The large-scale swine farm (~10,000 fattening pigs) is located in Sichuan province, China. The piglets were fed at 21–25°C, and the fattening pigs were fed at 18–23°C, which were vaccinated and in healthy condition. The piglets were fed with florfenicol or doxycycline to prevent diarrhea. A total of 450 fresh fecal swabs from different fattening pigs aged between ~1, 3, and 6 months (150 fattening pigs at each stage) were randomly collected between October and November 2021. Salmonella isolation was carried out as previously described (Yang et al., 2014). All culture mediums were purchased from Qingdao Hope Bio Technology, China. The swab samples were inoculated into 3 ml of buffer peptone water (BPW). Then, the 0.5 ml BPW-incubated culture was transferred to 10 ml of tetrathionate (TT) broth, and another 0.1 ml was transferred to 10 ml of Rappaport-Vassiliadis (RV) enrichment broth incubated at 42°C for 24 h. One loopful of each TT and RV broth culture was streaked onto xylose lysine tergitol 4 (XLT4) and brilliant green sulfa (BGS) agar and incubated at 37°C for 24 h. In the case of growth, one typical Salmonella colony was randomly selected from each plate and confirmed using the BD Phoenix-100 system (Sparks, MD, United States). Only one isolate from each sample was selected for further study.

Susceptibilities to 16 antimicrobial agents, including ampicillin (AMP), amoxicillin-clavulanate (AMC), ceftriaxone (CRO), ceftazidime (CAZ), meropenem (MEM), streptomycin (STR), gentamicin (GEN), amikacin (AMK), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), florfenicol (FFC), sulfamethoxazole (SUL), trimethoprim/sulfamethoxazole (SXT), tetracycline (TET), and polymyxin B (PMB), were determined using the standard Kirby–Bauer disk diffusion method according to Clinical and Laboratory Standards Institute guidelines (Clinical Laboratory Standards Institute, 2020). Escherichia coli ATCC25922 was used as a control strain.

Genomic DNAs of Salmonella isolates were extracted using a bacterial genomic DNA extraction kit (Tiangen, China). Whole-genome sequencing (WGS) was performed using the Illumina HiSeq 4000 platform (150 bp paired-end reads with ~200-fold average coverage). The raw reads were uploaded to the EnteroBase Salmonella database (http://enterobase.warwick.ac.uk/species/index/senterica) for genome assembly, serotyping, multilocus sequence typing (MLST; Maiden et al., 1998), ribosomal MLST (rMLST; Jolley et al., 2012), and core-genome MLST (cgMLST; Alikhan et al., 2018). The prokaryotic genome annotation software, RAST (Aziz et al., 2008), was used to identify the basic information of the draft genomes, such as the size of the genomes, GC content (%), and the number of contigs (above 200 bp). Antimicrobial resistance genes and plasmids were identified by CGE ResFinder 4.1 (Bortolaia et al., 2020) and PlasmidFinder 2.0 (Carattoli et al., 2014), respectively.

To interpret the phylogenetic relationship of the S. 4,[5],12:i:- strains in China, the phylogenetic tree based on SNPs was constructed using the EnteroBase Salmonella database combining 45 strains in this study and 241 strains in the database (accessed 3 January 2023). The phylogenetic tree was modified in Itol (Letunic and Bork, 2021). The presence of Salmonella genomic island 4 (SGI4) was identified by the BLAST program (Camacho et al., 2009), using the SGI4 sequence in S. Typhimurium strain SLK-532 (GenBank accession number MN730129) as the reference sequence (Mourao et al., 2020).

