Characterization of a Multidrug-Resistant Salmonella Enteritidis Clinical Strain Carrying a Novel Hybrid Plasmid

Salmonella enterica serotype Enteritidis is one of the most prevalent serotypes among Salmonella enterica and is commonly associated with human food poisoning worldwide (Jiang et al., 2018; Haley et al., 2019). Plasmids, insertion sequences (ISs), transposons (Tns), and integrons can facilitate resistance dissemination through horizontal gene transfer (McMillan et al., 2020). Hybrid plasmid formation by IS26 insertion has been reported previously (Wong et al., 2017; Du et al., 2020). Wong et al. (2017) reported that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 resistance plasmid through IS26, thus forming a novel hybrid plasmid pSE380T.

Here we report the identification of a novel hybrid plasmid in a multidrug-resistant (MDR) Salmonella Enteritidis clinical strain in China. Salmonella Enteritidis isolate SAL045 was recovered from a 1-year-old infant patient suffering from food poison symptoms including diarrhea and vomiting in September of 2020 in Wuhan city. Antimicrobial susceptibility testing was performed by the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (2019). E. coli ATCC 25922 was used as the quality control strain.

Salmonella Enteritidis SAL045 was found to be resistant to ampicillin, streptomycin, tetracycline, doxycycline, chloramphenicol, sulfisoxazole, trimethoprim, trimethoprim–sulfamethoxazole, nalidixic acid, ciprofloxacin, rifampicin, and polymyxin E. It was sensitive to ceftazidime, cefotaxime, cefazolin, cefoxitin, imipenem, gentamicin, and azithromycin.

Whole-genome sequencing was performed using Illumina HiSeq and Oxford Nanopore PromethION platforms through de novo assembly. Genome annotation was generated by Glimmer 3.02 and RAST 2.0, respectively. Antimicrobial resistance genes and virulence genes were identified using Resfinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/), CARD 2020 (Jia et al., 2017), and VFDB database (Chen et al., 2016). A total of 29 different resistance genes were located in the genome of Salmonella SAL045 (Table 1).

Polymyxin E is considered as last-line drug for MDR Gram-negative bacteria (Poirel et al., 2017). Resistance to polymyxin E has mostly been associated with lipopolysaccharide (LPS) modification. A large panel of genes and operons are involved in modification of the LPS, including transferable mcr-type genes and genes involved in the PmrAB and PhoPQ two-component systems (Poirel et al., 2017). The resistance breakpoint for polymyxin E for Enterobacteriaceae was determined to be minimum inhibitory concentration (MIC) >2 mg/L (CLSI, 2019).

But although SAL045 had a polymyxin E MIC of 4 mg/L, mcr, and mutations in genes encoding two-component systems, PhoPQ and PmrAB were not found. These genes and mutations can be responsible for polymyxin E resistance in Gram-negative bacteria (Sato et al., 2018). However, SAL045 possessed novel amino acid substitutions such as A159V, E415Q, G232S, and L77P in PmrC. The contributions of novel amino acid alterations in PmrC to polymyxin E resistance are currently unknown.

Two plasmids were identified in SAL045. The smaller plasmid (24,484 bp), named pS045B, carried only four resistance genes: bla _TEM-1B, strA, strB, and sul2. The larger plasmid, designated pS045A, was 318,041 bp in size and contained 22 different resistance genes. Analysis by using pMLST and MobileElementFinder found that pS045A could be grouped into IncHI2 type plasmid. A BLASTN search against the NCBI database showed that plasmid pS045A displayed 100% nucleotide sequence identity within 80% query coverage with E. coli strain ECCNB12-2 plasmid pTB-nb4 (CP033636.1) (Chang et al., 2019).

