Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.


Introduction
Non-typhoid strains of Salmonella enterica (NTS) contribute significantly to the incidence of intestinal infections worldwide [1]. Globally, there are more than 90 million cases of Salmonella gastroenteritis each year, resulting in 155,000 deaths [2]. Despite a trend towards a decrease in salmonellosis, it is still the most frequently recorded foodborne zoonosis in Russia. The annual incidence rate of salmonellosis in Russia decreased from 36.6 per 100,000 population in 2012 to 22.0 per 100,000 in 2017, but gradually increased in 2018 and 2019 up to 24.2 per 100,000. In 2020 and 2021, the annual incidence rate of salmonellosis decreased to 14.7 per 100,000 population [3]. During that time period, the prevailing serotypes in the etiological structure of morbidity remained Salmonella enterica subsp. enterica serotype Enteritidis and serotype Typhimurium [4].
The epidemiological and clinical relevance of NTS is determined by the genomic plasticity that underlies the adaptation to a wide host range, and the emergence of Salmonella strains with novel resistant profiles, in particular, the emergence of strains with plasmidmediated colistin resistance [5][6][7]. Importantly, in clinical practice, colistin is not used for

Bacterial Isolation and Identification
The collection included Salmonella strains collected by the All-Russia Reference Center of Salmonellosis during the national Salmonellosis surveillance program. In general, the collection of isolates (n = 586) included strains derived from humans (n = 293, 58.6%), food (n = 208, 35.4%), environmental samples (n = 81, 13.8%), and animals (n = 3, 0.5%). The Salmonella isolated from humans included 139 representative isolates from 59 salmonellosis outbreaks (approximately two isolates from each outbreak) and 154 isolates from sporadic cases. S. Enteritidis was the most prevalent serotype among the outbreak strains (74.8%).
All isolates were screened using McConkey medium. Individual colonies were used for re-identification and serotyping. Subsequently, each isolate was subjected to serological characterization according to the Kauffmann-White scheme using polyclonal (Petsal, St. Petersburg, Russia) and monoclonal antibodies (Sifin, Berlin, Germany).

Antibiotic Susceptibility Testing
Antibiotic susceptibility was tested by determining the minimum inhibitory concentration (MIC) for antibiotic susceptibility using GI and GII Mikrolatest ® SensiLaTest MIC panels (Erba Lachema, Brno, Czech Republic). Escherichia coli (ATCC 25922) was used as an internal quality control. Isolates were tested on 19 antibiotics grouped into ten classes. Ssusceptibility interpretation criteria were used in accordance with the European Committee on Antimicrobial Susceptibility (EUCAST) interpretative criteria [18]. The double-disk synergy test (DDST) was employed to confirm the extended-spectrum beta-lactamase (ESBL) production using antimicrobial disks of ceftazidime (30 µg), cefepime (30 µg), and amoxicillin-clavulanic acid (20/10 µg). Expansion of the indicator cephalosporin inhibi-Microorganisms 2021, 9,2515 3 of 13 tion zone towards the amoxicillin-clavulanic acid disk was considered indicative of ESBL production [19].

Real-Time PCR Assays for mcr-1
Salmonella isolates were tested for the presence of mcr-1 group genes. DNA extraction and real-time polymerase chain reaction (qPCR) was performed using the PCR-kit «AmpliSens ® MDR MCR-1-FL» (Central Research Institute of Epidemiology, Moscow, Russia). The qPCR assay was performed on a Rotor-Gene ® 6000 (Corbett Research, Mortlake, Australia). Data obtained with all used reagent kits was analyzed using the RotorGene 6000 v1.8 software, according to the manufacturer's instructions.

