Emergence of 16S rRNA Methylase Gene rmtB in Salmonella Enterica Serovar London and Evolution of RmtB-Producing Plasmid Mediated by IS26

This study aimed to characterize 16S rRNA methylase genes among Salmonella and to elucidate the structure and evolution of rmtB-carrying plasmids. One hundred fifty-eight Salmonella isolates from one pig slaughterhouse were detected as containing 16S rRNA methylase genes; two (1.27%) Salmonella London isolates from slaughtered pigs were identified to carry rmtB. They were resistant to gentamicin, amikacin, streptomycin, ampicillin, tetracycline, florfenicol, ciprofloxacin, and sulfamethoxazole/trimethoprim. The complete sequences of RmtB-producing isolates were obtained by PacBio single-molecule real-time sequencing. The isolate HA1-SP5 harbored plasmids pYUHAP5-1 and pYUHAP5-2. pYUHAP5-1 belonged to the IncFIBK plasmid and showed high similarity to multiple IncFIBK plasmids from Salmonella London in China. The rmtB-carrying plasmid pYUHAP5-2 contained a typical IncN-type backbone; the variable region comprising several resistance genes and an IncX1 plasmid segment was inserted in the resolvase gene resP and bounded by IS26. The sole plasmid in HA3-IN1 designated as pYUHAP1 was a cointegrate of plasmids from pYUHAP5-1-like and pYUHAP5-2-like, possibly mediated by IS26 via homologous recombination or conservative transposition. The structure differences between pYUHAP1 and its corresponding part of pYUHAP5-1 and pYUHAP5-2 may result from insertion, deletion, or recombination events mediated by mobile elements (IS26, ISCR1, and ISKpn43). This is the first report of rmtB in Salmonella London. IncN plasmids are efficient vectors for rmtB distribution and are capable of evolving by reorganization and cointegration. Our results further highlight the important role of mobile elements, particularly IS26, in the dissemination of resistance genes and plasmid evolution.


INTRODUCTION
Aminoglycosides such as gentamicin and amikacin have been widely used to treat infections caused by Gram-negative bacteria in clinical settings (human and animal), and they also used for growth promotion in animal husbandry. Resistance to aminoglycosides is due to enzymatic modification/inactivation of aminoglycosides, mutation or modification of aminoglycosidebinding site (16S rRNA of 30S ribosomal subunits), decreased permeability, and augmented efflux (Doi et al., 2016). Among them, 16S rRNA methylases are of great concern for conferring high-level resistance to all aminoglycosides used to treat systemic infections. Since the identification of armA in 2003, 10 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, and npmA) have been identified and globally disseminated in Enterobacteriaceae (Galimand et al., 2003;Doi et al., 2016). Although 16S rRNA methylase genes, especially armA and rmtB, are widely disseminated in Escherichia coli isolates from various sources, low prevalence is observed in Salmonella (Folster et al., 2009;Doi et al., 2016;Fang et al., 2019). Salmonella spp. are one of the leading causes of foodborne illness, and contaminated food, particularly animal-derived food products, are the main sources of Salmonella infections for humans (European Food Safety Authority and European Centre for Disease Prevention and Control [EFSA and ECDC], 2018; Marus et al., 2019). The dramatic increase of antibiotic resistance in Salmonella has been a global health concern.
Mobile genetic elements play a critically important role in the acquisition and dissemination of resistance genes in Gramnegative and Gram-positive bacteria (Partridge et al., 2018). Cointegration between plasmids via homologous recombination or IS26-mediated replicative or conservative transposition has been previously described, allowing plasmids to acquire more resistance or virulence genes (Sun et al., 2016;Wong et al., 2017;Hua et al., 2020).
In this study, we aimed to investigate the prevalence of 16S rRNA methylase genes among Salmonella isolates from one pig slaughterhouse in Jiangsu province, China, and to elucidate the structure and evolution of rmtB-carrying plasmids.

Antimicrobial Susceptibility Testing
The rmtB-positive isolates were determined for MICs of ampicillin, cefotaxime, meropenem, gentamicin, amikacin, streptomycin, tetracycline, florfenicol, ciprofloxacin, colistin, and sulfamethoxazole/trimethoprim using microbroth dilution method. The results were interpreted according to EUCAST 1 . The E. coli strain ATCC 25922 was used for quality control.

Plasmid Transferability and Stability
Conjugation experiments were performed using streptomycinresistant E. coli C600 as the recipient strain as previously described (Chen et al., 2007). Transconjugants were selected on MacConkey agar plates containing 3,000 mg/L streptomycin and 32 mg/L amikacin (pYUHAP5-2 and pYUHAP1) or chloramphenicol (pYUHAP5-1). Conjugal transfer frequencies were calculated as the number of transconjugants per recipient; experiments were performed in triplicate.
The stability of rmtB-carrying plasmids pYUHAP5-2 and pYUHAP1 in the original strains was investigated by passage in daily refreshed (100-fold dilution) antibiotic-free LB broth for 7 days. On the last day, the cultures were streaked on LB agar plates. 100 colonies were replica-plated on LB agar plates with amikacin (32 mg/L) and were randomly selected to confirm the presence of rmtB by PCR.

Whole Genome Sequencing and Analysis
The whole genome of rmtB-positive isolates was extracted and sequenced using PacBio single-molecule real-time sequencing (RSII platform) (Pacific Biosciences, Menlo Park, CA). Raw sequence data were introduced into the nonhybrid Hierarchical Genome Assembly Process (HGAP version 4). The plasmid sequences were analyzed and annotated by the RAST server 2 , ResFinder 3 , PlasmidFinder 4 , and BLAST 5 .

