Evaluation of Antibiotic Resistance of Salmonella Serotypes and Whole-Genome Sequencing of Multiresistant Strains Isolated from Food Products in Russia

Food products may be a source of Salmonella, one of the main causal agents of food poisoning, especially after the emergence of strains resistant to antimicrobial preparations. The present work dealt with investigation of the occurrence of resistance to antimicrobial preparations among S. enterica strains isolated from food. The isolates belonged to 11 serovars, among which Infantis (28%), Enteritidis (19%), and Typhimurium (13.4%) predominated. The isolates were most commonly resistant to trimethoprim/sulfamethoxazole (n = 19, 59.38%), cefazolin (n = 15, 46.86%), tetracycline (n = 13, 40.63%), and amikacin (n = 9, 28.13%). Most of the strains (68.75%) exhibited multiple resistance to commonly used antibiotics. High-throughput sequencing was used to analyse three multidrug-resistant strains (resistant to six or more antibiotics). Two of them (SZL 30 and SZL 31) belonged to S. Infantis, while one strain belonged to S. Typhimurium (SZL 38). Analysis of the genomes of the sequenced strains revealed the genes responsible for antibiotic resistance. In the genomes of strains SZL 30 and SZL 31 the genes of antibiotic resistance were shown to be localized mostly in integrons within plasmids, while most of the antibiotic resistance genes of strain SZL 38 were localized in a chromosomal island (17,949 nt). Genomes of the Salmonella strains SZL 30, SZL 31, and SZL 38 were shown to contain full-size pathogenicity islands: SPI-1, SPI-2, SPI-4, SPI-5, SPI-9, SPI-11, SPI-13, SPI-14, and CS54. Moreover, the genome of strain SZL 38 was also found to contain the full-size pathogenicity islands SPI-3, SPI-6, SPI-12, and SPI-16. The emergence of multidrug-resistant strains of various Salmonella serovars indicates that further research on the transmission pathways for these genetic determinants and monitoring of the distribution of these microorganisms are necessary.


Introduction
Food safety is a key problem worldwide, while the provision of pathogen-free food is an important social problem. Food pathogens are among the major food-related risk factors, affecting over 2 billion people per year [1]. Salmonella strains are among the major causes of foodborne diseases in many regions worldwide [2]. Salmonella is common among wild and domesticated animals, including the animals used by humans for food. While over

Genovar
Three of the studied isolates exhibited the highest resistance. SZL 38 was resistant to six antibiotics belonging to four groups of antimicrobial compounds, while SZL 30 and SZL 31 were resistant to eight of the antimicrobial compounds belonging to six groups.   Salmonella Rissen S10 CFZ-CHL 1 1 S18 AMP-AMK-STX-AZM 4 4 Salmonella Infantis

Sequencing
Sequencing was carried out for three multiply resistant strains: S. enterica serovar Infantis isolates SZL 30 and SZL 31, and S. Typhimurium SZL 38, which exhibited resistance to at least six antibiotics. The technologies used were Illumina (Illumina, San Diego, CA, USA) and single-molecular nanopore sequencing (Oxford Nanopore, Oxford, UK). The results for sequencing of genomic DNA libraries of the studied Salmonella strains are presented in Supplementary Table S1.
Assembling the contigs for each strain resulted in a complete circular chromosome sequence and a set of ring-shaped contigs representing the plasmids. Thus, the chromosome Antibiotics 2022, 11, 1 7 of 20 size of the sequenced strains was from 4.68 to 5.05 × 10 6 nt, while the number of plasmids varied from one to two, depending on the strain (Table 5).

General Characterization of the Genomes
Analysis of the genomes of Salmonella strains SZL 30, SZL 31, and SZL 38 revealed that they contained 5078 to 5209 protein-encoding genes, with 73% of them being genes with predicted function. All sequenced genomes contained eight copies of 5S rRNA, seven copies each of 16S rRNA and 23S rRNA, and 84 to 89 tRNA (Table 6).

Multilocus Sequence Typing of Strains
From analysis of the genomes of the Salmonella strains SZL 30, SZL 31, and SZL 38 using the Salmonella In Silico Typing Resource (SISTR) and SeqSero2, it was established that SZL 30 and SZL 31 belong to the serovar Infantis, and SZL 38 to the serovar Typhimurium.
Strain Salmonella SZL 38 possessed the plasmid pSZL38.1 belonging to the p0111 incompatibility group and containing the relB/relE toxin-antitoxin locus. Comparison of the nucleotide sequence of pSZL38.1 to GenBank sequences revealed 98.84% homology to pD72C of E. coli strain D72C [20], which may indicate horizontal gene transfer in the family Enterobacteriaceae.

