Characterization of a Novel Chromosomal Class C β-Lactamase, YOC-1, and Comparative Genomics Analysis of a Multidrug Resistance Plasmid in Yokenella regensburgei W13

Yokenella regensburgei, a member of the family Enterobacteriaceae, is usually isolated from environmental samples and generally resistant to early generations of cephalosporins. To characterize the resistance mechanism of Y. regensburgei strain W13 isolated from the sewage of an animal farm, whole genome sequencing, comparative genomics analysis and molecular cloning were performed. The results showed that a novel chromosomally encoded class C β-lactamase gene with the ability to confer resistance to β-lactam antibiotics, designated blaYOC–1, was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the β-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized β-lactamases with the highest amino acid identities to YOC-1 were CDA-1 (71.69%) and CMY-2 (70.65%). The genetic context of the blaYOC–1-ampR-encoding region was unique compared with the sequences in the NCBI nucleotide database. The plasmid pRYW13-125 of Y. regensburgei W13 harbored 11 resistance genes (blaOXA–10, blaLAP–2, dfrA14, tetA, tetR, cmlA5, floR, sul2, ant(3″)-IIa, arr-2 and qnrS1) within an ∼34 kb multidrug resistance region; these genes were all related to mobile genetic elements. The multidrug resistance region of pYRW13-125 shared the highest identities with those of two plasmids from clinical Klebsiella pneumoniae isolates, indicating the possibility of horizontal transfer of these resistance genes between bacteria of various origins.


INTRODUCTION
Yokenella regensburgei is a species of Enterobacteriaceae originally identified by Kosako et al. (1984) through DNA hybridization and biochemical tests in 1984. In 1985, Hickman-Brenner et al. (1985 proposed the name Koserella trabulsii for a new group of Enterobacteriaceae they had discovered that was previously called Enteric Group 45. However, it was subsequently hown by DNA hybridization that Y. regensburgei and K. trabulsii were the same species (Kosako et al., 1987). In 1991, the Centers for Disease Control and Prevention acknowledged that Y. regensburgei had priority upon the basis of prior publication; therefore, the use of K. trabulsii was discontinued in 1991 (McWhorter et al., 1991). Y. regensburgei has been found in a variety of environmental samples (Jain et al., 2013;Chi et al., 2017). This organism has also been sporadically isolated from clinical samples, thus demonstrating its ability to act as an opportunistic pathogen (Abbott and Janda, 1994;Bhowmick and Weinstein, 2013;Jain et al., 2013;Chi et al., 2017;Palmisano et al., 2019;Wright et al., 2019). Intriguingly, Y. regensburgei closely resembles Hafnia alvei and thus has possibly been misidentified as the latter by automated systems. It has been hypothesized that infections caused by Y. regensburgei were underestimated due to misidentification of the bacterium (Bhowmick and Weinstein, 2013).
Many studies have reported that Y. regensburgei possesses an ampC gene and that the expression of this gene is highly inducible (Chi et al., 2017). However, to date, there has been no detailed study of the AmpC β-lactamase in Y. regensburgei, and no complete genome sequence of Y. regensburgei is available in the public NCBI genome database. Y. regensburgei appears to be intrinsically resistant to colistin (Hickman-Brenner et al., 1985), azithromycin and some β-lactam antibiotics, and this resistance is likely related to chromosome-mediated AmpC (Jachymek et al., 1999). In addition to the production of ESBLs, resistance to third-generation cephalosporins can also be mediated by chromosomal and plasmid-encoded AmpCs. In general, AmpCs exhibit broad substrate specificity, including penicillins, narrow-spectrum cephalosporins, cephamycins and aztreonam (variable), and their expression can confer resistance to all these compounds. AmpCs are characterized by their greater capability of hydrolyzing cephalosporins and resistance to β-lactam-based β-lactamase inhibitors, such as clavulanate, sulbactam and tazobactam (Jacoby, 2009;Meini et al., 2019).
The acquisition of exogenous resistance genes is often associated with conjugative plasmids, which may carry multiple resistance genes (De Toro et al., 2015;Medaney et al., 2016). Studies have reported that plasmids are abundant in Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae (Decre et al., 2002;Doi et al., 2002;Gao et al., 2016). However, except for sporadic clinical case reports available in the literature, there have been no reports of the molecular characteristics of the Y. regensburgei genome. In this study, for the first time, we determined the complete genome sequence of the Y. regensburgei strain W13 isolated from the sewage of an animal farm. Based on sequence analysis, we identified and characterized a novel chromosomally encoded AmpC β-lactamase gene, bla YOC-1 , and a conjugative plasmid, pYRW13-125, carrying multiple resistance genes. The identification of the novel chromosome-encoded bla YOC-1 gene in this environmental bacterium can provide detailed information on the intrinsic resistance mechanism of this unusual opportunistic pathogen.

