EBR-5, a Novel Variant of Metallo-β-Lactamase EBR from Multidrug-Resistant Empedobacter stercoris

Carbapenemases are one of the greatest threats to clinical therapy, as they could confer resistance by hydrolyzing carbapenems and other β-lactam antimicrobials. In this study, we identified a novel metallo-β-lactamase EBR variant, namely, EBR-5, in Empedobacter stercoris. ABSTRACT A novel chromosome-encoded metallo-β-lactamase (MBL) EBR variant, namely, EBR-5, was identified in a multidrug-resistant Empedobacter stercoris strain SCVM0123 that was isolated from chicken anal swab samples. EBR-5 shared 82.13% amino acid identity with the previously known EBR-1. The expression of EBR-5 in Escherichia coli reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. Compared with blaEBR-1, the recombinant strain harboring blaEBR-5 exhibited higher minimum inhibitory concentrations of piperacillin, cefotaxime, and meropenem. Despite the genetic diversity, EBR-5 and EBR-1 possessed similar kinetic parameters, except for cefepime, cefotaxime, cefoxitin, cephalothin, and meropenem, which were hydrolyzed more by EBR-5. In addition to blaEBR-1, a whole-genome sequencing analysis of SCVM0123 also revealed a plasmid-mediated blaRAA-1 gene. This study underlines the importance of E. stercoris monitoring, as it could be a potential reservoir of these β-lactamase genes. IMPORTANCE Carbapenemases are one of the greatest threats to clinical therapy, as they could confer resistance by hydrolyzing carbapenems and other β-lactam antimicrobials. In this study, we identified a novel metallo-β-lactamase EBR variant, namely, EBR-5, in Empedobacter stercoris. The biochemical properties, substrate hydrolysis abilities, and inhibition profiles of EBR-5 were reported. Through whole-genome sequencing and bioinformatic analyses, we revealed for the first time that the ESBL gene blaRAA-1 was located on a plasmid. This study extends the database of class B metallo-β-lactamases. Meanwhile, E. stercoris could be a major reservoir of blaEBR-5 and blaRAA-1, which have potential to spread to pathogens.