Prophages
Analysis of the presence of prophages in the sequenced genomes using the PHASTER software package revealed 14 prophages in the genome of Salmonella SZL 30 and 13 prophages in the genomes of both Salmonella SZL 31 and Salmonella SZL 38 (Supplementary Table S2 Table S3).
Comparison of the sequences of these prophages with the GenBank data revealed most of them to be widespread in the genomes of previously sequenced Salmonella strains. The sequences of 32 out of 40 revealed prophages exhibiting over 99% homology to the database sequences at coverage of over 99% (Supplementary Table S3).

Discussion
Salmonellosis is considered a serious public healthcare issue worldwide [24]. The emergence of Salmonella strains with multiple drug resistance is an important problem in terms of human health and may result in inefficient treatment [25]. Salmonella spp. are among the main pathogens causing food poisoning in the Russian Federation and the third most important ones in the structure of the nidi of group diseases with faecal-oral transmission mechanisms. In 2020, 20 outbreaks of salmonellosis group infection were revealed (compared to 70 in 2019), which affected 422 people [4].
The overall prevalence of Salmonella in this study was 6.8%. In general, the high isolation rate of Salmonella spp. in chicken samples indicates that chicken is one of the most important sources of human salmonellosis.
In the present work, 32 bacterial isolates were obtained and identified as Salmonella strains based on their biochemical characteristics. The most common serovar was Infantis (28.13%, n = 9). S. Infantis is considered one of the most frequent causes for bacterial food poisoning worldwide [26,27]. Its high occurrence (25.8%) was revealed by serotyping of the Salmonella isolates from food carried out in Turkey [28].
The third most common serovar was Typhimurium. Among the major serovars, S. Typhimurium is mostly responsible for diarrhoea in humans [36]. In the present work, four isolates were identified as S. Typhimurium. This serotype was recently acknowledged as a new healthcare problem, since it has been isolated from various animals, environmental objects, and food; moreover, the frequency of human diseases caused by this organism in various countries is increasing [37][38][39].
These results are in complete agreement with the data from the report by the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, since in 2015-2020 the predominant serotypes in Russian territory were S. Enteritidis (64.7%), S. Typhimurium (4.8%), and S. Infantis (3.2%). S. Infantis was most common in chicken meat, S. Kentucky in turkey meat, and S. Typhimurium in environmental objects and pork [4].
In most cases, salmonellosis is cured by itself, with antimicrobial therapy required only for invasive or long-term infections. However, large-scale application of antibiotics results in the development of resistance to antimicrobial preparations and favours the emergence of strains possessing multiple drug resistance (MDR) [40][41][42].
The Salmonella strains isolated in our work were tested for their sensitivity to 13 antimicrobial preparations significant for healthcare and veterinary medicine. Only two of the studied strains (6.25%) were sensitive to all tested antimicrobial agents. In total, 93.75% of the isolates were resistant to at least one class of antimicrobial preparations, while 68.75% exhibited MDR to three or more classes of antimicrobial agents. Multiple resistance implies simultaneous resistance to three or more antibiotics. Other researchers have also reported a high percentage of antibiotic-resistant Salmonella strains isolated from food [43][44][45]. Animals are the main reservoir of Salmonella, and irresponsible application of antimicrobial preparations to cure productive animals, for disease prevention, and for growth stimulation results in the emergence of resistant pathogens. Increased occurrence of human salmonellosis caused by food infected with Salmonella strains resistant to antimicrobial agents has been reported [46,47].
Investigation of the sensitivity of our Salmonella isolates to antimicrobial preparations showed that the greatest number of isolates were resistant to folate pathway antagonists (trimethoprim/sulfamethoxazole), followed by cephems (cefazolin) and the tetracycline group (tetracycline). High resistance to these groups of antibiotics is in agreement with previous reports [48,49], which emphasized uncontrolled application of these antibacterial preparations. Resistance of 31 Salmonella strains isolated from poultry samples in Ethiopia was 60% [48]. Most studied isolates were sensitive to amoxicillin/clavulanic acid, imipenem, ampicillin, and streptomycin.
All S. Infantis isolates were resistant to at least one antibiotic, with the highest resistance observed for trimethoprim/sulfamethoxazole, tetracycline, chloramphenicol, and cefazolin. The S. Enteritidis isolates were most often resistant to trimethoprim/sulfamethoxazole, cefazolin, azithromycin, and tetracycline.
S. Typhimurium was most resistant to streptomycin, trimethoprim/sulfamethoxazole, tetracycline, and azithromycin. All studied S. Typhimurium and S. Enteritidis strains were sensitive to nitrofurantoin and amoxicillin + clavulanic acid.
Our findings of high resistance to trimethoprim/sulfamethoxazole are similar to the results obtained previously [50] for S. enterica serovars isolated in Egypt from broiler chickens and chicken carcasses, as well as for Salmonella isolates from chicken carcasses sold in Ibagué, Columbia [51].