Bacterial Strains
Y. regensburgei W13 was isolated from sewage discharged from an animal farm in Wenzhou, southeastern China. The strain was first identified using the bioMérieux VITEK 2 Compact Instrument (bioMérieux, Marcy L'etoile, France) and then verified by analysis of the 16S rRNA gene sequences and average nucleotide identity (ANI). The strains and plasmids used in this work are listed in Table 1.

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), and the susceptibility patterns were interpreted according to the CLSI breakpoint criteria (CLSI, 2019). E. coli ATCC 25922 was used as a reference strain for quality control.

Whole-Genome Sequencing (WGS) and Functional Annotation of the Genome Sequence
Bacterial DNA was extracted using the Generay Genomic DNA Miniprep kit (Shanghai Generay Biotech Co., Ltd., Shanghai, China) from a single colony subcultured in brain heart infusion broth at 37 • C for approximately 16 h. Genomic DNA was sequenced on a PacBio RS II instrument (Pacific Biosciences). The PacBio long reads were initially assembled by Canu v1.8 (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 (Li and Durbin, 2009) and the Genome Analysis Toolkit (McKenna et al., 2010). Genes were predicted and annotated by using Prokka v1.14.0 (Seemann, 2014); furthermore, the predicted proteins were searched against the NCBI nonredundant and Swiss-Prot databases using DIAMOND (Buchfink et al., 2015) with an e-value threshold of 1e-5. Annotation of the resistance genes was performed using ResFinder (Zankari et al., 2012) and Resistance Gene Identifier (RGI) software of Comprehensive Antibiotic Resistance Database version 4.0.3 1 (McArthur et al., 2013). GView was used to construct basic genomic features (Petkau et al., 2010). Annotation of MGEs was performed using ISfinder (Siguier et al., 2006) and INTEGRALL (Moura et al., 2009). Average nucleotide identity was calculated using OrthoANI version 0.93.1 (Lee et al., 2016). Easyfig v2.2.2 (Sullivan et al., 2011) software was used to generate the figure showing structural comparisons and the nucleotide identities of the different MDR regions. The molecular weight and pI value of YOC-1 were predicted using ProtParam 2 . The putative signal peptide cleavage site of YOC-1 was identified by SignalP 5.0 3 . Amino acid alignment and neighbor-joining phylogenetic tree construction of YOC-1 with other AmpCs were performed using MEGAX (Kumar et al., 2018).
Cloning of Bla YOC-1 , Expression, and Purification of YOC-1 The bla YOC-1 gene sequence with its promoter region and a pair of flanking restriction endonuclease adapters (EcoRI for the forward primer pro-bla YOC-1 -F and HindIII for the reverse primer pro-bla YOC-1 -R, respectively, Table 2) was PCRamplified, and the PCR product eluted from the agarose gel was digested with the corresponding restriction endonucleases and ligated into pUCP24 (digested with EcoRI and HindIII) with a T4 DNA ligase cloning kit (Takara Bio Inc., Dalian, China) ( Table 2). The resulting recombinant plasmid (pUCP24-probla YOC-1 ) was transformed into E. coli DH5α using the calcium chloride method (Suzuki et al., 2019). Transformants (pUCP24pro-bla YOC-1 /DH5α) were selected on Luria-Bertani (LB) agar plates containing 40 µg/mL gentamicin. The cloned PCR product was further confirmed by Sanger sequencing (Shanghai Sunny Biotechnology Co., Ltd., Shanghai, China). The resistance activity of the transformants containing bla YOC-1 to antibiotics was further determined. The predicted cleavage site of the YOC-1 β-lactamase is located after the Ala-22 residue. In order to obtain the β-lactamase YOC-1, the ORF of bla YOC-1 without a signal sequence was cloned with a pair of flanking restriction endonuclease adapters (BamHI and DDDDK for the forward primer orf-bla YOC-1 -F and HindIII for the reverse primer orfbla YOC-1 -R, Table 2) by similar procedures as described above. Using pCold I as a cloning vector and E. coli BL21 as the recipient, transformants (pCold I-bla YOC-1 /BL21) were selected on LB agar plates containing 100 µg/mL ampicillin. The overnight culture of the recombinant strain (pCold I -bla YOC-1 /BL21) was diluted 100-fold in 1 L of LB medium, incubated for 2-3 h at 37 • C with orbital shaking at 250 rpm until the OD 600 reached 0.6, and incubated with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma Chemicals Co., St. Louis, MO, United States) for 24 h at 16 • C (Choi and Geletu, 2018). The recombinant protein was purified by affinity chromatography using BeyoGold His-tag Purification Resin (Beyotime, Shanghai, China) according to the manufacturer's instructions. The histidine tag was removed by Enterokinase (GenScript, Nanjing, China) for 24 h at 23 • C.