Three S. 4,[5],12:i:- strains, namely 3M-23, 6M-54, and 6M-71, harboring IncHI2 plasmids and various resistance genes were further sequenced by Nanopore MinION using Rapid Sequencing Kit. The complete genome sequences were assembled by Unicycler v0.5.0 using nanopore sequencing data combined with Illumina sequencing data. The complete nucleotide sequences of IncHI2 plasmids and the mcr-1 region were analyzed using the BLAST program (Camacho et al., 2009). BLAST Ring Image Generator (BRIG; Alikhan et al., 2011) and Easyfig v2.2.2 (Sullivan et al., 2011) were used for comparative analyses of the genetic environments. Conjugation experiments were performed by using 3M-23, 6M-54, and 6M-71 as the donor strains and rifampin-resistant E. coli EC600 as the recipient strain with selection on nutrient agar plates containing 300 mg/l rifampin and 8 mg/l florfenicol. The positive transconjugants were further determined by 16S rRNA gene sequencing and detection of antimicrobial resistance profiles.

The genomes of the 54 S. enterica isolates reported in this study have been deposited in National Center for Biotechnology Information and registered BioProject number PRJNA912362. The complete nucleotide sequences of three IncHI2 plasmids and the mcr-1 region in 6M-54 have been deposited in GenBank and assigned accession numbers OP970992-OP970994 and OP978507.

A total of 54 S. enterica strains were isolated and identified from 450 samples, with an isolation rate of 12%. Most of the strains (83.3%, 45/54) were recovered from the 6-month fattening pigs, and only two strains came from the 3-month fattening pigs. Serotyping based on the genome sequences indicated that those strains belonged to three serovars, 4,[5],12:i:- (the monophasic variant of Typhimurium; n = 45), Enteritidis (n = 7), and Derby (n = 2; Table 1). All 7 S. Enteritidis strains came from the 1-month pigs, and 43 out of 45 S. 4,[5],12:i:- strains came from the 6-month fattening pigs. Detailed information on the 54 S. enterica strains reported in this study is presented in Supplementary Table 1.

In total, 45 S. 4,[5],12:i:- strains belonged to ST34 and were further divided into two different rSTs, namely rST1369 and rST60594. It was worth noting that 43 strains from the 6-month fattening pigs belonged to rST1369, and the other two strains from the 3-month fattening pigs belonged to rST60594. The 45 S. 4,[5],12:i:- strains consisted of nine different cgSTs, highlighting the genetic diversity and presence of different lineages of S. 4,[5],12:i:- in the same farm. The cgST303338 (n = 35) was the predominant lineage. All seven S. Enteritidis strains belonged to ST11 and shared the same rST1425 and cgST303335, indicating that those strains were clonally related. The two S. Derby strains belonged to ST40 and rST3735 and were divided into two different cgSTs, namely 303905 and 303918 (Supplementary Table 1).

To investigate the phylogenetic relationship of these 45 S. 4,[5],12:i:- isolates, a maximum likelihood core-genome phylogenetic tree was constructed using 286 S. 4,[5],12:i:- strains (241 from EnteroBase Salmonella database and 45 from this study), with different sources (human, n = 179; swine, n = 87; food, n = 11; and poultry, n = 9) in China based on 4,243 SNPs (between 0 and 646 pairwise SNPs; median = 59; Figure 1). The pairwise SNP distance of 45 S. 4,[5],12:i:- strains from this study was between 0 and 560; however, the SNP distance of 39 strains was between 0 and 5. Furthermore, these 39 strains were divided into six cgSTs (cgST303338, n = 33, token the majority). The minimum SNPs distance between human isolates and pig isolates was 12 (6M-71/6M-78 from pigs and SSHZZ21-117 from humans). All 286 S. 4,[5],12:i:- strains belonged to ST34 and were divided into 22 rSTs, in which rST1369 (87.4%, 250/286) was the predominant rST (Supplementary Table 2). In total, 45 strains in this study were located in four different positions of the phylogenetic tree, especially strains 3M-23 and 3M-26 that belonged to rST60594 were in the root position (Figure 1). It was found that 90.6% (259/286) strains harbored SGI4, an integrative and conjugative element that carried heavy metal tolerance genes showing resistance to silver, arsenic, and copper (Branchu et al., 2019). The IncQ1 replicon gene was detected in 76.2% (218/286) strains; on the other hand, IncHI2 and IncHI2A replicon genes were detected in 37.4% (107/286) strains. The most commonly acquired AMR gene detected among these strains was aac(6′)-Iaa (286/286, encoding resistance to tobramycin and amikacin), followed by sul2 (246/286, sulfamethoxazole), tet(B) (244/286, tetracycline), aph(6)-Id (235/286, streptomycin), aph(3′')-Ib (222/286, streptomycin), and bla_{TEM − 1B} (178/286, beta-lactam). Three bla_{CTX − M} subtypes were identified among these strains, including bla_{CTX − M−14} (13/286), bla_{CTX − M−55} (26/286), and bla_{CTX − M−65} (28/286). fosA3 encoding for fosfomycin resistance was detected in seven strains. In addition, mobile colistin resistance gene mcr-1 was detected in 26 strains, 14 (53.8%) of them were of pig origin. It was also found that all of the mcr-1 positive strains detected IncHI2 and IncHI2A replicon genes.

