI value of OXA-830 was predicted to be 9.18. The closest relative of OXA-830 was OXA-725, a class D β-lactamase with detectable hydrolytic activity against oxacillin, penicillin and ampicillin as well as carbenicillin, which was previously described in a clinical A. jandaei (formerly A. sobria) isolate from the Hammersmith Hospital, London (Walsh et al., 1995). Screening for bla OXA−830 -homologous genes (>70% amino acid sequence identity) in the NCBI nucleotide database showed that most of the close relatives (89.19%, 66/74) were derived from the genus Aeromonas, except 4 genes from Enterobacteriaceae and 4 of undetermined origin, suggesting the importance of Aeromonas as the reservoir for bla OXA−830 -like genes. Additionally, OXA-830 was distantly related to other class D β-lactamases in amino acid sequence identity, i.e., OXA-12 and its close variants (77.1% with OXA-12; 78.9% with OXA-726; and 78.6% with OXA-724). Moreover, it shared identities of respective 72.2, 71.1, and 70.7% with OXA-427, OXA-780, and OXA-504 (Figure 3), and possessed less than 50% identities to all of the other DBLs.

Genetic Context of bla OXA−830 Gene
A comparative genomics analysis of the genetic environment of the bla OXA−830 gene in A6 with that of closely related DBLencoding genes in three other Aeromonas strains showed that glmU (encoding the bifunctional protein GlmU) together with upstream sequences shared a conserved syntenic architecture in gene content and gene order (Figure 5). However, the genes downstream of glmU in the bla OXA−830 -related region were highly distant to those of all the other bla OXA genes. In fact, the region from bla OXA−830 to a large hypothetical gene in the bla OXA−830 -related region was flanked by a pair of perfect 9-bp inverted repeats (IRs), suggesting these genes might be transferred from the other strain through horizontal gene transfer. Furthermore, an approximately 25-kb genomic island including two phage integrase-encoding genes was found upstream of the large hypothetical gene, which was enwrapped by a pair of perfect 10-bp direct repeats (DRs). This indicated that the mobilization of this genomic island onto the genome of A6 might have resulted from the integrase-mediated site-specific recombination. Altogether, these potential lateral gene transfer events may well explain the discrepancy of gene content between the bla OXA−830 -related region and other bla OXA -related regions.
The kinetic parameters of the OXA-830 β-lactamase purified from the extract of E. coli BL21 harboring the recombinant plasmid pET-OXA-830 demonstrated that OXA-830 had a strong hydrolytic activity against penicillins (including oxacillin, cloxacillin, benzylpenicillin, ampicillin, piperacillin, and ticarcillin) and cefazolin (k cat /K m ratios were ≥0.73 µM −1 ·s −1 ), while very poor hydrolytic activities were detected for cefoxitin and ceftazidime. Moreover, no measurable hydrolytic activities were observed for cefepime, aztreonam and imipenem (Table 3). Nevertheless, this finding was not entirely consistent with the above MIC changes for E. coli DH5α recombinant clones (pUCP24-bla OXA−830 /DH5α) in the antibiotic susceptibility assay, as the hydrolytic activities against cefazolin, cefoxitin and ceftazidime did not result in significant changes in the MICs for E. coli DH5α recombinant clones. It may be that the activity in vitro was simply too low to contribute to activity in vivo. A similar phenomenon was observed from OXA-436 which showed hydrolytic activity against ceftazidime but not confer resistance to ceftazidime (Samuelsen et al., 2018). OXA-830 exhibited an approximately 2-fold increase in the turnover rate (k cat ) for oxacillin and cloxacillin compared to that for benzylpenicillin. The highest catalytic efficiency was observed with ampicillin (k cat /K m ratio was 2.36 µM −1 ·s −1 ). Of note, the catalytic efficiencies of OXA-830 against oxacillin and ampicillin matched or surpassed those of several clinically important DBLs in Gram-negative pathogens, such as OXA-48 and OXA-58 (Docquier et al., 2009;Verma et al., 2011). Compared to OXA-12 (Rasmussen et al., 1994), OXA-830 exhibited significantly lower affinity for penicillins used in both studies. Although susceptible to cephalosporins, the extended hydrolysis spectrum of OXA-830 indicated that it could be exceptionally classified into subgroup 2de, a new subgroup of DBLs including the oxacillin-or cloxacillin-hydrolyzing β-lactamases exhibiting hydrolytic activity against oxyimino-cephalosporins but not carbapenems (Bush and Jacoby, 2010). Interestingly, most of the members of this subgroup originated from OXA-10, with a small number of amino acid substitutions described as previously (Bush and Jacoby, 2010). However, OXA-830 exhibited only 22.6% global amino acid identity with OXA-10. In contrast, OXA-830 shared much higher global amino acid identity (72.2%) with OXA-427, which could hydrolyze imipenem and exhibit resistance to extended-spectrum cephalosporins (mostly ceftazidime) (Bogaerts et al., 2017).

CONCLUSION
The complete genome sequence of A. simiae was firstly reported in this work. We identified a novel OXA DBL named OXA-830 that was encoded on the chromosome of A. simiae A6.
The new enzyme exhibited high amino acid sequence divergence from all currently available DBLs and showed the top amino acid identities (77.1-79.3%) with OXA-12 and OXA-12-like β-lactamases. Therefore, OXA-830 could be characterized as the first member of a new lineage of DBLs. OXA-830 showed some common functional properties with other OXA β-lactamases such as OXA-12, but significant differences were also observed. It possessed extended-spectrum hydrolytic properties which was strongly inhibited by both sulbactam and tazobactam. These findings revealed the importance of OXA-830 as a new model to study the structure/function relationships among OXA β-lactamases. Paired terminal IRs at both ends of the bla OXA−830related region suggested that the possibility of dissemination of bla OXA−830 in the environment is still existent, although it is not associated with any known MGE.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the GenBank database as GenBank: CP040449 for the A. simiae A6 genome sequence, GenBank: MK926981 for the OXA-830 gene.

AUTHOR CONTRIBUTIONS
QC, KS, XZ, DZ, HL, WL, and KL collected the strains and performed the experiments. WZ, ZS, and QC analyzed the experimental results. WZ, QC, and TX performed the bioinformatics analysis. WZ, JL, and QB co-led the writing of the manuscript. JL and QB designed the work. All authors read and approved the final version of the manuscript.