Conjugation Experiment
E. coli C600 (MIC to rifampicin > 2048 µg/mL) was used as the recipient in conjugation experiments to detect the transferability of plasmids carried by Y. regensburgei W13 using the broth dilution method. The transconjugant was selected on a brain heart infusion plate supplemented with 2048 µg/mL rifampicin and 16 µg/mL florfenicol. The candidate transconjugant was first tested by plasmid profiling and then indole biochemical property and 16S rRNA gene sequence analyses, which could distinguish whether the candidate transconjugant was the donor, Y. regensburgei W13, or the recipient, E. coli C600 (Y. regensburgei W13 is negative but E. coli C600 is positive for the indole test) (Abbott and Janda, 1994). The plasmid of the transconjugant (pYRW13-125/EC600) was further analyzed by PCR and sequencing for the presence of resistance genes.

Enzyme Kinetic Analysis
Kinetic parameters for hydrolysis of β-lactams by the purified β-lactamase YOC-1 were examined using a UV-VIS spectrophotometer (U-3900, HITACHI, Japan) at 37 • C in 10 mM phosphate buffer (pH 7.0) in a final reaction volume of 200 µL. The steady-state kinetic parameters (k cat and K m ) were determined by nonlinear regression of the initial reaction rates with the Michaelis-Menten equation in Prism (version 7) software (GraphPad Software, CA, United States) (Chen et al., 2019).
β-Lactamase inhibition was studied with nitrocefin (100 µM) as the substrate. The β-lactamase inhibitors avibactam, GMP (disodium 5 -guanosine monophosphate) and clavulanic acid at various concentrations were preincubated with purified YOC-1 pro-bla YOC-1 -R CCAAGCTTTTACTGTAACGCGTTATCACACATC HindIII a Primers with "orf" were used to clone the ORF of the bla YOC-1 gene, and primers with "pro" were used to clone the bla YOC-1 gene with its promoter region. b The underlined sequences represent the restriction endonuclease sites and their protective bases.
at 37 • C for 5 min before the addition of substrate. The inhibitor concentration required to reduce the hydrolysis of 100 µM nitrocefin by 50% was determined by nonlinear regression with the log (inhibitor) vs. response -Variable slope equation in Prism (version 7) software (GraphPad Software, CA, United States) (Chen et al., 2019).

Nucleotide Sequence Accession Numbers
The complete chromosome and plasmid sequences of Y. regensburgei W13 have been submitted to GenBank, and the accession numbers of the chromosome, pYRW13-125 and pYRW13-131 are CP050811, CP050812, and CP050813, respectively.