The high levels of antibiotic resistance of Salmonella food isolates revealed in the present work indicate non-selective and constant application of higher than therapeutic doses of antibiotics to animals.
Antimicrobial resistance (AMR) poses a colossal threat to global health and incurs high economic costs to society. This ever-growing problem not only threatens public health, but also incurs a huge toll on a nation's economic growth by delayed hospitalizations, lengthening recovery time, expensive medicines, and specialized care for patients [10][11][12]. Authors have estimated the economic cost of resistance per antibiotic by drug class and compared those in developing and developed countries, like in Thailand and the United States [52]. The total economic cost of AMR due to resistance in five pathogens, S. aureus, E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa was $0.5 billion and $2.9 billion in Thailand and the US, respectively. So, resistance has a significant impact on the cost of treatments [11]. Increased illness coupled with limited options for treatment further strains low-income settings already struggling with low resources.
Antibiotic-resistant infections also affect animals, and the increasing rates of resistance also mean that it becomes more difficult to treat such infections. Death to livestock can further damage the finances of both individual citizens and society. Novel treatments for MDR infections can cost up to tens of thousands of US dollars, which ultimately makes the medicines unreachable for many [6]. Understanding the depth of the problem of the prevalence of resistance, especially multiple resistance, primarily among pathogenic microorganisms can result in better-informed policy recommendations regarding interventions that affect antimicrobial consumption and those aimed specifically at reducing the burden of AMR.
In the second stage of the work, Salmonella strains selected in the previous stage (SZL 30, SZL 31, and SZL 38) were sequenced. Genomic analysis of the sequenced strains revealed the genes responsible for antibiotic activity, and their localization was determined.
Aminoglycoside resistance in Salmonella is also related to the expression of drugmodifying enzymes. These enzymes are classified into three main groups, according to the type of reaction they catalyse: acetyltransferases, encoded by the aac genes, phosphotransferases encoded by aph genes, and nucleotidyltransferases, encoded by aadA genes. Many variations of genes in each class are described [53]. In this study, all the strains had at least one of the three classes of genes described.
Strain SZL 38 had two different genes associated with resistance to aminoglycosides localized in chromosomes. Previously, it has been reported that there are different variants of resistance to aminoglycosides in the same strains isolated from the poultry production chain [42,51]. This may be due to the intense selection pressure in these production lines.
Resistance of Salmonella strain SZL 30 was probably caused by the aadA1 gene localized in the plasmid pSZL30.2 and encoding aminoglycoside-3 -adenylyl transferase, which inactivates streptomycin or spectinomycin by adenylation ( Table 7). The nucleotide sequence of aadA1 was 100% identical to the previously described aadA1 gene (JQ414041.1) from Pseudomonas aeruginosa strain K7 [54]. Streptomycin resistance of Salmonella strain SZL 31 may be due to the presence of the aadA2b gene localized in the plasmid pSZL31.1 and also encoding aminoglycoside-3 -adenylyl transferase. The nucleotide sequence of the previously described aadA2b gene (D43625.1) from the plasmid pSA1700 of P. aeruginosa strain ST1700, which provides Escherichia coli cells with streptomycin resistance [55], differs by one pointwise replacement from the aadA2b gene of the plasmid pSZL31.1. Streptomycin resistance of Salmonella strain SZL 38 may be caused by the presence of the aph(3")-Ib and aph(6)-Id genes, encoding aminoglycoside phosphotransferases, which inactivate a number of aminoglycoside antibiotics by phosphorylation, in the chromosome of this strain. The nucleotide sequence of the aph(3")-Ib gene was 100% identical to that of the previously described aph(3")-Ib gene (AF321551.1) from the plasmid pSTR1 of Shigella flexneri strain 2731 [56]. The nucleotide sequence of the aph(6)-Id gene was 100% identical to that of the previously described aph(6)-Id gene (M28829.1) from the plasmid pRSF1010 of an E. coli strain [57]. The formation of genetic resistance to streptomycin in all three strains may be associated with the intensive use of streptomycin in animal husbandry, as evidenced by the results of a study of 66 meat samples of slaughter animals, in which a relatively high concentration of streptomycin was detected-5 × 10 −1 (from 3 × 10 −1 to 8 × 10 −1 ) mg/kg at a limit of 0.2 mg/kg in the territory of the Russian Federation.
