ABSTRACT
We detected the colistin resistance gene mcr-1 in four Salmonella serovars isolated from humans and animals with diarrhea. The resistance gene was carried on different plasmids. One mcr-1-carrying conjugative plasmid, a variant of pHNSHP45, was disseminated among Salmonella isolates recovered from humans, pigs, and chickens.
TEXT
Colistin is considered the last resort for treatment of multidrug-resistant Gram-negative bacterial infections. Colistin resistance in bacteria is mediated by several subtle mechanisms that mainly result from mutations in chromosomal genes (1). Recently, Liu et al. (2) found a plasmid-borne colistin resistance gene—mcr-1, which encodes a phosphoethanolamine transferase—in Escherichia coli from humans and animals (pigs and chickens) in China. The resistance gene was soon detected worldwide in several Enterobacteriaceae species, including E. coli, Klebsiella pneumoniae, Shigella sonnei, and several Salmonella serovars, either carried by various plasmids (3–5) or located in the chromosomes (6). However, few reports show the dissemination of mcr-1-carrying plasmids in zoonotic pathogens between humans and animals. We investigated mcr-1 and the plasmids that carry it in Salmonella isolates from humans and food-producing animals, collected between 2010 and 2015 in Taiwan.
We conducted antimicrobial susceptibility testing on 6,386 Salmonella isolates from humans (5,178) and diseased animals (1,208), namely, chickens (450), pigs (279), ducks (206), turkeys (170), geese (88), and other animals (15) (see Table S1 in the supplemental material), using a custom-made 96-well Sensititre MIC panel (Trek Diagnostic Systems Ltd., West Grinstead, England). The isolates from humans were collected from 52 hospitals across the country, and the isolates from animals were recovered by the Veterinary Teaching Hospital of National Chiayi University from sick animals in central and southern Taiwan. Salmonella serotypes were determined by use of the typing scheme developed by Chiou et al. (7). Colistin resistance was identified in 1,917 isolates, grouped into 28 Salmonella serovars, with MIC values of ≥4 mg/liter (8). Of the colistin-resistant isolates, 1,792 (93.4%) were Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis), and 125 belonged to the other 27 serovars (see Table S2 in the supplemental material). Among the 1,917 colistin-resistant isolates, we selected 556 (431 colistin-resistant S. Enteritidis isolates and all resistant isolates of the other 27 serovars) for detection of mcr-1 using the PCR primers designed by Liu et al. (2). The tests identified 19 mcr-1-carrying isolates (S. enterica subsp. enterica serovars Typhimurium [14], Anatum [3], Albany [1], and Newport [1]) recovered from 10 humans, 7 pigs, and 2 chickens between 2012 and 2015 (Fig. 1). Notably, S. Enteritidis displayed a high colistin resistance rate, but mcr-1 was not detected in all of the 431 colistin-resistant isolates tested. The resistance mechanism for these colistin-resistant S. Enteritidis isolates has not been identified. The mcr-1-carrying isolates, except for one, were multidrug resistant, with resistance to three or more classes of antimicrobial agents (Fig. 1). The 9 mcr-1-carrying isolates from animals were collectively more resistant than the 10 isolates from humans (see Table S3 in the supplemental material). We conducted whole-genome sequencing of 3 cefotaxime-resistant isolates (C215, P111, and P165) and found a blaCMY-2 gene in P165. No other β-lactamase gene was found in C215 or P111. The blaCMY-2 gene was then detected by PCR in P163, P164, NG14.043, and C214 but not in P110. These 3 cefotaxime-resistant isolates (C215, P110, and P111) should have different resistance mechanisms from the 5 blaCMY-2-carrying isolates.
Genetic relationships among 19 mcr-1-carrying Salmonella isolates built using PFGE patterns, their corresponding antimicrobial resistance patterns, and relative information. Antimicrobials: FTX, cefotaxime; CAZ, ceftazidime; IMP, imipenem; NAL, nalidixic acid; CIP, ciprofloxacin; GEN, gentamicin; AMP, ampicillin; CHL, chloramphenicol; STR, streptomycin; SUL, sulfamethoxazole; TCY, tetracycline; SXT, trimethoprim-sulfamethoxazole; COL, colistin; EPT, ertapenem; FOX, cefoxitin. Red boxes, resistance; yellow boxes, intermediate resistance; green boxes, susceptibility. The plasmid types are defined on the basis of the BglII and AccI restriction profiles of the mcr-1-carrying conjugative plasmids from the isolates. ND, plasmid type not determined.
We characterized Salmonella isolates, using the standardized PulseNet pulsed-field gel electrophoresis (PFGE) protocol with XbaI (9), and investigated the genetic relatedness among Salmonella isolates by comparing PFGE patterns, using the cluster analysis program provided in BioNumerics version 6.6 software (Applied Maths, Sint-Martens-Latem, Belgium). The PFGE analysis identified 10 patterns for the 14 mcr-1-carrying S. Typhimurium isolates and 5 patterns for the 5 remaining mcr-1-carrying isolates. Cluster analysis of PFGE patterns for the mcr-1-carrying and 3,084 non-mcr-1-carrying S. Typhimurium isolates collected between 2010 and 2015 revealed that the 14 mcr-1-carrying isolates fell into 2 major clusters, and each clonal cluster contained isolates recovered from humans, pigs, and chickens. These studies indicated that the mcr-1 resistance gene in the strains should have derived from multiple origins.
