A Multidrug-resistant Monophasic Salmonella Typhimurium Co-harboring mcr-1, fosA3, bla_CTX-M-14 in a Transferable IncHI2 Plasmid from a Healthy Catering Worker in China

Introduction

Salmonella enterica serovar Typhimurium is well known to be associated with foodborne diseases and outbreaks, as well as causing infectious diseases in animals and humans.¹ In recent year, the Salmonella Typhimurium monophasic variant (Salmonella 4,[5],12:i:-) was frequently reported from human salmonellosis in both Europe and the US.^2,3 Salmonella 4,[5],12:i:- is described as a variant of Salmonella Typhimurium lacking the second phase flagellum, which mostly belonged to the pandemic ST34 clone with an antibiotic resistance pattern of ASSuT (ampicillin, streptomycin, sulphonamides and tetracycline).⁴ In addition, the mcr-1 gene responsible for the colistin resistance has frequently occurred in ST34 Salmonella Typhimurium and Salmonella 4,[5],12:i:-.^4–7 In China, Salmonella 4,[5],12:i:- has increased to being one of the most predominant serovars causing human salmonellosis, while ST34 is the most common ST type of Salmonella 4,[5],12:i:-.⁸ ESBL-producing Salmonella isolates have raised concern due to their resistance to cephalosporins, which have been widely applied for treating severe salmonellosis.⁹ The ESBL-producing Salmonella strains showed a higher prevalence (8.58%) on retail chicken in China than in the US.^10,11 Meanwhile, fosfomycin has recently been reintroduced into clinical treatment for ESBL-producing Enterobacteriaceae. The occurrence of multidrug resistance in Salmonella 4,[5],12:i:- isolated from animal origin food products has increased the potential infection risk while treatment options are becoming limited.

In this study, we identified a MDR ST34 Salmonella 4,[5],12:i:- isolate carrying an IncHI2 plasmid with mcr-1, fosA3, bla_CTX-M-14 genes, which was from a healthy catering industry worker in Jiangsu province, China. To our best knowledge, this is the first report of the multidrug-resistant Salmonella 4,[5],12:i:- isolate harboring mcr-1, bla_CTX-M-14 and fosA3 from a human in China.

Materials and Methods

Bacterial Isolate

A surveillance study for foodborne pathogens was performed toward food catering workers and diarrheal patients by Nantong CDC, Jiangsu, China.¹² The initial aim of this project was to evaluate the role of food handler as a vehicle for transmitting foodborne pathogens by screening human fecal samples. In order to selectively enrich Salmonella, samples were added to the selenite cysteine broth and incubated at 35°C for 24 h. The 10 µL of each incubated sample was inoculated onto xylose lysine deoxycholate (XLD, OXOID, England) and Salmonella Shigella (SS) agar plate, and cultured for 24 h at 37°C. Two or more presumptive Salmonella colonies were subcultured in the triple sugar-iron-agar medium. The serotyping of Salmonella was based on Kauffmann–White (KW) scheme. During the process, we identified Salmonella Typhimurium and its monophasic variant by multiplex-PCR for the fliB-fliA intergenic region and the fljB gene.¹³

Antimicrobial Susceptibility Testing

Minimum inhibitory concentrations (MICs) were determined by the agar dilution method. Antibiotics used in this study were listed in Table 1. MICs were interpreted by CLSI breakpoints,^14,15 except that florfenicol (>16 mg/L) was interpreted according to EUCAST epidemiological cutoff values (http://mic.eucast.org/Eucast2).

Table 1 The Minimum Inhibitory Concentration (MIC) Values of the Donor (Salmonella 4,[5],12:i:- YZU1189), Transconjugant (E. coli C600-pYZU1189) and Recipient (E. coli C600)

Plasmid Conjugation Experiments

Conjugation experiments were performed between donor Salmonella 4,[5],12:i:- and recipient streptomycin-resistant E. coli C600, and transconjugants were selected on the Luria–Bertani agar plate containing streptomycin (3000 mg/L) and colistin (2 mg/L). The mcr-1 and mdh genes were used to distinguish donor, recipient and transconjugant based on the PCR analysis.^16,17

Whole Genome Sequencing Analysis

To understand the genetic background of Salmonella 4,[5],12:i:- YZU1189, the strain YZU1189 was sequenced using the Hiseq 2500 platform. Raw sequencing reads were deposited in the European Nucleotide Archive database under the accession number PRJEB38934. The raw reads were trimmed and filtered by NGSQC toolkit (v2.3.3), and subjected to de novo assembly by SPAdes 3.6.¹⁸ The assembled genome was annotated by Prokka version 1.12.¹⁹ The acquired antimicrobial resistance genes and chromosomal mutations was identified by ResFinder 3.2.²⁰ The multilocus sequence type of the strain YZU1189 was obtained by MLST 2.0.²¹ The virulence factors of strain YZU1189 was detected by BLASTn based on seven typical virulence genes in Salmonella Typhimurium, of which four genes located on prophages including gipA (encoding a Peyer’s patch-specific virulence factor), sspH1 (Salmonella-type III effector protein), sodC1 (putative Cu/Zn superoxide dismutase) and sopE1 (Salmonella-type III effector protein), and three genes (spvC, pefA, rck) in plasmids.²²

Ethical Approval

The study protocol was performed following the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committee of the Chinese Centers for Disease Control and Prevention (CDC). Fresh feces of all healthy catering workers were sampled after obtaining written informed consents.