Resistance of Salmonella strains SZL 30, SZL 31, and SZL 38 to β-lactam antibiotics may be caused by the presence in the genomes of these strains of the bla TEM-1B gene, which is localized in the plasmids pSZL30.2 and pSZL31.2 and in the chromosome of strain SZL 38 ( Table 7). The bla TEM-1B gene encodes β-lactamase, which provides bacterial resistance to β-lactam antibiotics by hydrolysing them. The nucleotide sequences of all revealed bla TEM-1B genes were 100% homologous to each other and to the previously described bla TEM-1B gene (HM769901.1) from the plasmid pZM3 of an S. enterica subsp. enterica serovar Wien strain [58]. β-Lactamase hydrolyses the β-lactam ring, resulting in the formation of β-amino acids that do not have antimicrobial activity [59]. The presence of genetic resistance to lactamases, which are often used to treat humans, among Salmonella strains isolated from meat products indicates their use as a prophylaxis or for the treatment of animals [60]. Such strains circulating in animals can infect humans and transmit antibiotic resistance to other pathogens [61].
Resistance of Salmonella strains SZL 30, SZL 31, and SZL 38 to amikacin and tobramycin was probably caused by the presence of the aac(6')-Iaa gene in the chromosomes of these strains. The aac(6')-Iaa gene encodes aminoglycoside-6 -acetyl transferase, which provides bacterial resistance to a number of aminoglycoside antibiotics by their acetylation. The nucleotide sequences of the aac(6')-Iaa genes located in the chromosomes of the Salmonella strains SZL 30 and SZL 31 were 100% identical and 97.5% homologous to the previously described aac(6')-Iaa gene (NC_003197.2) from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 [62]. The sequence of the aac(6')-Iaa gene localized in the chromosome of Salmonella strain SZL 38 was 100% identical to that of the previously described aac(6')-Iaa gene (NC_003197.2), which is known to provide E. coli cells with resistance to amikacin, tobramycin, and kanamycin [63].
Sulphonamide resistance in Gram-negative bacilli usually results from the acquisition of one of three sul1, sul2, and sul3 genes encoding forms of dihydropteroate synthase that are not inhibited by the drug. Resistance of the Salmonella strains SZL 30 and SZL 31 to sulfamethoxazole was due to the presence of the sul3 gene localized in the plasmids pSZL30.1, pSZL31.1, and pSZL31.2 and encoding the sulfamethoxazole-resistant dihydropteroate synthase (Table 7). Nucleotide sequences of the sul3 located in the plasmids pSZL30.1, pSZL31.1, and pSZL31.2 were 100% homologous to each other and to the previously described sul3 gene (AJ459418) from the pVP440 plasmid of E. coli strain rl0044, providing E. coli cells with sulfamethoxazole resistance [64]. Sulfamethoxazole resistance of Salmonella strain SZL 38 may be due to the presence of the sul2 gene encoding dihydropteroate synthase in the chromosome of this strain. The spread of the sul2 gene has increased over the years in other European countries [2,9], as it has often been reported that this gene is more widespread among clinical isolates of E. coli than the sul1 gene. The nucleotide sequence of the sul2 gene was 100% homologous to that of the previously described sul2 gene (HQ840942.1) for sulfamethoxazole resistance from the plasmid pSRC27-H of S. enterica strain SRC27 [65].
Trimethoprim resistance of Salmonella strains SZL 30 and SZL 31 was probably due to the presence of the dfrA14 gene localized in the plasmids pSZL30.1 and pSZL31.1 and encoding the trimethoprim-resistant dihydrofolate reductase, which is responsible for synthesis of tetrahydrofolate from dihydrofolate ( Table 7). The nucleotide sequences of the dfrA14 located in the plasmids pSZL30.1 and pSZL31.1 were 100% homologous to each other and to the previously described dfrA14 gene (AJ313522.1) from the plasmid pSTOJO1 of a uropathogenic E. coli strain, which provides trimethoprim resistance to E. coli cells [66].
Resistance of the Salmonella strains SZL 30 and SZL 31 to tetracycline may be caused by the presence of the tet(A) gene localized in the plasmids pSZL30.1 and pSZL31.1 and encoding the MSF transporter, providing resistance to the tetracycline group antibiotics by removing these compounds from the cell ( Table 7). The tet(A) and tet(B) genes were more associated with tetracycline resistance, which is consistent with previous studies [6,14].
The nucleotide sequences of the tet(A) genes localized in the plasmids pSZL30.1 and pSZL31.1 were 100% homologous to each other and to the previously described tet(A) gene (AJ517790.2) from the plasmid pRAS1 of Aeromonas salmonicida strain 2402, which provides E. coli with resistance to the tetracycline group antibiotics [67,68]. Tetracycline resistance of Salmonella strain SZL 38 was probably caused by the presence in its chromosome of the tet(B) gene, which also encodes the MSF transporter, providing resistance to tetracycline group antibiotics by removing these compounds from the cell. The nucleotide sequence of the tet(B) gene differed by one pointwise replacement from the previously described tetA(B) gene (P02980.1) of tetracycline resistance from the Tn10 transposon [69].
Salmonella strains SZL 30 and SZL 31, unlike strain SZL 38, were found to be resistant to chloramphenicol (Table 7). Chloramphenicol resistance of the Salmonella strains SZL 30 and SZL 31 was probably caused by the presence of the cmlA1 gene located in the plasmids pSZL30.1, pSZL31.1, and pSZL31.2 and encoding the MSF transporter of the Bcr/CflA family, which is responsible for bacterial resistance to chloramphenicol due to its release from the cell. The nucleotide sequences of the cmlA1 genes localized in the plasmids pSZL30.1, pSZL31.1, and pSZL31.2 were 100% homologous to each other and to the previously described cmlA1 gene (U12338.3) from the plasmid R1033 of a P. aeruginosa strain, which provides E. coli cells with chloramphenicol resistance [70].
