Genetic Relationships among Multidrug-Resistant Salmonella enterica Serovar Typhimurium Strains from Humans and Animals

ABSTRACT

We identified 20 to 22 resistance genes, carried in four incompatibility groups of plasmids, in each of five genetically closely related Salmonella enterica serovar Typhimurium strains recovered from humans, pigs, and chickens. The genes conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, fluoroquinolones, extended-spectrum cephalosporins and cefoxitin, and azithromycin. This study demonstrates the transmission of multidrug-resistant Salmonella strains among humans and food animals and may be the first identification of mphA in azithromycin-resistant Salmonella strains in Taiwan.

TEXT

Salmonella enterica serovar Typhimurium is a prevalent zoonotic pathogen with a broad host range and causes infections in humans mainly via fecal-oral routes. In Taiwan, S. Typhimurium was the second most frequently isolated serovar to cause human salmonellosis (1). Of S. Typhimurium isolates recovered from humans during 1998 to 2002, 72.7% were found to be ACSSuT-resistant (resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline), but no isolate resistant to third-generation cephalosporins was found (2). Isolates recovered during 2009 and 2010 maintained high ACSSuT resistance, and more than 10% of the isolates developed resistance to cefotaxime. Isolates from Taiwan displayed a significantly higher level of resistance to all the tested antimicrobials (except two carbapenems) than those recovered in Denmark during the same period (3). Genetic analysis of the isolates revealed four multidrug-resistant (MDR) clusters: C (ASSuT-resistant clone, resistant to ampicillin, streptomycin, sulfisoxazole, and tetracycline), E and F (ACSSuT-resistant clone), and G. Cluster G showed high levels of resistance to cefotaxime, ceftazidime, nalidixic acid, gentamicin, ACSSuT, and trimethoprim (3). The ASSuT-resistant clone and clone G were found to be the primary S. Typhimurium clones to infect pigs during 2011 and 2012, and almost all the clone G strains were resistant to the third generation of cephalosporins, cefotaxime and ceftazidime (1). ASSuT- and ACSSuT-resistant clones were widespread among continents, and the resistance mechanisms have been well elucidated (4, 5). However, the resistance mechanism for the clone G strains remains to be investigated.

Our previous study found that clone G was transmitted among humans and food animals and exhibited relatively varied resistance patterns (3). In the present study, we selected five highly resistant strains of clone G and identified the resistance genes and the vehicles for the resistance genes to investigate the genetic relationships among MDR strains recovered from humans and food animals.

We obtained Salmonella isolates from hospitals and the Department of Veterinary Medicine at National Chia-Yi University that had been recovered from humans, pigs, and chickens in central and southern Taiwan between 2009 and 2013. The strains were subjected to genotyping using the PulseNet standardized pulsed-field gel electrophoresis (PFGE) protocol (6), serotype determination using both PFGE pattern comparison and the traditional method (7), and antimicrobial susceptibility testing using the broth microdilution method and custom-made 96-well Sensititre MIC panels (Trek Diagnostic Systems, Ltd., West Essex, United Kingdom). We selected for whole-genome analysis five highly resistant S. Typhimurium strains (BL10, BL33, CS058, SD09.161, and CD13.012), which were recovered from humans, pigs, and chickens between 2009 and 2013 and were resistant to 12 of the 14 antimicrobials tested (Fig. 1).

FIG 1.

Dendrogram of S. Typhimurium strains constructed with whole-genome SNP (wgSNP) profiles using the categorical (value) similarity coefficient and the single-linkage clustering algorithm. The complete genomic sequence of S. Typhimurium strain LT2 was used as the reference for SNP calling by using the option of strict SNP filtering (closed SNP set). Red boxes, resistance; green boxes, susceptibility.

We obtained whole-genome sequence data for the five strains by using the Illumina MiSeq platform (2 by 300 bp) and obtained sequence data of long reads for strain BL10 by using the PacBio RSII platform (Pacific Bioscience, Inc., Menlo Park, CA). We used the CLC Genomics Workbench software (Qiagen Bioinformatics, Germany) to assemble the sequence reads for all strains, assembled the PacBio long reads for strain BL10, mapped the scaffold sequences of the chromosome and plasmids for strain BL10 with the Illumina reads and contigs, and filled the gaps using PCR and Sanger sequencing techniques.

We identified the resistance genes and the incompatibility groups of the plasmids using the ResFinder and PlasmidFinder tools provided by the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/). We annotated the complete chromosome and plasmid sequences of strain BL10 by using RAST (http://rast.nmpdr.org/) (8), and we employed the complete plasmid sequences of strain BL10 as references to compare the plasmid sequences of the other four strains by using BRIG (http://brig.sourceforge.net/) (9). We determined the sequence type (ST) and constructed a dendrogram for S. Typhimurium strains with whole-genome single nucleotide polymorphism (wgSNP) profiles by using the analysis pipelines provided by BioNumerics version 7.6.2 (Applied Maths, Inc.).