Whole-Genome Sequencing and Assembling
Salmonella isolates were subjected to HiSeq (Illumina, San Francisco, CA, USA) and MinION (Oxford Nanopore, Oxford, UK) sequencing technology. Genomic DNA purification was performed using a DNeasy ® Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The purified genomic DNA was used for both whole genome sequencing technologies (Illumina and Oxford Nanopore). The libraries for Illumina sequencing were prepared using the Nextera XT DNA Library Prep Kit (Illumina, USA), Index Kit (Illumina, USA), and Reagent Kit (Illumina, USA). The sequencing was performed using a HiSeq 2500 instrument that produced 2 × 250 base pair (bp) paired-end reads.
The DNA library for MinION sequencing was prepared using the Native Barcoding Kit 1D (EXP NBD104, Oxford Nanopore) and Ligation Sequencing Kit 1D (SQK-LSK109, Oxford Nanopore, Oxford, UK), according to the manufacturer's protocol, then sequenced using an R9 SpotON flow cell (FLO-MIN106). The raw FAST5 reads were basecalled using the Guppy Basecalling Software v3.4.4. The reads were then subsequently trimmed by Porechop (https://github.com/rrwick/Porechop) (accessed on 1 November 2020) with the flag 'discard_middle,' and filtered on the quality and read length by Nanofilt. The quality threshold was defined as the median quality of initial sets of raw long reads of a particular isolate, while the minimum read length was set to 1000 bp. Accession numbers for the short-read and long-read data for each isolate are available under the following accession numbers: SLR1_8250 − SRR12342988 (long-reads), SRR12342993 (short-reads), SLR1_8245 − SRR15212457, SRR15212459, SLR1_7627 − SRR12342987, SRR12342992, SLR1_8094 − SRR12342986, SRR12342991.

Bioinformatic Analysis
Unicycler v0.4.2 [20] was used to generate a hybrid assembly, combining the long reads (Oxford Nanopore) and short reads (Illumina). Independently, Canu v1.8 [21] assemblies using only the Oxford Nanopore long reads, and subsequent dot-plot analyses using Flexidot [22], were performed to compare assembly results (Unicycler versus Canu). The comparison parameter was the topology and approximate length of the assembled chromosomes and plasmids.
The sequenced genomes were submitted to GenBank and annotated by the prokaryotic annotation pipeline (PGAP). These genomes were automatically imported to the NCBI Pathogen Detection system (https://www.ncbi.nlm.nih.gov/pathogens/) (accessed on 29 November 2021) and to EnteroBase (https://enterobase.warwick.ac.uk/species/index/ senterica) (accessed on 29 November 2021). Using EnteroBase's information, we could identify the sequence types (ST) defined by Achtman's seven gene MLST scheme, as well as the eBURST groups (eBG) for each strain [23]. These systems also provided results by searching for clonally-related isolates via incorporated bioinformatic pipelines. The NCBI Pathogen Detection system used an SNP-based approach, while EnteroBase used a core genome MLST (cgMLST). It should be noted that in the SNP-based and core genome MLST comparative analysis we also included an isolate of S. Enteritidis SLR1_8239 (accession numbers for the short-read SRR15212458), which originated from the same outbreak as SLR1_8245, to demonstrate clonal relationships between isolates.
Each assembled genome was submitted for analyses using the MOB-suite v3.0.0 [24] and PlasmidFinder v1.3 [25] to define replicon types, and to predict mobility for the assembled plasmids.
To determine whether differences may exist between the plasmids harboring mcr genes, annotated sequences of plasmids were visualized and compared in the Easyfig software v.2.1 [28] or BLAST Ring Image Generator (BRIG) [29].

Isolates Description and Antimicrobial Susceptibility Testing
During microbiological monitoring of 85 federal subjects of the Russian Federation between 2018 and 2020 by the All-Russian Salmonella Reference Laboratory, NTS isolates (n = 586) were collected from the environment, food, and patients. All isolates were subjected to antibiotic susceptibility testing, as well as screened for the presence of mcr-1-type by a specific qPCR. In this screen, only three isolates (S. Enteritidis (n = 2), S. Bovismorbificans (n = 1)) were shown to carry an mcr-1 gene (Table 1). In our study, we also included an isolate, SLR1_8094, representing a S. Typhimurium with an mcr-9 gene, which was isolated from a sporadic case in 2019. The presence of an mcr-9 gene in its genome was discovered during a whole genome sequencing study of Salmonella isolates circulating in Russia, which were characterized as a multidrug-resistant phenotype. While the mcr-1 positive S. Enteritidis SLR1_8250 and S. Bovismorbificans SLR1_7627 strains were isolated from sporadic cases, it should be noted that the mcr-1 positive strain SLR1_8245 (S. Enteritidis) was a representative isolate from an outbreak of salmonellosis with 239 affected people in El'ban village in the Khabarovsk Territory.
Isolates carrying the mcr-1 gene are characterized by high MIC values to colistin (MIC 8 mg/L) in antimicrobial susceptibility testing (AST). On the contrary, the isolate with the mcr-9 gene showed no resistance to colistin (MIC 1 mg/L) ( Table 2). SLR1_8094 produced ESBL which was confirmed by DDST. Moreover, according to our data, only isolate SLR1_8250 was resistant to a single drug-namely colistin-while the other three isolates showed a multidrug-resistant (MDR) phenotype defined as resistance to drugs of at least three different antimicrobial classes. An extended spectrum of resistance to 11 antimicrobial agents, representing seven antimicrobial classes, was detected in the monophasic variant S. Typhimurium SLR1_8094 ( Table 2). Table 2. Antimicrobial susceptibility testing of four Salmonella isolates containing an mcr gene based on MIC and AMR genes associated with resistance to particular antimicrobial agents.