Nucleotide Sequence Accession Number
The whole genome sequences of strains HA3-IN1 and HA1-SP5 have been deposited in the GenBank database under accession number PRJNA648279.

Identification of rmtB and Antimicrobial Susceptibility
Among the 158 Salmonella isolates, two ST155 Salmonella London isolates from carcass swab samples (Zhou et al., 2017) were positive for rmtB. None of the other 16S rRNA methylase genes were detected in this study. Salmonella London was rarely reported before, but it has begun to emerge in various sources (patients, pigs, wild birds, meat products, and the environment) and spread worldwide over the past decades (Shipp and Dickson, 2011;Bonardi et al., 2016;Jurado-Tarifa et al., 2016;Trimoulinard et al., 2017;Chen et al., 2019b). To our knowledge, this is the first report of 16S rRNA methylase genes in Salmonella London.

Characterization of rmtB-Positive Salmonella London Strains
To better understand the genetic features of rmtB, complete sequences of the two rmtB-bearing Salmonella London strains were obtained. The isolate HA1-SP5 consisted of a 4,747,268-bp chromosome and two plasmids pYUHAP5-1 and pYUHAP5-2 ( Table 1). The isolate HA3-IN1 consisted of a 4,746,179-bp chromosome and a single plasmid pYUHAP1 ( Table 1). Both of them carried the aminoglycoside resistance gene aac(6')-Iaa in the chromosome and mutation in parC (T57S) which could partly explain the ciprofloxacin resistance ( Table 1).
pYUHAP1 Was Generated by Plasmid Fusion Between pYUHAP5-1-Like and pYUHAP5-2-Like Plasmids The sole plasmid in HA3-IN1, namely pYUHAP1, was 176,767 bp with an average G+C content of 53.14%. It harbored three replicons, including IncN1, IncX1, and IncFIB K ( Table 1). pYUHAP1 was a cointegrate plasmid comprised of sequence from pYUHAP5-1-like and pYUHAP5-2-like plasmids (Figure 3). The IncN1-X1 RmtB-producing plasmid region of pYUHAP1 was similar to pYUHAP5-2, except that the first and second parts of variable region in pYUHAP5-2 ( ble-aph(3')-IIa-IS50-IS1294-IS26-stbD/E-pir-repX-bis-ddp3-IS1-IS26-Tn2-rmtB) was in the opposite orientation with a shorter 157-bp IS1 in pYUHAP1 ( Figure 2B). This could be readily explained by homologous recombination between IS26 located in inverse orientations. Additionally, the pYUHAP5-1derived region in pYUHAP1 was 119,756 bp, the size difference was due to the acquisition of 5,466-bp ISCR1-qnrB6 segment within the class 1 integron, and the presence of one copy of ISKpn43 (1,374 bp) located between IS4321 and IS903-like in pYUHAP1 ( Figure 3A). The presence of additional plasmidmediated quinolone resistance gene qnrB6 could easily explain the difference of MIC of ciprofloxacin between HA1-SP5 and HA3-IN1 ( Table 1). The regions of pYUHAP1 derived from the different ancestor plasmids were separated by a pair of IS26, the cointegrate formation was likely generated by IS26 ( Figure 3B). We did not observe additional copies of IS26 or 8-bp target-site duplication, suggesting that this cointegration may occur by IS26-mediate homologous recombination or conservative transposition rather than replicative transposition (He et al., 2015;Harmer and Hall, 2016). The insertion, deletion, or recombination events mediated by mobile elements (e.g. IS26, ISCR1, ISKpn43) have occurred to form plasmids as ancestors for pYUHAP1 or occurred after cointegration, but the precise series of events cannot be determined with the available data. Similar cointegration events mediated by IS26 have occurred between IncN1-F33:A-:B-plasmids and mcr-1-carrying phagelike plasmid in E. coli (He et al., 2019), IncN, F33:A-: B-, and rolling-circle plasmids in Proteus mirabilis (Hua et al., 2020), or virulence plasmid and IncHI2 resistance plasmid in Salmonella Enteritidis (Wong et al., 2017). IS26 elements play a vital role in the dissemination of resistance genes, formation of mosaic resistance regions, and reorganization of plasmids in Gram-negative bacteria (He et al., 2015;Partridge et al., 2018). They also indicate that cointegration formation between plasmids is an important but not rare way for plasmids to evolve and capture more resistance genes, virulence genes, or other beneficial genes for their dissemination and maintenance. In this study, pYUHAP1 acquired more resistance genes through cointegration between two resistance plasmids, but it remained stable (100%) and conjugative with frequency of 1.03 × 10 −4 transconjugants/recipient.

CONCLUSION
This study revealed the emergence of rmtB in Salmonella London of swine origin in China. The fusion of rmtB-carrying IncN plasmid and IncFIB K multiresistance plasmid was mediated by IS26. IncN plasmids have become efficient vectors for rmtB transmission not only in E. coli, but also in Salmonella, and are able to evolve by reorganization and co-integration. Our results further confirm the critical role of mobile elements, particularly IS26, in the mobilization of resistance genes, formation of resistance regions, and diversity of plasmid structures.

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 in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
Z-MP, XJ, and JW conceived the study. YW, Z-YW, FS, HW, P-CS, and JW carried out the experiments. JW, Z-YW, and WL analyzed the data. JW wrote the manuscript. Z-MP and XJ revised the manuscript. All authors read and approved the final manuscript.