RESULTS AND DISCUSSION
Genome Characteristics and the Resistance Profile of Y. regensburgei W13 The 16S rRNA gene similarity analysis showed that Y. regensburgei W13 had the closest relationship with Y. regensburgei CIP 105435 (NR_104934.1) at 99.00% coverage and 98.17% identity. The average nucleotide identity (ANI) analysis results revealed that W13 shared high identity (99.05%) with Yokenella regensburgei ATCC 43003 (NZ_AGCL00000000), and this isolate was finally designated Y. regensburgei W13. The whole genome of Y. regensburgei W13 consisted of a chromosome and two circular plasmids. The chromosome was approximately 4.96 Mb in length and encoded 4,802 ORFs. One plasmid, pYRW13-131, was 131,292 bp in length and encoded 184 ORFs, of which 124 were predicted to encode proteins with known functions ( Table 3). The other plasmid, pYRW13-125, encoding eleven resistance genes against six classes of antibiotics, was 125,281 bp in length and encoded 150 ORFs (Figure 1). Similar to most Yokenella regensburgei, the in vitro susceptibility test showed that wild-type Y. regensburgei W13 exhibited resistance to penicillin G, ampicillin, cefoxitin, tetracycline and streptomycin ( Table 4).

Homologs to YOC-1 Are Present in Other Enterobacterales Species
To investigate whether any novel β-lactamase gene was encoded in the Y. regensburgei W13 genome, we analyzed the predicted β-lactamase genes and found one containing conserved motifs of an Ambler class C β-lactamase; the predicted protein shared the highest amino acid identity (71.69%) with a functionally characterized AmpC, CDA-1 (Ammenouche et al., 2014) (Figure 2). We cloned the potential β-lactamase gene, and the results of the antibacterial susceptibility test showed that it was functional ( Table 4). The novel β-lactamase gene, designated bla YOC-1 , is 1,158 bp and encodes a preprotein of 385 amino acids, ca. 41.5 kDa. A signal peptide cleavage site was predicted to be between an alanine and asparagine at amino acid residues 22 and 23, respectively. Furthermore, the pI value of YOC-1 was predicted to be 8.50. Even though a hypothetical class C β-lactamase (EHM51798.1) showing the highest amino acid identity (99.48%, 383/385) with bla YOC-1 was predicted to be encoded in a draft genome of Yokenella regensburgei ATCC 43003 (NZ_AGCL00000000), the closest functionally characterized relatives to YOC-1 were β-lactamases CDA-1 (AID52933.1) and CMY-2 (X91840.1). They share only 71.69% (274/385) and 70.65% (272/385) amino acid sequence identity with YOC-1, respectively. When searching for YOC-1like proteins (≥70% aa identity and ≥90% coverage) in the NCBI nucleotide database, it was found that more than half of the close relatives (52.04%, 51/98) were derived from Enterobacter species. YOC-1 had a close relationship with the YOC-1-like AmpCs (Figure 2).