Multilocus Sequence Typing and WGS-Typing
The genomes of the sequenced isolates related to the ST and eBURST groups that correlated with the serotyping results, according to the analysis in EnteroBase using Achtman's seven gene MLST scheme [23]. The S. Enteritidis isolates SLR1_8250 and SLR1_8245 belonged to ST-11 and eBG-4; the S. Bovismorbificans isolate SLR1_7627 belonged to ST-142 and eBG-34; and the monophasic variant of S. Typhimurium, isolate SLR1_8094, was related to ST-34 and eBG-1.
Although the seven gene MLST scheme [23] enables the determination of serovars of Salmonella isolates, it is not suitable for the determination of the phylogenetic positions of, and relatedness between, isolates. For such analyses, core genome analyses (SNP analyses) and core genome MLST are better suited. Thus, to further investigate the phylogenetic positions of our isolates in a global context, we used data publicity available in the NCBI Pathogen Detection system (https://www.ncbi.nlm.nih.gov/pathogens/), which contains 398,518 genomes of Salmonella (accessed on 10 October 2021), as well as data in EnteroBase (https://enterobase.warwick.ac.uk/species/index/senterica), which contains 326,421 genomes of Salmonella (accessed on 10 October 2021). It should be noted that historical Russian isolates of S. Enteritids collected from outbreaks and sporadic cases during 2012-2020 were previously submitted to the NCBI Pathogen Detection system under bioproject PRJNA484865 (including SRL1_8239). Although the new Russian isolates investigated in the present study matched the ST of the seven gene MLST scheme (see above), the more detailed core genome analyses (SNP analyses and core genome MLST) did not reveal any close relationships to each other or to other isolates, except for the match between isolates SLR1_8245 and SLR1_8239 (which originated from the same outbreak); all other studied sporadic and outbreak isolates formed unique clades (orphans) differing by at least 50 SNP to other isolates in the NCBI Pathogen Detection system, or showed at least 10 allele differences by core genome MLST in EnteroBase. (Supplementary Figures S1-S4).

Related Genetic Determinants of Salmonella Isolates to Antimicrobial Susceptibility
These four isolates were subjected to whole genome sequencing (WGS) using Illumina and Oxford Nanopore sequencing technology. Combining different WGS platforms provides the opportunity to reconstruct accurately all genomic elements, and to define the locations of plasmid-encoded genes responsible for colistin resistance, as well as additional sets of resistance genes (Table 3). Thus, we applied the PlasmidFinder and MOB-suite pipelines and the results are detailed below (Table 3).

