Clonal spread of mcr-1 in PMQR-carrying ST34 Salmonella isolates from animals in China

Since initial identification in China, the widespread geographical occurrence of plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae has been of great concern. In this study, a total of 22 Salmonella enterica were resistant to colistin, while only five isolates which belonged to ST34 Salmonella enterica serovar Typhimurium (S. Typhimurium) were mcr-1 positive. Four of them shared nearly identical PFGE type, although they were from different host species and diverse geographical locations. All the mcr-1-positive S. Typhimurium exhibited multi-resistant phenotypes including ampicillin, streptomycin, gentamicin, florfenicol, nalidixic acid, tetracycline, trimethoprim-sulfamethox, in addition to colistin. The oqxAB and aac(6′)-Ib-cr genes were present alone or in combination in four (80.0%) and five (100%) isolates, respectively. The mcr-1 gene was located on a transferable IncI2 plasmid in the four genetically related strains. In the other one strain, mcr-1 was located on an approximately 190 kb IncHI2 plasmid. In conclusion, we report five mcr-1-positive S. Typhimurium/ST34 isolates. Both clonal expansion and horizontal transmission of IncI2-type plasmids were involved in the spread of the mcr-1 gene in Salmonella enterica from food-producing animals in China. There is a great need to monitor the potential dissemination of the mcr-1 gene.

Salmonellosis is one of important global public health zoonoses, causing life-threatening infections. Each year, there are an estimated 1.0 million Salmonella infections in the United States 1 . By contrast, 30 millions of infections every year in China, approximately 75% of the food-borne diseases, are attributed to this bacterium 2 . Unpublished data from the China CDC surveillance system indicated that the carriage rate of human salmonellosis is 549 per 100,000 people in 2013, which is higher than that in the USA in 2012 (16.4 per 100,000) 3 . Salmonella enterica, especially non-typhoidal Salmonella (NTS), is a leading cause of food-borne disease of humans and livestock worldwide 3,4 . Various animal species, such as poultry, pigs, cattle, and reptiles, are reservoirs for NTS. Human NTS infections are frequently due to the consumption of contaminated cooked or raw meat, milk, eggs, seafood, and other fresh products derived from animals 5 .
In recently year, emerging fluoroquinolone resistance prevalence has been identified in several Salmonella serovars and the resistance rate to fluoroquinolone has increased dramatically both in clinical and food-borne Salmonella isolates around the world 6 . Resistance toward quinolone and fluoroquinolone antimicrobials is mainly attributed to mutations of quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) mechanism including Qnr peptides (QnrA, QnrB, QnrS, QnrD and QnrC), AAC(6′ )-Ib-cr and the efflux pumps QepA and OqxAB 7 . In our previous study, we have characterized a high prevalence of oqxAB (31.7%) in Salmonella enterica serotype Typhimurium (S. Typhimurium) isolated from food-producing animals in China. The oqxAB gene was present alone or in combination with other PMQR genes such as aac(6′ )-Ib-cr and qnrS1 genes. Interestingly, the S. Typhimurium isolates carrying oqxAB were clonally related as determined by PFGE and also defined as ST34 by MLST type 7 . A high prevalence of oqxAB-positive S. Typhimurium/ ST34 was also detected in human clinical and food samples in Hong Kong in the same period 8 . It aroused a possibility that the oqxAB-positive S. Typhimurium/ST34 transmitted from food animals to humans via food chain.
More recently, Salmonella species have been isolated that carried PMQR, can occur as a multiple drug resistance phenotype, which has caused international concern because it brought an even greater challenge for clinical treatment 9,10 .
Colistin (polymyxin E) is a cationic, multi-component, lipopeptide antibacterial agent discovered in the 1940s with significant activity against Gram-negative bacteria. Colistin has been used both in human and veterinary medicine for more than 50 years, although their parenteral usage in humans has been limited because of concerns about nephrotoxicity and neurotoxicity 11 . However, with a global increase in Gram-negative bacteria that are multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), colistin has been re-introduced as a last-resort drug for infections with such bacteria, which are frequently the cause of healthcare-associated infections 12,13 . In veterinary medicine, colistin is more widely used, mainly to treat Gram-negative infections of the intestinal tract 14 . In addition, colistin is used as a growth promoter in some countries duo to its great growth performance in pig and poultry production 14,15 .
Resistance to colistin in Gram-negative bacteria has been characterized by chromosomal mutations and was generally thought non-transferable by mobile genetic elements 11 . Specific regions mutations like pmrAB and phoPQ, which were related to structural changes (ParR-ParS two-component system) of the LPS at both the cytosol and periplasmic site of the cell membrane, could decrease colistin activity in Klebsiella, E. coli and Salmonella enterica 14 .
Recently, a plasmid-mediated colistin resistance gene (mcr-1) was firstly reported in food animals, food and humans in China 15 . The mcr-1 gene was proved subsequently to be disseminated worldwide, mainly found in E. coli, K. pneumonia and Salmonella spp 15,16 . In China, mcr-1 was dominantly identified in E. coli, K. pneumonia, Enterobacter aerogenes, and Enterobacter cloacae in many regions (Fig. 1 24 , whereas data on the transmission of mcr-1-mediated colistin resistance in Salmonella spp. are lacking. Although, during the preparation of this study, the prevalence of mcr-1 among ESBL-positive Salmonella spp. isolates was investigated, but the presence of this gene in particular successful resistant clone has not been demonstrated 25 . In this study, we did a retrospective study to examine the emergence of the mcr-1 gene in Salmonella enterica isolates from food-producing animals during 2007 to 2015.