Functional Characterization of the YOC-1 β-Lactamase
Chromosomal AmpC enzymes, usually inducible, are often expressed at low levels and therefore may not contribute to clinical β-lactam antibiotic resistance (Wu et al., 1999). To assess the potential resistance activities to β-lactams, the coding sequence of bla YOC-1 together with its promoter region was cloned into the pUCP24 vector and then transformed into E. coli DH5α. The MICs of penicillin G, ampicillin and several firstto fourth-generation cephalosporins against the recombinant strain containing bla YOC-1 (pUCP24-bla YOC-1 /DH5α) increased 4-and 256-fold compared with the control strains (DH5α and DH5α carrying the vector pUCP24). The MIC of benzylpenicillin increased more than 64-fold, the MICs of ceftriaxone, ampicillin and cefoxitin increased at least 256-, 64-, and 32-fold, respectively, and the MICs of cefazolin, ceftazidime and cefoperazone increased 128-fold ( Table 4). The results showed that the increase in the MIC of ceftazidime was higher than that of cefotaxime, and ceftazidime is documented to induce AmpC cephalosporinases, which may be the cause of the phenomenon (Jones, 1998). bla YOC-1 did not confer resistance to carbapenems, which is different from CDA-1; this result may be due to some mutations in conversed residues associated with the hydrolysis of carbapenem. The antibiotic resistance spectrum of YOC-1 is consistent with most members of the family Enterobacteriaceae with an inducible chromosomal ampC gene, which are usually not resistant to extended-spectrum cephalosporins, unless the ampC gene is expressed at a very high level. The antibiotic resistance spectrum of YOC-1 is similar to that of LHK-5, a β-lactamase from a clinical L. hongkongensis isolate (Lau et al., 2005). Similar to other chromosome-and plasmid-encoded class C β-lactamases, the catalytic efficiency (k cat /K m ) of the purified β-lactamase was high against first-and second-generation cephalosporins, such as cefazolin and cefoxitin, but low against extended-spectrum cephalosporins, such as cefepime (Horii et al., 1993;Raimondi et al., 2001;Lau et al., 2005;Zioga et al., 2008). The highest catalytic efficiency was observed with penicillin G (k cat /K m rate of 191.5), which is similar to CMY-2 (Kotsakis et al., 2015). YOC-1 was more active against cefoxitin and ceftazidime than CMY-2 because of its much higher k cat , which was partially compensated for by higher K m values. The turnover rate (k cat ) of YOC-1 for ceftazidime was higher than that of CDA-1, which shared 71.69% global amino acid identity with YOC-1 (Ammenouche et al., 2014;Kotsakis et al., 2015). However, the Michaelis constant (K m ) of YOC-1 for cefepime was much lower than that of CDA-1. In addition, no measurable hydrolytic activities were observed for aztreonam or imipenem, which is different from CDA-1 (Ammenouche et al., 2014; Table 5).
The hydrolysis of aztreonam was not detectable for the purified enzyme, which was contradicted by the increase in the MIC value of aztreonam against the recombinant FIGURE 2 | A phylogenetic tree showing the relationship of YOC-1 with other chromosome-borne AmpC β-lactamases. YOC-1 from our study is shown with a red dot. carrying bla YOC-1 , which may have been an experimental error in MIC determination. This discrepancy between phenotypic and biochemical approaches could also result from the low but not zero deacylation rate of AmpC β-lactamases with those compounds. A similar phenomenon was observed for CDA-1, whose host showed resistance to ertapenem, but the β-lactamase did not have hydrolytic activity against ertapenem (Ammenouche et al., 2014). The results of β-lactamase activity inhibition analysis, as measured by the IC 50 (50% inhibitory concentration), showed that YOC-1 was poorly inhibited by clavulanic acid (IC 50 : 57.33 ± 0.03 µM) and GMP (IC 50 : 81.73 ± 0.02 µM) and was strongly inhibited by avibactam (IC 50 : 0.001172 ± 0.0002 µM), which is consistent with the majority of class C β-lactamases (Bonnefoy et al., 2004;Na et al., 2017), especially CMY-2 (Bauernfeind et al., 1996;Na et al., 2017), which is the most prevalent AmpC (Doi et al., 2009).