IMPORTANCE Carbapenemases are one of the greatest threats to clinical therapy, as they could confer resistance by hydrolyzing carbapenems and other b-lactam antimicrobials. In this study, we identified a novel metallo-b-lactamase EBR variant, namely, EBR-5, in Empedobacter stercoris. The biochemical properties, substrate hydrolysis abilities, and inhibition profiles of EBR-5 were reported. Through whole-genome sequencing and bioinformatic analyses, we revealed for the first time that the ESBL gene bla RAA-1 was located on a plasmid. This study extends the database of class B metallo-b-lactamases. Meanwhile, E. stercoris could be a major reservoir of bla EBR-5 and bla RAA-1 , which have potential to spread to pathogens. KEYWORDS E. stercoris, carbapenemase, EBR-5, novel variant E mpedobacter stercoris (E. stercoris), a Gram-negative, rod-shaped bacterium that belongs to the Weeksellaceae (formerly classified as Flavobacteriaceae) family (1). It is usually recovered from fecal samples, and it has been reported as a major reservoir of the tigecycline resistance gene tet(X14) (2). However, few publications report its phenotype or its resistance mechanism toward b-lactams in E. stercoris.
A carbapenem-resistant E. stercoris SCVM0123 was isolated from chicken anal swab samples that were obtained in Chongqing, China, in 2019. Antimicrobial susceptibility tests were performed via a modified Clinical & Laboratory Standards Institute (CLSI)-based method (11). The minimum inhibitory concentration (MIC) results showed that SCVM0123 was resistant to almost all of the tested antibiotic categories, including aminoglycosides, phenicols, fluoroquinolones, macrolides, lincosamides, tetracyclines, expanded-spectrum cephalosporins, and carbapenems (Table 1). To understand the resistance mechanism, the complete genome of SCVM0123 was sequenced using an Illumina HiSeq 4000 system and a PacBio RS II platform. Genome annotation and gene prediction were performed using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) (12). The sequencing results indicated that the genome of SCVM0123 consisted of a circular chromosome (3.03 Mb; guaninecytosine [GC] content of 31.76%) and three plasmids: pLPY01 (10,226 bp; GC content of 34%), pLPY02 (15,774 bp; GC content of 30%), and pLPY03 (20,509 bp; GC content of 32%). A wholegenome sequencing (WGS) analysis based on NCBI's AMRFinderPlus (13) revealed 15 putative resistance genes, more than those that were previously reported in E. stercoris strain ES183 (2) (Table S1). Furthermore, these resistance genes were correlated with the antibiotic resistance profiles of SCVM0123. Among these, the most prominent genes were those encoding b-lactamases, including the plasmid-mediated extended-spectrum b-lactamase (ESBL) bla RAA-1 and a novel, chromosomally-located, metallo-b-lactamase (MBL) bla EBR -like gene.
The bla RAA-1 was recently identified in the genome of Riemerella anatipestifer RCAD0122, and it conferred high-level resistance to extended-spectrum cephalosporins (14). At present, this gene was not found in any clinical pathogens, except for RCAD0122. However, in this  study, we discovered that it was located on the pLPY03 plasmid of E. stercoris SCVM0123. The GC content of bla RAA-1 (32%) was the same as that of pLPY03, but it was slightly different from that of the RCAD0122 genome (35.03%). An analysis of the genetic environment revealed that the SCVM0123-harbored bla RAA-1 gene was embedded in a putative composite transposon in pLPY03 that was bracketed by two copies of IS982 family insertion sequences (IS) (Fig. S1). When the whole sequence of pLPY03 was aligned to the approximately 20 kb region of RCAD0122 that harbored the bla RAA-1 gene, the results indicated that only the bla RAA-1 region that was flanked by IS had 100% identity (14). As E. stercoris and R. anatipestifer possessed the same transposon structure, we speculate that the putative composite transposon has contributed to the dissemination of the bla RAA-1 gene among these strains. Moreover, the bla RAA-1 genes that were found in these strains might be all from a common origin. The WGS and polymerase chain reaction (PCR)-confirmed sequence both revealed a novel bla EBR -like gene. This gene was located at positions 786783 to 787487 on the genome of SCVM0123 (CP104209, locus_tag NZD85_03605), encoding a 234-amino-acid protein (pro-tein_id: UWX67704). The results of an amino acid sequence alignment that was performed using the NCBI Basic Local Alignment Search Tool (BLAST) showed that 7 hits were obtained with an identity of .94% and a coverage of 100%, including 1 amino-acid sequence, 2 complete genome sequences, and 4 draft genome sequences (Table S2) (accessed 20 September 2022). The alignment results also showed that this protein barely exceeded 80% identity to the previously reported EBR alleles EBR-1 to EBR-4 (which are nearly identical to each other) (15). The apparent amino acid sequence changes indicated that this MBL enzyme was a new variant, and as such, it was designated EBR-5 (Fig. 1). Interestingly, EBR-5 was exclusively detected in E. stercoris. A phylogenetic analysis showed that EBR-5 belonged to Ambler subclass B1 MBL and that it was branched separately from the previously reported EBR variants (Fig. 2). The cleavage site for the leader peptide of EBR-5 was predicted to be located between positions 17 and 18 (AFG-QI) (Fig. 1).
To characterize the function of bla EBR-5 , the intact gene was cloned into pET24a and transformed into E. coli BL21(DE3) ( Table S3). Compared with a negative control, a 2-to 512-fold increase in the MIC was observed for the bla EBR-5 recombination clones, except for aztreonam, suggesting that bla EBR-5 was active against penicillins, cephalosporins, cephamycins, and carbapenems ( Table 1). The MICs of b-lactams were not lowered by the addition of clavulanic acid, sulbactam, or tazobactam, indicating that these inhibitors could not inhibit the b-lactams' hydrolyzing activity of EBR-5. Compared with the previously reported EBR-1 (10), EBR-5 exhibited similar susceptibility to b-lactams, with the exceptions of piperacillin, cefotaxime, and meropenem, which were 4-, 16-, and 8-fold decreased, respectively. These results indicated that EBR-5 was a carbapenemase and conferred resistance to expandedspectrum cephalosporins and carbapenems.
In summary, this study reports the discovery of a novel, chromosomally-located bla EBR-5 and a plasmid-mediated bla RAA-1 in a multidrug-resistant E. stercoris strain SCVM0123. Our study extends the database of class B carbapenemases and highlights the need to monitor E. stercoris, as it might play an essential role in the dissemination of b-lactamase genes.
Data availability. The complete sequences of the E. stercoris SCVM0123 chromosome and plasmids (pLPY01, pLPY02, and pLPY03) were deposited at GenBank under the accession numbers CP104209 to CP104212.

ACKNOWLEDGMENTS
This work was supported by a grant from the National Natural Science Foundation of China (grant no. 31902340) and by the National Center of Technology Innovation for Pigs [NTCIP-XD/B11].
We have no competing interests to declare.