Salmonella Enteritidis SLR1_8250 and SLR1_8245
S. Enteritidis SLR1_8250 carried three plasmid types (Table 3), namely, a non-mobilizable 60 kb plasmid pS8250-1 of the IncFII(S)/IncFIB(S) type, a mobilizable small 2 kb plasmid pS8250-3 of the ColpVC type, and the conjugative plasmid pS8250-2 of IncX4 type (33 kb). The last one contains the phosphoethanolamine transferase gene (mcr-1.1, red arrow in Figure 1), which was located upstream (4-5 kb) of a mobile genomic element of the IS26family. However, there was no copy of the ISApl1 insertion sequence flanking the mcr-1.1 gene. The sequence of a PAP2 family protein that is associated with mcr-1.1 was located directly downstream of mcr-1.1 (green arrow in Figure 1). This IncX4 plasmid also has a standard plasmid backbone containing sequences that are involved in plasmid replication, maintenance, and transfer, as well as genes responsible for the toxin-antitoxin system. has a standard plasmid backbone containing sequences that are involved in plasmid replication, maintenance, and transfer, as well as genes responsible for the toxin-antitoxin system. Figure 1. Comparison of the IncX4 plasmids from SLR1_8245, SLR1_8250, and Salmonella isolates with previously sequenced IncX4. GenBank accession numbers for these plasmids are listed in Supplementary Table S1. Arrows represent the position of open reading frames. Regions of >99% identity are indicated in dark grey between adjacent plasmid sequences. Genes associated with the tra and pil loci are colored orange, replication associated genes are colored turquoise, antibiotic resistance genes are colored red, the PAP2 gene is colored green, insertion sequences are colored blue and black, and other genes are colored gray. The previously defined variable regions I, II, and III [30] were marked by square boxes.
Compared to the non-redundant collection of sequences (NT) representing genomes in the NCBI GenBank database the complete IncX4 sequence was 99% identical to and had a coverage from 92-100% of the IncX4 plasmids of E. coli (78 BLAST hits), K. pneumonia (12 BLAST hits), and S. enterica (11 BLAST hits), showing the prevalence of such a type of IncX4 plasmid among different species of Enterobacteriaceae. Comparative analysis with a set of previously sequenced IncX4 plasmids showed a high similarity level (100% coverage and 99.98% sequence identity) of pS8245-3 and pS8250-2 IncX4 plasmids to a so-called epidemic IncX4 plasmid pCSZ4 [30]. The similar IncX4 plasmids pCFSA231, pNG14043, and pTB602 have also been found in other NTS isolates [31,32] (Figure 1, Supplementary Table S1).

Salmonella Bovismorbificans SLR1_7627
The MDR colistin-resistant strain S. Bovismorbificans SLR1_7627 isolated from human samples in 2018 carried four plasmids of which only two carried resistance genes  Supplementary Table S1. Arrows represent the position of open reading frames. Regions of >99% identity are indicated in dark grey between adjacent plasmid sequences. Genes associated with the tra and pil loci are colored orange, replication associated genes are colored turquoise, antibiotic resistance genes are colored red, the PAP2 gene is colored green, insertion sequences are colored blue and black, and other genes are colored gray. The previously defined variable regions I, II, and III [30] were marked by square boxes.
Compared to the non-redundant collection of sequences (NT) representing genomes in the NCBI GenBank database the complete IncX4 sequence was 99% identical to and had a coverage from 92-100% of the IncX4 plasmids of E. coli (78 BLAST hits), K. pneumonia (12 BLAST hits), and S. enterica (11 BLAST hits), showing the prevalence of such a type of IncX4 plasmid among different species of Enterobacteriaceae. Comparative analysis with a set of previously sequenced IncX4 plasmids showed a high similarity level (100% coverage and 99.98% sequence identity) of pS8245-3 and pS8250-2 IncX4 plasmids to a so-called epidemic IncX4 plasmid pCSZ4 [30]. The similar IncX4 plasmids pCFSA231, pNG14043, and pTB602 have also been found in other NTS isolates [31,32] (Figure 1, Supplementary Table S1).