We conducted conjugation experiments to transfer the mcr-1-carrying plasmids to E. coli C600 recipients and used LB medium with 1 mg/liter colistin and 2,000 mg/liter streptomycin for the selection of transconjugants. The mcr-1-carrying plasmids in S. Albany, S. Newport, and S. Typhimurium were transferable, but transfer was not achieved in S. Anatum. We extracted plasmids of transconjugants, using the Qiagen Plasmid Midi kit (Hilden, Germany), and treated purified plasmids with BglII and AccI. Four restriction patterns were identified for the plasmids (see Fig. S1 in the supplemental material). Plasmid type I (∼60 kb) was identified in 5 S. Typhimurium isolates from humans, pigs, and chickens and 1 S. Albany and 1 S. Newport isolate from humans (Fig. 1). Three isolates (P110, P111, and C215) with the type I plasmid recovered from pigs and chickens shared an indistinguishable PFGE pattern. Plasmid type II (∼43 kb) was carried by 2 S. Typhimurium isolates from pigs and chickens, and type III (∼33 kb) and type IV (∼60 kb) plasmids were harbored in 7 S. Typhimurium isolates from humans.
We determined the sequences of the plasmids derived from strains P111 (type I), C214 (type II), NG14.043 (type III), and R15.0626 (type IV) using the Illumina MiSeq platform (2 × 300 bp), and we assembled the sequence reads and analyzed the complete sequences using the CLC Genomics Workbench (Qiagen Bioinformatics). The BglII, AccI, and AflII restriction profiles predicted for the complete plasmid sequences were concordant with those generated from purified plasmids digested with each of the enzymes. We determined the plasmid incompatibility types using the PlasmidFinder tool provided on the website of the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/ ) and annotated the four plasmid complete sequences with the Prokka program (10). The genetic maps for the four plasmids are listed in Fig. S2 in the supplemental material. pP111 (60,960 bp, type I) and pR150626 (59,651 bp, type IV) belonged to IncI2; pC214 (42,941 bp, type II) and pNG14043 (33,308 bp, type III) belonged to IncX4. All of the plasmids contained one copy of mcr-1, and the sequence of mcr-1 on these plasmids shared 100% sequence identity with the first one published by Liu et al. (2). pP111 had 89% sequence sharing with pR150626, and both had the same genetic structure surrounding mcr-1 (Fig. 2). pR150626 was a close variant of the first reported mcr-1-carrying plasmid, pHNSHP45, in China (2); its whole sequence could be found in pHNSHP45. However, compared to pHNSHP45, pR150626 and pP111 lacked an ISAPl1 insertion element upstream of mcr-1. pC214 was a novel mcr-1-carrying plasmid; it contained two copies of ISAPl1 elements surrounding mcr-1, harbored additional sulII and floR resistance genes (see Fig. S2B), and shared a 25-kb backbone with a non-mcr-1-carrying plasmid identified in an S. enterica subsp. diarizonae strain (accession no. CP011293.1 ). pNG14043 displayed a genetic structure surrounding mcr-1 that was distinct from that of the other 3 plasmids, and nearly 100% of its whole sequence was found in 6 plasmids (accession no. KX772777.1 , KX236309.1 , KU761327.1 , KU743383.1 , CP015977.1 , and CP016550.1 ) in the NCBI nucleotide database, which were recovered from geographically dispersed countries, including China, Italy, the Netherlands, Estonia, and Brazil.
Genetic structures surrounding the mcr-1 gene in pP111, pR150626, pC214, and pNG14043 compared with that in pHNSHP45. Open reading frames and functions: red arrow, antimicrobial resistance and metal transporter; yellow arrow, transposition of mobile element; green arrow, virulence; blue arrow, plasmid replication; white arrow, unknown function.
In conclusion, mcr-1 was found in diverse Salmonella strains of four serovars causing infections in humans and food-producing animals in Taiwan. The resistance gene was carried on distinctly different plasmids. Among the four mcr-1-carrying conjugative plasmids characterized, pP111, a variant of pHNSHP45 (2), had disseminated in four Salmonella serovars recovered from humans, pigs, and chickens. mcr-1 was recently reported in E. coli isolates from humans and retail beef, chicken, and pork in Taiwan (11). Thus, mcr-1 may have been widespread and become prevalent in zoonotic pathogens in this country.
ACKNOWLEDGMENT
This study was funded by the Ministry of Health and Welfare, Taiwan (grant no. MOHW105-CDC-C-315-123301).
FOOTNOTES
- Received 15 February 2017.
- Returned for modification 10 March 2017.
- Accepted 10 April 2017.
- Accepted manuscript posted online 17 April 2017.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00338-17 .
- Copyright © 2017 American Society for Microbiology.