Comparative Genomic Analysis of the Genetic Context of the Bla YOC-1 -Encoding Region
To compare the genetic environments of bla YOC-1 in Y. regensburgei W13 with those of the closest homologous  ampC-type genes (identity > 80%), the chromosome sequences of three Raoultella spp. strains and one Klebsiella aerogenes strain were retrieved from the NCBI nucleotide database. The ampC-ampR genetic locus was conserved but located in quite different genetic environments, and there were no obvious sequence similarities between the bla YOC-1 -ampR-encoding fragment and each of the other four fragments. Moreover, no sequence showing similarity with the bla YOC-1 -ampR-encoding fragment among the completely sequenced bacterial genomes was retrieved from the NCBI nucleotide database. Furthermore, the region encoding pdeG-bla YOC-1 -ampR-nimR-hp was flanked by a pair of perfect 7 bp direct repeats (DRs), indicating that the gene array might be mobile between strains of different species or genera through horizontal gene transfer (HGT). This exclusive gene content of the bla YOC-1 -ampR-encoding region was probably ascribed to the scarcity of completely sequenced genomes of Yokenella or other closely related species.
Nevertheless, it could not be ruled out that this might be caused by HGT events (Figure 3).
To better characterize the MDR region, the MDR region of pYRW13-125 was comparatively analyzed with other related sequences. The results showed that the MDR region of pYRW13-125 shared the highest nucleotide sequence similarities with a plasmid from a clinical K. pneumoniae strain AR_0152 (CP021946, 99.80% identity and 84% coverage) and a plasmid from the clinical K. pneumoniae strain 130411-38618 (MK649826, 99.88% identity and 61% coverage). The MDR region of pYRW13-125 was a complex mosaic structure consisting of 3 mobile genetic element-related units (Figure 4), including a truncated IS26-bla LAP-2 -qnrS1-tetA-tetR-IS26 unit, an atypical class 1 integron, an ISCR2-floR-sul2 unit. The plasmid tig00000195 harbors a complete IS26-bla LAP-2 -qnrS1-tetA-tetR-IS26 unit, and the presence of TnAs1 remnant indicated that this unit might be a result of recombination of two subregions (IS26-bla LAP-2 -qnrS1-IS26 unit and IS26-tetA-tetR-IS26 unit) mediated by TnAs1. Furthermore, the atypical class 1 integron (organized with a 5 -conserved segment [5 -CS: intI1], a variable region [VR: arr-2, cmlA5, bla OXA-10 , ant(3 )-IIa and dfrA14]) without a 3 -CS on pYRW13-125 was identical to that on p130411-38618_1. The integron did not contain the 3 -CS (qacE 1/sul1) usually found in clinical class 1 integrons, suggesting that this could be a preclinical integron (Ghaly et al., 2017). The integron in pYRW13-125 was immediately flanked by a pair of IS26s, suggesting the potential of its mobility. ISCR2 was found upstream of floR and downstream of sul2 on pYRW13-125. These elements are known to move by a process called rolling-circle replication, and a function of this process is the concomitant movement of accessory sequences found upstream of their transposase genes (Toleman and Walsh, 2010). The presence of ISCR2 makes the transmission of these two genes possible. The genetic relatedness of the MDR region of pYRW13-125 with those of the two plasmids of clinical strains indicated that the environmental strain W13 might be closely related to multidrug resistant bacteria of human clinical sources.

CONCLUSION
In this study, the first complete genome sequence of Y. regensburgei was reported. Apart from a novel chromosomeencoded AmpC β-lactamase gene, bla YOC-1 , the strain also harbored a plasmid (pYRW13-125) encoding multidrug resistance gene arrays that conferred resistance to a series of antibiotics, including tetracycline, amphenicols (chloramphenicol and florfenicol) and streptomycin. Among all of the recently reported functionally characterized β-lactamases, the novel β-lactamase YOC-1 shares the highest amino acid identities of 71.69 and 70.65% with CDA-1 and CMY-2, respectively. The newly identified AmpC β-lactamase encoded in Y. regensburgei W13, which carries a multidrug-resistant plasmid, will be helpful for understanding the intrinsic resistance mechanism of this unusual opportunistic pathogen and developing treatments for human (or animal) infections caused by Y. regensburgei.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih. gov/genbank/, MT271603.

ACKNOWLEDGMENTS
We would like to acknowledge all study participants and individuals who contributed to the study.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020. 02021/full#supplementary-material FIGURE S1 | Amino acid alignment of YOC-1 with carbapenem-resistant AmpCs. Dots indicate the absence of amino acid residues. The accession numbers of the cephalosporinases (from up to down) are as follows: QJD09144.1, AID52933.1, AIM49619.1, AAK59969.1, WP_023331841.1. The logo size indicates the degree of conservation of the amino acid residues. The amino acids in proteins with ≥80% identity are shaded in blue. The length of each AmpC protein is marked at the end of the alignment. Conserved motifs are indicated at the top of the sequence alignment. The red star indicates the unique residue in YOC-1, and the predicted signal sequence cleavage site for YOC-1 is indicated by a vertical arrow.