Salmonella Bovismorbificans SLR1_7627
The MDR colistin-resistant strain S. Bovismorbificans SLR1_7627 isolated from human samples in 2018 carried four plasmids of which only two carried resistance genes ( Table 3). The IncI2 conjugative 64 kb plasmid (pS7627-2) included mcr-1.1, while additional resistance genes were located on the 233 kb non-mobilizable plasmid pS7627-1 of the IncHI2/IncQ1 type.
The results of the AST correlated well with identified genes responsible for resistance to antibiotics (Tables 2 and 3). Trimethoprim-sulfamethoxazole resistance is explained by the presence of sul1, sul2, and dfrA1 genes. The resistance of the isolate to tetracycline is confirmed by presence of a tet(A) gene. As well, the identified blaTEM-1B gene enables resistance to penicillins. However, the roles of genes aadA1, aph(6)-Id, and gene aph(3")-Ib, which encode proteins for streptomycin and spectinomycin resistance and resistance to kanamycin, respectively, could not be confirmed due to the absence of these antimicrobial agents in the panel.
A BLASTN comparison of the complete sequence of plasmid IncI2 to the NCBI Gen-Bank database revealed similar plasmids in E. coli, S. Typhimurium, and K. aerogenes isolates with an identity of 99% and a query cover from 97-56%. No further isolates of the serotype S. Bovismorbificans carrying similar plasmids were identified. Comparisons with previously published Salmonella and E. coli IncI2 plasmids (Supplementary Table S1), carrying mcr-1.1 gene, are shown in Figure 2. The IncI2 plasmid pS7627-2 has a similar plasmid backbone and a similar genetic environment surrounding the mcr-1.1 gene (nikBmcr-1.1-PAP) compared to other IncI2 plasmids (pCESA664-3, pCESA244-2, pK18JST013) previously isolated in China and Korea [33]. The ISApl1 insertion sequence belonging to the IS30 family was not identified near mcr-1.1.  Supplementary Table S1. Regions of >99% identity are indicated in dark grey in the bar between compared plasmid sequences. The RepA gene is colored turquoise, genes associated with the plasmid backbone are colored orange, antibiotic resistance genes are colored red, the PAP2 gene is colored green, insertion sequences are colored black, genes encoding toxin-antitoxin (TA) systems of type I (TA-I) and type II (TA-II) are colored yellow, and other genes are colored gray.

Discussion
The novelty of our study is the detection and description of four Russian NTS isolates at a genomic level, revealing details about plasmids carrying mcr genes. Three isolates belong to S. Enteritidis (n = 2) and S. Bovismobificans (n = 1), and all of them were characterized by the presence of an mcr-1.1 gene and had phenotypical resistance to colistin. Colistin is one of the so-called "last-resort" antimicrobials used to treat MDR infections cloacae), and p505108-MDR (C. sakazakii) generated by the BLAST Ring Image Generator tool. The pS80904-1 plasmid was used as a reference. The outer circle denotes the annotation of the reference plasmid. The AMR genes are highlighted by red boxes, genes encoding hypothetical proteins highlighted by grey boxes, IS-elements highlighted by blue boxes, and other genes highlighted by black boxes. The backbone (tra1 and tra2 region), two accessory resistance regions (MDR region 1, MDR region 2), and mcr-9 cassette (rcnR-rcnA-pcoE-pcoS-IS5-mcr-9-wbuC-IS6) are indicated by black curves.