They shared multiple genes that confer resistance including the quaternary ammonium compound resistance gene qacH. Plasmid pS045A harbored an extra 65,859 bp region, which was not present in plasmid pTB-nb4 (Fig. 1). The region was 100% identical to the Salmonella enterica plasmid pFORC51 (CP017233.1). The spv ABCD operon and rck and the pef fimbrial operon that may increase virulence (Silva et al., 2017) were present in the region (Fig. 1). Two copies of IS26 were present at the end of this virulence region, indicating that pS045A was generated by incorporation of an entire virulence plasmid into an IncHI2 resistance plasmid through IS26 insertion.

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FIG. 1.

Diagram of plasmid pS045A and comparison with pTB-nb4 and pFORC51. Structures of MDR-1 and MDR-2 in plasmid pS045A are marked. Locations of resistance genes (red), transfer and replicon genes (blue), mobile genetic elements including IS (green) and virulence genes (purple) are shown. Representative genes are shown as arrows and the direction of transcription is indicated by the arrowheads. Figure illustrates direct insertion of virulence plasmid pFORC51 into an IncHI2 plasmid pTB-nb4. MDR-1 is flanked by IS6 and Tn21, whereas MDR-2 is flanked by IS1006 and ISKpn8. IS, insertion sequence; MDR, multidrug resistant. Color images are available online.

In pS045A, IS, Tn, and class 1 integron (intI1) frequently inserted and generated MDR loci (Fig. 1). Two MDR regions were found in pS045A (MDR-1 corresponds to bases 86,796–118,944 and MDR-2 corresponds to bases 196,292–270,611 in the plasmid). The resistance genes were located in gene cassettes bounded by various ISs. For example, a rare gene cassette IS26-intI1-aadA22-lnuF-IS26 was present in the MDR-1 region. The MDR-2 region was flanked by IS1006 and ISKpn8, which was followed by transposase encoding gene cassette tnpA-rve. These ISs captured resistance genes including △bla _TEM-1B (456 bp deleted and truncated by IS26) and allowed them to be integrated into pS045A (Fig. 1).

The MDR-2 region in plasmid pS045A contained the origin of transfer oriT and genes encoding a relaxase TraI, the type IV secretion system TraD and TraGHF. The conjugative transfer of pS045A was tested as previously described (Lu et al., 2021). Conjugation was assessed by the broth mating method using E. coli EC600 as the recipient. Transconjugants were selected on MacConkey agar supplemented with tetracycline (128 μg/mL) and streptomycin (32 μg/mL).

The results showed that the transfer frequency was 1.76 × 10⁻⁶ transconjugants per donor after an 18-hour mating period at 37°C. The resistance to tetracycline, streptomycin, ampicillin, and ciprofloxacin was measured by the broth microdilution method. MICs of EC600 for tetracycline, streptomycin, ampicillin, and ciprofloxacin were 32, 8, 8, and 0.25 μg/mL, respectively.

The transconjugants had increased resistance, with tetracycline MIC of ≥128 μg/mL, streptomycin MIC of ≥128 μg/mL, ampicillin MIC of 64 μg/mL, and ciprofloxacin MIC of 1 μg/mL. Previous research indicated that an IncHI2 plasmid pSTM6-275 of Salmonella enterica transferred to EC DH5α at a frequency of 1.3 × 10⁻⁵ transconjugants per donor at 27°C, whereas no transfer occurred at 37°C (Billman-Jacobe et al., 2018). In comparison with pSTM6-275, pS045A was transferable even at 37°C, indicating the risk of dissemination of resistance among Enterobacteriaceae.

In addition to pTB-nb4, BLASTN searches of the GenBank database failed to find that any other plasmids were a better match to pS045A (≥99% nt identity at >80% coverage). Therefore, in this study we identified a novel hybrid plasmid pS045A carrying MDR genes. To our knowledge, plasmid pS045A contains the greatest number of resistance genes found in a Salmonella hybrid plasmid to date and may be able to disseminate these and associated virulence genes with new hosts. It may pose strengthened hazards in dissemination of resistance and virulence.