Thus, as a result of our study, it was found that strains SZL 30 and SZL 31 are resistant to the following antibiotics: STR, AMP, AMK, TM, STX, IPM, TET, and CHL, which correlated with the identified genetic elements fixed in the genome or localized in plasmids. However, the revealed phenotypic resistance to imipenem has not been confirmed at the genetic level; it is possible that the genetic determinants of resistance to imipenems may be non-specific carrier genes. We have shown the phenotypic resistance of strain SZL 38 to the following antibiotics: streptomycin (STR), ampicillin (AMP), amikacin (AMK), tobramycin (TM), trimethoprim/sulfamethoxazole (STX), and tetracycline (TET). There was a 100% correlation between the phenotypic and genotypic data for strain SZL 38. From analysis of the genomes of these strains, genetic determinants were identified, presumably responsible for resistance to STR, AMP, AMK, and TET. The gene dfrA14, responsible for resistance to trimethoprim, was not found; it is possible that the genetic determinants of resistance to trimethoprim may be non-specific transporter genes.
The high prevalence of aadA1, aadA2b, aph(3")-Ib, bla TEM-1B , tet(A), tet(B), and cmlA1 genes in Salmonella isolates may be due to the common use of aminoglycosides, phenicols, β-lactams, sulphonamides, and tetracycline group antibiotics in animal husbandry to combat bacterial infections and stimulate growth [6]. The presence of these genes in mobile genetic elements, such as transposons and plasmids, can facilitate their transfer [33].
Comparison of the nucleotide sequences of the plasmids pSZL30.2 and pSZL31.2 with GenBank data revealed that they exhibited the highest homology (99.9%) at 66% coverage with an unnamed plasmid from Shigella flexneri strain 95-3008 (CP026773.1).
All antibiotic resistance genes detected in Salmonella strain SZL 38 were localized in the chromosome. Five genes, bla TEM-1B , aph(6)-Id, aph(3")-Ib, sul2, and tet(B), were localized in a 17,949 nt resistance island ( Figure 2C). Comparison of the nucleotide sequences of this island with GenBank data revealed that it differed by one pointwise replacement from a similar island (CP019649.1) in the chromosome of S. enterica strain TW-Stm6 [72].
Comparison of the nucleotide sequences of the plasmids pSZL30.2 and pSZL31.2 with GenBank data revealed that they exhibited the highest homology (99.9%) at 66% coverage with an unnamed plasmid from Shigella flexneri strain 95-3008 (CP026773.1).
All antibiotic resistance genes detected in Salmonella strain SZL 38 were localized in the chromosome. Five genes, blaTEM-1B, aph(6)-Id, aph(3″)-Ib, sul2, and tet(B), were localized in a 17,949 nt resistance island ( Figure 2С). Comparison of the nucleotide sequences of this island with GenBank data revealed that it differed by one pointwise replacement from a similar island (CP019649.1) in the chromosome of S. enterica strain TW-Stm6 [72]. (C) An island of resistance located on the chromosome of Salmonella S38. Red arrows indicate genes for resistance; orange genes are involved in the mobile transfer of genetic material. aadA1, aadA2b, aph(3")-Ib, and aph(6)-Id-genes for streptomycin resistance, bla TEM-1B -ampicillin resistance gene, cmlA1-chloramphenicol resistance gene, dfrA14-trimethoprim resistance gene, mefB-gene for resistance to macrolide antibiotics, qacH-gene for resistance to quaternary ammonium compounds, sul2 and sul3-genes for resistance to sulfamethoxazole, sat1-gene for resistance to streptothricin, tetA and tetB-genes for resistance to tetracycline, tetR-transcription regulator of genes tetA and tetB. merRTPCADE-mercury resistance operon. intl1 and intl2-integrases, IS26 and IS1R-insertion elements, orf1, orf2, orf3, and orf4-genes coding proteins with unknown function, sdr-short chain dehydrogenase, repA and repC-replication proteins, tnpTn21-transposase. ∆IS440, ∆tetC, ∆tnpM, and ∆resTn3-different variants of insertion element, transcription regulator, transposase, and resolvase, respectively. AMR is a serious and growing problem for S. enterica and other Gram-negative pathogens [73]. Overuse of antibiotics in medicine and agriculture is the most important factor contributing to the emergence of bacteria resistant to various antibiotics [74]. Poultry, cattle, and pigs can act as effective carriers of Salmonella to humans [75][76][77].
AMR can occur as a result of point mutations in the bacterial genome or as a result of the horizontal transfer of genetic elements carrying resistance genes. Probably the most effective way of transferring antibiotic resistance genes between microorganisms is the horizontal transfer of mobile genetic elements-integrons, transposons, and plasmids containing one or more genes that determine antibiotic resistance [78,79]. In our work, it was shown that strains SZL 30 and SZL 31 contain plasmids pSZL30.1, pSZL30.2, pSZL31.1, and pSZL31.2, carrying various genes for antibiotic resistance (Figure 2). Integrons that are part of these plasmids and carry genes for antibiotic resistance are widespread in various strains of Salmonella, Proteus mirabilis, and E. coli. It is likely that these antibiotic resistance genes can be transmitted to other Gram-negative microorganisms, especially as a result of selective pressure caused by the use of antibiotics in agriculture.