Results
Antimicrobial susceptibility and detection of resistance genes. These isolates showed the minimal inhibitory concentrations (MIC) for colistin of 0.25 to 16 mg/L. A total of 22 Salmonella isolates were resistant to colistin with MIC ≥ 8 mg/L. The mcr-1 gene was detected in only five colistin-resistant S. Typhimurium isolates with diverse origins (Table 1). Susceptibility testing showed that all the mcr-1-positive isolates in this study were resistant to nalidixic acid, olaquindox, ampicillin, streptomycin, gentamicin, florfenicol, tetracycline and trimethoprim-sulfamethox. All the five mcr-1-containing strains carried aac(6′ )-Ib-cr, and four of them also carried oqxAB and floR (Table 1). Interestingly, all strains showed increased MIC values and exhibited intermediate resistance phenotype (strain GDS 79, GDS82, and GDS141) or resistance phenotype (strain S01) but except strain GDS78 retained sensitivity to ciprofloxacin according to the cutoff of CLSI, although most of them harboured one or more PMQR genes ( Table 1).
Transfer of mcr-1 gene. Four transconjugants were successfully obtained from the five mcr-1-positive isolates. Conjugation and transformation tests were not successful for strain S01 despite repeated attempts. All transconjugants showed 32-fold increase in the MICs of colistin, in comparison with the recipient E. coli C600 (0.125 mg/L). However, other antibiotic-resistant phenotypes could not be co-transfered with colistin except for strain GDS78. The transconjugant of GDS78 was multidrug-resistant and showed resistance to more than five antibiotics, in addition to colistin (Table 1).
Plasmid analysis. PCR-based replicon typing (PBRT) including IncHI2, IncI2, IncFIB and IncFII were detected in the original strains. While, only IncI2 was found in the three transconjugants. For GDS78T, both IncI2 and IncFII were identified (Table 1). S1-PFGE and Southern blot showed that mcr-1 in all the four transconjugants was located in an IncI2 plasmid with size of 70 kb, approximately. In strain S01, the mcr-1 was located in a round 190 kb IncHI2 plasmid ( Fig. 2A). Restriction Fragment Length Polymorphism (RFLP) indicated that pattern of GDS79T was different from GDS82T and GDS141T (Fig. 2B). After 14 days of passage without colistin, the plasmids carrying mcr-1 were stable both in the parent strains and transconjugants. PCR-mapping and sequencing showed that an approximately 2,600 bp long fragment designed as the mcr-1 cassette 26 , was inserted between nikB and ydfA on the backbone of IncI2 plasmid in the four transconjugants. The mcr-1 cassette encompasses the likely promoter sequences, the mcr-1 gene and a hypothetical protein 26 . Nevertheless, compared with pHN-SHP45 (KP347127), the firstly identified mcr-1-carrying IncI2 plasmid, the ISApl1 is absent from the upstream  of the mcr-1 gene (Table 2). In the original strain S01, we then confirmed that a 3,679 bp length of ISApl1-mcr-1 fragment was inserted in approximately 8,500 bp downstream of terY2 on pHNSHP45-2 (KU341381), which is in accordance with pMR0516mcr (KX276657) (Fig. 3).