We conducted conjugation experiments to transfer the resistance gene-carrying plasmids from strain BL10 into Escherichia coli C600 recipients and used LB medium with 2,000 mg/liter streptomycin and 50 mg/liter ampicillin for the selection of p113k-harboring transconjugants or 2,000 mg/liter streptomycin and 50 mg/liter chloramphenicol for the selection of p220k-harboring transconjugants.

The five S. Typhimurium strains with resistance to 12 of 14 antimicrobials tested (Fig. 1), namely, BL10, BL33, CS058, CD13.012, and SD09.161, were identified with 20 to 22 resistance genes and 4 to 5 incompatibility plasmid types from the whole-genome sequence data. The resistance genes were aac(3)-IId, aac(3)-IVa, aadA1, aadA2, aadA22, aph(3′)-IIa, aph(4)-Ia, sat, strA, strB, cmlA1, floR, sul1, sul2, sul3, dfrA12, tetA, qnrS1, oqxA, oqxB, bla_CMY-2, and mphA, which conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, nalidixic acid, fluoroquinolones, cefoxitin and extended-spectrum cephalosporins, and azithromycin (Table 1). Strains BL10 and BL33 recovered from pigs harbored all 22 resistance genes, whereas CS058 recovered from chickens harbored 21 genes (missing floR); moreover, CD13.012 and SD09.161 from humans harbored 20 genes [missing aac(3)-IId and aadA22]. No mutation was found in the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE. The five strains belonged to ST36 and differed from each other by 11 to 41 SNPs; they were genetically distant from the ACSSuT-resistant (ST19) clone and the ASSuT-resistant (ST34) clone (Fig. 1).

TABLE 1.

Plasmids and resistance genes present in five S. Typhimurium strains

Plasmid and resistance gene	Antimicrobial(s) to which strain is resistant	Presence of plasmid and type of resistance in straina:
		BL10	BL33	CS058	CD13.012	SD09.161
p220k (IncHI2)		+	V	V	V	V
aac(3)-IVa	Gentamicin, tobramycin	+	+	+	+	+
aadA1	Streptomycin, spectinomycin	+	+	+	+	+
aadA2 (2)*	Streptomycin, spectinomycin	+	+	+	+	+
aph(3')-IIa	Kanamycin, neomycin, paromycin, gentamicin B	+	+	+	+	+
aph(4)-Ia	Hygromycin B	+	+	+	+	+
cmlA1	Chloramphenicol	+	+	+	+	+
dfrA12	Trimethoprim	+	+	+	+	+
floR	Chloramphenicol	+	+	−	+	+
mphA	Azithromycin	+	+	+	+	+
oqxA	Quinolones	+	+	+	+	+
oqxB	Quinolones	+	+	+	+	+
sat	Streptothricin	+	+	+	+	+
strA [aph(3'')-Ib]	Streptomycin	+	+	+	+	+
strB [aph(6)-Id]	Streptomycin	+	+	+	+	+
sul1	Sulfonamides	+	+	+	+	+
sul3	Sulfonamides	+	+	+	+	+
p113k (IncI1)		+	V	V	V	V
aac(3)-IId	Gentamicin, tobramycin	+	+	+	−	−
aadA22	Streptomycin, spectinomycin	+	+	+	−	−
blaCMY-2	Cefotaxime, ceftazidime, cefoxitin	+	+	+	+	+
p11k (IncQ1)		+	V	V	V	V
strA [aph(3'')-Ib]	Streptomycin	+	+	+	+	+
strB [aph(6)-Id]	Streptomycin	+	+	+	+	+
sul2	Sulfonamides	+	+	+	+	+
tetA	Tetracycline	+	+	+	+	+
p10k (ColRNAI-like)		+	V	V	V	V
qnrS1	Quinolones	+	+	+	+	+
p3.8k (ColRNAI-like)		+	V	−	−	V

^aV, variant; +, present; −, absent.

The complete whole-genome sequence of strain BL10 was determined. The chromosome was 4,835,517 bp in size; no horizontally transferable resistance gene was carried in the chromosome. Strain BL10 harbored five plasmids, which were designated p220k (220,023 bp; incompatibility group IncHI2), p113k (112,565 bp; IncI1), p11k (11,020 bp; IncQ1), p10k (10,047 bp; putative ColRNA1), and p3.8k (3,830 bp; putative ColRNA1). Plasmid p220k harbored 17 (16 different) resistance genes, which were distributed across 3 distinct regions (Fig. 2A; Table 1). The locations of resistance genes were accompanied by complete and partial sequences of insertion sequence elements—including IS10, IS26, IS440, IS1294, IS6100, ISEc57, ISCR2 (partial), and ISEcp1 (partial)—and partial sequences of class I transposon Tn5 and class II transposons Tn1721 and Tn5393. Plasmid p220k was an IncHI2 plasmid and contained a number of conjugation genes. Conjugation experiments proved that p220k and p113k could be transferred into E. coli strain C600. Plasmid p220k shared 72% sequence identity with an IncHI2 plasmid, pC629 (GenBank accession no. CP015725.1), which harbored 21 resistance genes, 9 of which were found in p220k. Strains BL33, CS058, CD13.012, and SD09.161 each carried a p220k variant with a few deletions (Fig. 2A). The sequence region with floR was missing in the p220k variant in strain CS058.