Strains Used in the Work
In total, 32 Salmonella strains were included in the study. This study concerns 32 Salmonella strains isolated from 443 samples (112 samples from different types of poultry, 91 samples of pork, 103 samples of beef, and 168 samples of minced meat) collected in 2019 (Supplementary  Table S4).

Isolation and Confirmation of Salmonella Isolates
The preparation of samples, isolation, and identification of Salmonella was done according to techniques recommended by the International Organization for Standardization ISO 6579-1: 2017 [80]. Twenty-five grams from the skin of each sample was aseptically excised using a sterile scalpel then weighed and aseptically homogenized with 225 mL of sterile buffered peptone water (Oxoid, Basingstoke, UK) in a laboratory blender for 1 min and incubated at 37 • C for 24 h. One hundred microlitres from each pre-enrichment broth was inoculated into 10 mL of Rappaport-Vassiliadis broth (RV; Obolensk, Russian Federation) and incubated at 42 • C for 24 h. Then, 1 mL from each pre-enrichment broth was inoculated into 10 mL of Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn; Oxoid, Basingstoke, UK) and incubated at 37 • C for 24 h. A loopful of each enriched broth was streaked onto two selective solid media: xylose-lysine-desoxycholate (XLD) agar (Oxoid, Basingstoke, UK) and bismuth sulphite agar (Oxoid, Basingstoke, UK) then the inoculated plates were incubated at 37 • C for 24 h. All presumptive colonies on XLD and bismuth sulphite agar were picked up and cultured onto nutrient agar plates (FBUN SSC PMB, Obolensk, Russian Federation) and incubated at 37 • C for 24 h to be subjected to further confirmation by biochemical and serological identifications. Specific colonies were confirmed by biochemical reactions using API 20E tests in accordance with the manufacturer's instructions (BioMérieux, Craponne, France) and frozen at −86 • C in collections of the Gorbatov Federal Centre of Food Systems. When the work started, strains were revitalized and characterized bacteriologically and biochemically to support their identities again.