Molecular typing.
All of the five isolates were successfully typed by pulsed-field gel electrophoresis (PFGE), and two different PFGE clusters designated A and B were obtained (Fig. 4). Cluster A contains four isolates, three of which were isolated from different pig farms in 2008 and 2009, while the remaining one was isolated from duck in 2010. The single strain of cluster B was isolated from chicken in 2007. Multi-locus sequence typing showed that the five mcr-1-positive S. Typhimurium belongs to ST34, though they were classified into two PFGE clusters (Fig. 4).    8 , which was also frequently detected in human clinical and food samples in Hong Kong in the same period 8 . The dissemination of this clone carrying mcr-1 from food-producing animals to humans via food chain might expedite colistin resistance in Salmonella strains. The mcr-1 gene was so far associated with diverse plasmids belonging to the IncI2, IncHI2, IncP, IncX4, IncFII and IncF replicon types 15,[35][36][37][38][39] . Among them, IncHI2 and IncX4 rather than IncI2 plasmids, which were firstly identified to harbour the mcr-1 gene in E. coli, were the most common replicons in Salmonella spp 10,32,36 . However, the mcr-1 gene was located on the IncI2 plasmid in all S. Typhimurium/ST34 strains except strain S01 in this study, which indicated that the IncI2 plasmid harbouring the mcr-1 gene has circulated between E. coli and Salmonella spp. Interestingly, the mcr-1-positive IncI2 plasmid was co-occurrence with the other plasmids with diverse replicon types such as IncHI2 and IncFII in the four clonal mcr-1-positive strains. Unlike the original E. coli SHP45, which carried two mcr-1-positive plasmids including an IncI2 type pHNSHP45 and an IncHI2 type pHNSHP45-2 15,40 , the mcr-1 gene was only found on IncI2 plasmid in this study. This indicated that other mobile elements were probably involved in the mobilization of the mcr-1 gene, in addition to plasmids. Indeed, ISApl1 was present upstream of the mcr-1 gene on both mcr-1-carrying plasmids in E. coli SHP45, while it was absent on all the mcr-1 positive IncI2 plasmids in this study.
Only five mcr-1-positive strains were identified in 276 isolates, indicating that the mcr-1-positive Salmonella isolates are sporadic in animals in China. It is possible that the transferable mcr-1 gene here appears in the form lacking the insertion sequence ISApl1 41 . In addition, all the mcr-1-positive Salmonella enterica were isolated from the disease animals, whereas most of the mcr-1-negative colistin resistant strains were from healthy animals. Although the prescription was not recorded during the sampling, existing evidence suggested that exposure to colistin was closely related to its resistance rates 14 . The high rate of colistin resistance and low mcr-1 positive rates showed that the plasmid-mediated colistin resistance was not the main way conferred colistin resistance among Salmonella isolates. It is of possibility that other colistin-resistant mechanisms or even novel mcr-1 type exisit 35 , which needs to be evaluated in future study.
In conclusion, we reported five mcr-1-positive Salmonella isolates from animals in China between 2007 and 2015. Clonal spread of PMQR-carrying ST34 Salmonella isolates and horizontal transmission of IncI2 plasmids were the main way to disseminate mcr-1 gene in this study. Colistin should be used more prudently in food-producing animals to prevent mcr-1 gene spreading between different specials.

Materials and Methods
Bacterial strains. A total of 276 nonduplicate Salmonella enterica isolates (246 from avian and 30 from swine), isolated from faecal swabs of healthy or sick animals at poultry farms, swine farms and two diagnostic laboratories in Guangdong and Shandong province in China among 2007 and 2015, were used in this study (Table 3). Among them 127 were S. Typhimurium, 79 were Salmonella enterica serotype Indiana, six were Salmonella enterica serotype Enteritidis, two were Salmonella enterica serotype Meleagridis, two were Salmonella enterica serotype Bredeney, one was Salmonella enterica serotype Abaetetuba and 59 were non-typeble. The details of these strains have been described in our previous works 7,42 . Conjugation and transformation analysis. E. coli C600 was used as the recipient for the conjugation experiment of MCR-producing Salmonella isolates. The transconjugants were selected on MacConkey agar containing colistin (2 mg/L) and streptomycin (2,000 mg/L), and finally confirmed by PCR and ERIC-PCR 45 . Plasmids that are not transferable by conjugation were studied by transformation assay. Plasmid DNA was extracted using a QIAGEN Prep Plasmid Midi Kit. Purified plasmids were used in electroporation experiments with E. coli DH5α following the manufacturer's instructions. Transformants were incubated at 37 °C for 1 h and were then selected on LB agar containing 2 mg/L colistin. Plasmid Characterization. Incompatibility (Inc) groups were assigned by PBRT. To analyze the location of the mcr-1 gene, S1 nuclease-PFGE and Southern blot analysis were performed. Briefly, whole-cell DNA of the donor strains and the transconjugants harbouring mcr-1 were extracted and embedded in agarose gel plugs. Subsequently, the agarose gel plugs were treated with S1 nuclease (TaKaRa, Dalian, China) and the DNA fragments were separated by PFGE. Southern blot hybridization was then performed with DNA probes specific for the mcr-1 gene, which was non-radioactively labeled with a DIG High Prime DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany). Transconjugants containing one plasmid were extracted and analyzed by RFLP using ApaLI (TaKaRa, Dalian, China) digestion. The genetic context surrounding the mcr-1 gene was investigated by PCR mapping and sequencing according to the plasmids sequences which had been submitted to GenBank. The primers used to determine the regions upstream and downstream of the mcr-1 gene are listed in Table 2. To assess the stability of mcr-1-positive plasmids, the original strains and transconjugants were cultured by in daily serial passages with the absence of colistin for two weeks. Molecular typing. Genomic DNA of the mcr-1-positive isolates was analyzed by PFGE following digestion with XbaI 46 . Salmonella enterica serotype Braenderup H9812 standard was used as size marker. Comparison of PFGE patterns was performed by BioNumerics ® v6.6 (Applied Maths, Ghent, Belgium) with a cut-off at 90% of the similarity values to indicate identical PFGE types. Multi-locus sequence typing (MLST) was performed by using the primers and protocol specified at the Salmonella enterica MLST website (http://mlst.warwick.ac.uk/ mlst/dbs/Senterica).