Results and Discussion

In the surveillance study, we observed a Salmonella 4,[5],12:i:- strain YZU1189 harboring mcr-1, fosA3 and bla_CTX-M-14 from a healthy female catering worker. This MDR isolate was defined using the criteria established by Magiorakos et al.²³ MICs of 15 antibiotics for the strain YZU1189 showed that YZU1189 was resistant to antibiotics including aminoglycosides, beta-lactams, polymyxins, fosfomycins, phenicols, trimethoprims, sulfonamides, tetracyclines and fluoroquinolones (Table 1). Compared to the typical ASSuT resistance pattern in most of Salmonella 4,[5],12:i:- isolates, this strain lacked the phenotypic streptomycin resistance, while showed resistance to colistin and fosfomycin.

According to the MLST analysis, Salmonella 4,[5],12:i:- strain YZU1189 belonged to sequence type (ST) 34. Virulence factors analysis of seven genes showed that this isolate carried virulence factors gipA and sodC1, which were prevalent in Salmonella 4,[5],12:i:- isolates from foods in China.⁸ Strain YZU1189 contained several antimicrobial resistance (AMR) genes including aminoglycosides (aac(3)-IV, aph(4)-Ia, aph(3ʹ)-Ia, aac(6ʹ)-Iaa), β-lactamase (bla_CTX-M-14), polymyxins (mcr-1), fosfomycins (fosA3), phenicols (floR), sulphonamides (sul2 and sul3), tetracycline (tet(B)), and trimethoprims (drfA12). In addition, the mutation of codon 87 (D87N) in the GyrA protein causing the quinolone resistance was also detected in this strain. The mcr-1 gene was previously observed in ST34 Salmonella Typhimurium from pigs in China, which located mainly on IncHI2-like plasmids.⁵ The PCR analysis showed that the transconjugant was positive for the mcr-1 gene and negative for the Salmonella Typhimurium specific mdh gene, indicating the successful transmission of mcr-1 positive plasmid into the recipient strain E. coli C600. The plasmid conjugation experiment showed that the mcr-1 gene in YZU1189 could be co-transferred with bla_CTX-M-14 and fosA3 genes (Table 1).

By whole genome sequencing analysis, we confirmed several antimicrobial resistance genes located on an IncHI2 plasmid (246Kb), named pYZU1189 (accession number ERS4951189), which harbored mcr-1, fosA3, floR, sul2, sul3, drfA12, aac(3)-IV, aph(4)-Ia, aph(3ʹ)-Ia, bla_CTX-M-14 genes (Figure 1). The mcr-1 gene was located in the common genetic background of ISApl1-mcr-1-orf, which was also detected in pECHN-15–61(IncI2), pECFJ-B42-63(IncI2), and pECJS-B60-267(IncHI2) plasmids,²⁴ while the fosA3 gene had the common genetic background of ΔISEcp1-bla_CTX-M-14-IS903B-fosA3-orf located in pYZU1189. IncHI2 plasmids harboring antimicrobial genes found in this study were also reported in Enterobacteriaceae, such as E. coli and Salmonella spp.^6,7,24 The sequence alignment showed that an IncHI2-type plasmid pHNSHP45-2 harboring a similar genetic background with mcr-1, bla_CTX-M-14 and fosA3 in E. coli (Figure 1). Five Salmonella Typhimurium isolates carrying co-transferred fosA3, bla_CTX-M-14, mcr-1, oqxAB and floR genes from food animals between 2016 and 2017 in China have been previously reported.⁶ More importantly, mcr-positive Salmonella 4,[5],12:i:- isolates with multiple antimicrobial resistance genes have been frequently reported from different sources in multiple countries worldwide,^25–32 indicating the increasing risk potential of this pathogen transmitting worldwide. Also, mcr-positive Salmonella 4,[5],12:i:- isolates from difference sources showed a potential dissemination between food animals and humans, which remains a significant threat to human health.

Figure 1 Sequence alignment of pYZU1189 and pHNSHP45-2 (GenBank no. KU341381). The pHNSHP45-2 was used as a reference to compare with mcr-1-bearing plasmid which possess the IncHI2 replicon in this study. Red arrows represent the plasmid pHNSHP45-2.

Conclusion

In conclusion, this study reported an ESBL-producing Salmonella 4,[5],12:i- isolate of human origin carrying a colistin resistance gene mcr-1, a fosfomycin resistance gene fosA3 and a rare sulfonamide resistance gene sul3. The occurrence of mcr-1 and fosA3 genes in a transferable IncHI2 plasmid pYZU1189 from a ESBL-producing Salmonella 4,[5],12:i:- isolate brings great considerable public health threat. Great concern should be taken for the spread of multidrug-resistant ESBL-producing Salmonella isolates from food catering workers to consumers. In the “One Health” aspect, quick and thorough action should be taken to reduce the use of colistin in food-producing animals in the farming industry. Interventions of restricting antibiotic use in food-producing animals should also be applied to reduce the prevalence of MDR bacteria.