Serotyping of Salmonella Isolates
Serotyping of Salmonella isolates was carried out by the molecular genetic method, using Check & Trace Salmonella (Check-Points B.V., Wageningen, The Netherlands) according to the manufacturer's method (details available at http://www.checkandtrace.com/, accessed on 21 July 2021).

DNA Isolation and Sequencing
LB medium (5 mL) was inoculated with the material from a single colony of Salmonella strain SZL 30, SZL 31, or SZL 38 and incubated for 16-20 h at 30 • C in a New Brunswick C-24 shaker incubator. The cells were separated on an Eppendorf MiniSpin centrifuge for 5 min at 10,000 rpm. Total DNA was isolated using a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.
Genomic DNA (200 ng) was homogenized in a Bioruptor UCD 200 sonicator (Diagenode, Denville, NJ, USA) for 10 min at the maximal power (5 cycles of 30 s on/90 s off). Paired DNA libraries (300 × 2) were obtained using a NEBNext ® Ultra™ II DNA Library Prep Kit (NEB) according to the manufacturer's conditions. The number and quality of the libraries thus obtained were determined using a Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA). The DNA libraries were sequenced on MiSeq (Illumina) using a MiSeq Reagent Kit v3 (600-cycle; Illumina).
Genomic DNA was also sequenced using a MinION device (Oxford Nanopore, Oxford, UK). DNA libraries were obtained using a Ligation Sequencing Kit 1 D (SQK-LSK109) system (Oxford Nanopore), omitting the initial stage of genomic DNA fragmentation and then following the manufacturer's recommendations. Genomic libraries were sequenced on a MinION Flow Cell FLO-110.

Bioinformatic Techniques
The contigs from all Illumina and Nanopore reads were assembled using Unicycler v. 0.4.8 [82].
In the next stage, pairwise intersecting reads obtained using MiSeq (Illumina) were combined using flash [83], and low-quality read ends were removed with Sickle. Complete genome sequences were assembled from the reads obtained on MiSeq and MinION using Flye 2.7 [84] and were twice corrected by Illumina reads with Pilon 1.22 [85].
Identification of genes and theoretical prediction of their functions were carried out using the RAST server [86,87].
The search for genes homologous to genes of antibiotic resistance was carried out using Comprehensive Antibiotic Resistance Database (CARD) [88] and ResFinder 4.1 (80% identity and 60% gene coverage) [89]. The search for insertion sequences was carried out using the ISfinder server [90]. The plasmid type was determined using the Plasmid Finder 2.1 database [91]. The search for prophages was carried out using the Phage Search Tool Enhanced Release (PHASTER) server [92]. SPI presence and completeness were determined by comparison of the SPI nucleotide sequences [22,23] with the sequences of the genomes of Salmonella strains SZL 30, SZL 31, and SZL 38.

Nucleotide Sequence Accession Number
Whole genomes for Salmonella enterica were submitted in BioProject PRJNA774121, and BioSamples SAMN22551429, SAMN22551430, and SAMN22551506. The version described in this paper is the first version.

Conclusions
In conclusion, this study found a high incidence of MDR in various serotypes of Salmonella isolated from various types of food, including those commonly seen in infections in humans. Three strains with multiple AMRs (more than six) had different resistance genes with a heterogeneous distribution in the bacterial genome, which may indicate intense selection pressure during rearing and treatment of animals. Therefore, it is necessary to implement a plan to combat the abuse of antibiotics in veterinary medicine, as well as a system of epidemiological surveillance, including one based on whole-genome sequencing.