FIG 2.

Genetic maps of plasmids p220k (A) and p113k (B) in S. Typhimurium strain BL10 and their variants in strains BL33, CS058, CD13.012, and SD09.161.

Plasmid p113k carried three resistance genes: aac(3)-IId, aadA22, and bla_CMY-2 (Table 1). Plasmid p113k shared 90% sequence identity with plasmids pEC974-1 (accession no. CP021841.1) and pEC1515-1 (accession no. CP021841.1) and 70% sequence identity with plasmid pCMY2 (accession no. LC019731.1) found in E. coli. These four plasmids all carried bla_CMY and shufflon genes (10). Strains BL33, CS058, CD13.012, and SD09.161 each harbored a p113k variant. The p113k variants in strains CD13.012 and SD09.161 displayed a large sequence deletion that resulted in the loss of aac(3)-IId and aadA22. In addition, bla_CMY-2, encoding an AmpC-type β-lactamase, was located in a conserved ISEcp1-bla_CMY-2-blc-sugE segment; this ISEcp1-mediated transposon-like element is named Tn6092 (11). Research has associated bla_CMY-2 with an increase in ceftriaxone resistance in salmonellae in Taiwan and has reported it to be widespread among other Enterobacteriaceae (12, 13).

Plasmid p11k carried four resistance genes, namely, strA, strB, sul2, and tetA, and p10k carried qnrS1. All five strains carried both p11k and p10k, and each plasmid in the five strains shared nearly 100% sequence identity. This qnrS1-carrying plasmid and its variant have been found in several countries (14–16).

In a previous study, we found that an MDR S. enterica serovar Braenderup clone displayed relatively varied resistance patterns resulting from deletions in regions with resistance genes in a large plasmid (17). We previously found that strains of the cluster G group displayed diverse resistance patterns (3); therefore, we expected that the resistance genes could be carried by plasmids. As expected, we found that all the resistance genes in the five S. Typhimurium strains were carried by plasmids in this study.

The resistance genes found in the five strains were concordant with the results of antimicrobial susceptibility testing (Table 1 and Fig. 1). Because the strains harbored an AmpC gene, bla_CMY-2, they were resistant to the tested antimicrobials, including ampicillin, cefoxitin, ceftazidime, and cefotaxime. The strains also displayed a low level of ciprofloxacin resistance (MIC, 1 to 4 mg/liter), because they had no mutation on gyrase and topoisomerase IV and harbored only qnrS1 and oqxAB, which confer only low levels of resistance to fluoroquinolones.

Notably, the five strains harbored sat and mphA, which confer resistance to streptothricin and azithromycin. Streptothricin has been used as a growth promoter in animal feed. A streptothricin, nourseothricin, was used as in East Germany as a growth promoter from 1983 to 1990, which resulted in the emergence and spread of a transposon-mediated streptothricin resistance gene, sat (18). In Taiwan, an integron-mediated gene, namely, sat, was previously identified in S. enterica serovar Choleraesuis (19). The prevalence of sat and the reason for its emergence in salmonellae in Taiwan warrant further investigation. mphA encodes a macrolide 2′-phosphotransferase that can deactivate azithromycin. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug for the treatment of invasive Salmonella infection (20). Salmonella strains resistant to azithromycin associated with mphA have been reported in many countries (21–23), but not in Taiwan. This is the first mphA detection in Salmonella strains in this country.

In conclusion, our study indicates that five highly resistant S. Typhimurium strains, despite recovery from humans and different food animals, are genetically closely related and harbor 20 to 22 common resistance genes located in 4 incompatibility types of plasmid in each strain. These strains are resistant to the principal drugs for treatment of invasive Salmonella infections, including chloramphenicol, fluoroquinolones, extended-spectrum cephalosporins, and azithromycin. Because the highly resistant strains may have propagated, intensive surveillance and control measures are required in this country.

Accession number(s).

The complete chromosome and plasmid sequences of S. Typhimurium strain BL10 have been submitted to the GenBank database of the National Center for Biotechnology Information under the following accession numbers: CP024619.1 (chromosome), CP025340.1 (p220k), CP025339.1 (p113k), CP025338.1 (p11k), CP025337.1 (p10k), and CP025336.1 (p3.8k).