Antimicrobial susceptibility testing showed that the 45 S. 4,[5],12:i:- strains displayed four different resistance profiles, and all exhibited ASSuT resistance patterns (ampicillin, streptomycin, sulfonamides, and tetracycline; Hopkins et al., 2010). In addition, six strains were resistant to SXT, GEN, CHL, and FFC. Three strains showed resistance to CRO, one of which was also resistant to PMB. The seven S. Enteritidis and two S. Derby strains were susceptible to all 16 antimicrobial agents tested. Five different antimicrobial resistance genotypes were identified in S. 4,[5],12:i:- strains (Table 1). Comparative genomic analysis showed that the IncQ1 plasmid carrying bla_{TEM − 1B}, aph(3′')-Ib (strA), aph(6)-Id (strB), sul2, and tet(B) was inserted in the chromosomal fljBA operon. However, three strains lost the tet(B) gene. Two strains (3M-23 and 3M-26) harbored ESBLs gene bla_{CTX − M−65}, and one strain (6M-54) harbored ESBLs gene bla_{CTX − M−14}, fluoroquinolone resistance gene aac(6′)-Ib-cr, and colistin resistance gene mcr-1 (Supplementary Table 1).

Three different IncHI2 plasmids, namely p3M-23, p6M-71, and p6M-54, were characterized in this study (Figure 2). The sizes of the three plasmids varied from 236,551 to 253,164 bp. BLAST analysis showed that they shared ~96-kb conservative backbone region carrying functional genes including replication gene, tra/trh genes associated with the conjugative transfer, and tellurium resistance gene cluster (Figure 2). p3M-23 shared 99.99% nucleotide identity with 99% cover to pS304_1 (CP061127), an IncHI2 plasmid in S. Typhimurium strain recovered from the stool of a patient in China. A comparative analysis of the antimicrobial resistance regions in the three IncHI2 plasmids is shown in Figure 3. The mosaic antimicrobial resistance regions consisted of various resistance genes, insertion sequences, and transposons. Compared with pS304_1, p3M-23 lost class I integron region intI1-estX-cmlA-aadA1-qacL-sul3, and the IS26-lun(F)-aadA22-IS1-IS26 region was instead of IS26-bla_{TEM − 1B}-IS1-IS26 region (Figure 3). p3M-23 and p6M-71 shared the same 16 resistance genes, but p3M-23 supplemented bla_{CTX − M−65} gene flanked by IS26 and IS903B. The antimicrobial resistance regions in p6M-54 showed a high difference from those in p3M-23 and p6M-71. Conjugation experiments showed that the three IncHI2 plasmids reported in this study could be easily conjugated to E. coli.

The mcr-1 and bla_{CTX − M−14}, as well as a copper resistance gene cluster, were co-located on the chromosome of 6M-54 (Figure 4). The bla_{CTX − M−14} gene was flanked by ISEcp1 and IS903. The integration of this 27.3-kb region into the chromosome resulted in a deletion of the 7.4-kb chromosomal segment. BLAST analysis showed that it shared the 26.2-kb region with IncHI2 plasmid p143-mcr (CP091564) with 100% nucleotide identity, speculating that this region harboring mcr-1 and bla_{CTX − M−14} might come from the IncHI2 plasmid.