Discussion
The novelty of our study is the detection and description of four Russian NTS isolates at a genomic level, revealing details about plasmids carrying mcr genes. Three isolates belong to S. Enteritidis (n = 2) and S. Bovismobificans (n = 1), and all of them were characterized by the presence of an mcr-1.1 gene and had phenotypical resistance to colistin. Colistin is one of the so-called "last-resort" antimicrobials used to treat MDR infections caused by members of the Enterobacteriaceae family. The fourth isolate belonged to monophasic S. Typhimurium and, while carrying an mcr-9 gene, was susceptible to colistin. The successful combination of the genome sequencing approaches based on short and long reads allowed us to accurately reconstruct the complete sequences of the chromosomes and plasmids for each isolate, permitting detailed analyses of the plasmid's structure that carries the mcr genes. To the best of our knowledge, this is the first study that describes cases of NTS strain isolation with mcr-mediated resistance to colistin in Russia with a detailed analysis of both AMR genes and phenotypical resistances.
Based on the data obtained, we conclude that the prevalence rate of mcr-1.1 in Salmonella isolates is very low (0.5%), which correlates well with previous observations [38,39]. Nevertheless, three of the studied isolates were phenotypically resistant to at least three classes of antimicrobial drugs. These MDR phenotypes were accurately confirmed by the presence of corresponding AMR genes in the genomes, corroborating the predictive power of WGS [40]. Based on the WGS data, we show that the sequenced isolates are genetically unique by core genome features (SNP and alleles of core genes) among all available genomes sequenced to date for Salmonella. Moreover, we did not find any similarity to previously isolated S. Enteritidis representatives from the outbreaks and sporadic cases circulating in Russia.
Based on the analyzed genomic sequences, we found that the mcr-1.1 genes of two S. Enteridis isolates were localized on IncX4 plasmids, whilst in the S. Bovismorbificans isolate the mcr-1.1 was on the IncI2 plasmid. An mcr-9 gene variant was detected on the IncHI2 plasmid in the monophasic human isolate of S. Typhimurium. The data obtained are consistent with previous observations, where it was shown that plasmid types predominantly harboring mcr genes are IncX4, IncI2, and IncHI2 [39,41,42]. Interestingly, only the IncHI2 plasmid was associated with additional antibiotic resistance genes, while the IncX4 and IncI2 plasmids carried only one mcr gene. The absence of a flanking ISApl1 near the mcr cassettes on the IncX4 and IncI2 plasmids suggest loss of the ISApl1 elements after the integration of the transposon. It further suggests that the loss of ISApl1 anchors the mcr-1 gene on the plasmid, which probably indicates the stability of the mcr genes on plasmids [6].
By comparing the plasmid sequences with previously published data, we found that the IncX4 plasmids in the two S. Enteritidis strains in this study showed high levels of similarity to an epidemic pCSZ4-like IncX4 plasmid [30]. The IncX4 epidemic plasmid pCSZ4 contains an identical mcr cassette with absence of the ISApl1 transposon. It should be noted that IncX4 is one of the most widespread plasmid types among E. coli and other Enterobacteriaceae. This plasmid is characterized by a high frequency of self-transfer between different species of Enterobacteriaceae, indicating its high epidemiological significance [43]. It was also interesting to note that mcr-1.1 positive isolates were found among the Salmonella isolates belonging to the serotype Enteritidis, which is the dominant etiological serotype for morbidity worldwide [44,45]. In the present study, S. Enteritidis were isolated during an outbreak of salmonellosis and from a poultry product, which indicates the opportunity for successful circulation of epidemic pCSZ4-like IncX4 plasmids in S. Enteritidis populations. To date, there are only a few cases of S. Enteritidis described with plasmids carrying mcr genes, but none of these studies detected the epidemic IncX4 plasmid in S. Enteritidis isolates [38,46].
Remarkably, the isolate carrying the mcr-9 gene (monophasic S.Typhimurium) did not show phenotypical resistance to colistin. According to earlier studies [47,48], this phenomenon was previously observed, although colistin-resistant Salmonella enterica carrying the mcr-9 gene was also discovered [49]. Obviously, mcr-9 is widely distributed among various Enterobacteriaceae species, and the most common mcr-9 carrying plasmid is shown to be IncHI2 [37]. Moreover, co-occurrence of the mcr-9 gene with various antibiotic resistance genes was shown, indicating the epidemiological significance of IncHI2 plasmids in spreading of AMR genes [50]. Most often, and consistent with our data, the structure of the mcr-9 cassettes in Salmonella plasmids presents as IS5-mcr-9-wbuC-IS6 and lacks the downstream regulatory genes qseB and qseC [48,50].
The data provided here expand the global picture fordissemination of mobilized colistin resistance in Enterobacteriaceae. However, future retrospective studies from different sources over a longer period of time are urgently needed to assess the prevalence of plasmid-mediated colistin resistance among NTS strains in Russia.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/microorganisms9122515/s1. Table S1: Detailed information of IncX4, IncI2 plasmids used for comparative analysis. Figures S1-S4: Defining the uniqueness of isolates using the NCBI Pathogen Detection system and EnteroBase.  Institutional Review Board Statement: Ethical review and approval were waived for this study since the human samples were routinely collected and patients' data remained anonymous.
Informed Consent Statement: Patient consent was waived since the human samples were routinely collected, and patients' data remained anonymous. Data Availability Statement: Raw sequencing reads of strains as well as complete genome assemblies have been deposited in the NCBI BioProject database under accession numbers PRJNA484865 (SAMN15666299 = SLR1_8250, SAMN15666300 = SLR1_7627, SAMN15666301 = SLR1_8094, SAMN20345806 = SLR1_8245, SAMN20345807 = SLR1_8239).

Conflicts of Interest:
The authors declare no conflict of interest.