Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany.

ABSTRACT

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

INTRODUCTION

Foodborne salmonellosis caused by nontyphoidal Salmonella spp. is the second most commonly reported gastrointestinal infection in Europe (1, 2). The acquisition of antimicrobial resistance (AMR) genes in zoonotic Salmonella spp. might impair antimicrobial treatment and therapy in humans and animals (3). A potential reservoir of AMR genes for pathogenic bacteria persists in livestock and the environment (4). The emergence and spread of mobile resistance against the last-resort antibiotic colistin in Enterobacteriaceae are alarming and of major public health concern (5).

Mobile colistin resistance (MCR) is known to be caused by phosphoethanolamine transferases. So far, eight mcr genes termed mcr-1 to mcr-8 have been described (6–14). The mcr-5.1 gene was initially identified in d-tartrate-fermenting Salmonella enterica subsp. enterica serovar Paratyphi B variant Java (S. Paratyphi B dTA+) from Germany (10). In these strains, mcr-5 was associated with a Tn3 family transposon, later termed Tn6452 (Transposon Registry; see http://transposon.lstmed.ac.uk/tn-registry), which was either located on multicopy ColE-like plasmids or in the bacterial chromosome (10). In silico analysis of whole-genome sequencing (WGS) data provided in the NCBI database (10) and recent publications reporting the presence of mcr-5 and its variants mcr-5.2 (15) and mcr-5.3 (16) indicate a global spread of mcr-5 in various classes of bacteria from different sources (10, 15–23). So far, five mcr-5 plasmid types have been published in GenBank. Nevertheless, little is known about the transfer of mcr-5-harboring plasmids.

The aim of this study was to screen colistin-resistant Salmonella isolates from the strain collection of the German National Reference Laboratory for Salmonella (NRL-SALM) for the presence of mcr-5, to identify novel mcr-5-harboring plasmids, and to characterize these plasmids regarding their ability to spread through conjugation.

PCR screening of 315 colistin-resistant Salmonella isolates revealed that mcr-5 was harbored by eight Salmonella strains isolated from pigs and meat in five German federal states (Table 1). Based on WGS data analysis, all strains could be assigned to sequence type 34 (ST34). Serovars were predicted as biphasic or monophasic Salmonella enterica subsp. enterica serovar Typhimurium (Table 1). Particularly worrying is the presence of mcr-5 plasmids observed in three monophasic S. Typhimurium strains assigned to phage type DT193. This lineage has emerged in several European countries (24, 25). Assessment of genetic similarity using single-nucleotide polymorphism (SNP) analysis showed that with two exceptions (12-SA04158 and 13-SA00596), mcr-5-harboring Salmonella isolates are not closely (<10 SNPs) related (see Fig. S1 in the supplemental material).

TABLE 1.

Characteristics of mcr-5-harboring Salmonella Typhimurium isolates

Strain no.	Source	SeqSero serovar; seroformula	Phage type	Phenotypic resistance profilea	Corresponding resistance genes (ResFinder 3.0)	CST MIC (mg/liter)
11-03155	Pig feces	Typhimurium; 4:i:1,2	DT120	AMP, CST, SMX, TET, TMP	blaTEM-1B, mcr-5.1, sul2, sul1, tet(A), dfrA1-like	4
11-03671	Pig feces	Monophasic variant of Typhimurium; 4:i:-	DT193	AMP, CST, SMX, TET	blaTEM-1B, mcr-5.1, sul2, tet(B)	4
12-02284	Pork	Monophasic variant of Typhimurium; 4:i:-	DT193	AMP, CST, SMX, TET, TMP	blaTEM-1B, mcr-5.1, sul2, tet(B), dfrA5	4
12-02784	Pig	Typhimurium; 4:i:1,2	DT120	AMP, CST, SMX, TET, TMP	blaTEM-1B, mcr-5.1, sul2, sul1, tet(A), dfrA1-like	4
12-SA04158	Pig feces	Typhimurium; 4:i:1,2	DT120	AMP, CST, SMX, TET, TMP	blaTEM-1B, mcr-5.1, sul2, sul1, tet(A), dfrA1-like	4
13-SA00569	Pig feces	Typhimurium; 4:i:1,2	Not determined	AMP, CST, SMX, TET, TMP	blaTEM-1B, mcr-5.1, sul2, sul1, tet(A), dfrA1-like	4
13-SA02717	Gyro meat	Monophasic variant of Typhimurium; 4:i:-	Not determined	AMP, (CST),b SMX, TMP	blaTEM-1B, mcr-5.1, sul2, dfrA1	2/4b
16-SA02404	Pig feces	Monophasic variant of Typhimurium; 4:i:-	DT193	AMP, CHL, CST, SMX, TET, TMP	blaTEM-1B, cmlA1-like, mcr-5.1, sul2, sul3, tet(B), dfrA12	4

^aAMP, ampicillin; CST, colistin; SMX, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim; CHL, chloramphenicol.

^bMIC testing was performed twice, leading to different results. The original CST MIC testing result in 2013 was 4 mg/liter. The CST MIC retesting result in 2018 was 2 mg/liter. CST is therefore given in parentheses in the phenotypic resistance profile column.

The results of S1 pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and DNA-DNA hybridization experiments showed that mcr-5 was located on plasmids of various sizes (Fig. S2). Plasmid sequences extracted either from WGS data or sequencing of plasmid extractions confirmed that five different mcr-5-carrying plasmid types were observed (Table 2 and Fig. 1).

TABLE 2.

mcr-5-harboring plasmids in German Salmonella Typhimurium strains

mcr-5 plasmid designation	Reference or source	Plasmid derivatives (this study)	Strain no.	Plasmid size, Inc type, AMR gene(s)	mcr-5 locationa	GenBank accession no. (plasmid)	Transformant/transconjugant data
							Designation, conjugation frequency	COL MIC (mg/liter)
pEC0674-like	Hammerl et al. (15)	pSE11-03155, pSE12-02784	11-03155, 12-02784	6,268, not typeable, mcr-5.1	pMIC	MF684783 (pEC0674)	DH5α(pSE11-03155)	2
pSE11-03671b	This study		11-03671	8,936, ColE, mcr-5.1	pMIC	MK360094 (pSE11-03671)	DH5α(pSE11-03671)	2
pSE12-02284b	This study		12-02284c	13,989, ColE, mcr-5.1	Tn6452	MK360095 (pSE12-02284)	DH5α(pSE12-02284)	2
pSE13-SA01718-like	Borowiak et al. (10)	pSE12-SA04158, pSE13-SA00569, pSE16-SA02404	12-SA04158, 13-SA00569, 16-SA02404	12,201, ColE, mcr-5.1	Tn6452	KY807921 (pSE13-SA01718)	J53(pSE16-SA02404), 5.8 × 10−2	4
pSE13-SA02717d	This study		13-SA027171c	50,928, IncX1, mcr-5.1, intI2-dfrA1-sat2-aadA1	Tn6452 (with deletions)	MK360096 (pSE13-SA02717)	J53(pSE13-SA02717), 1.54 × 10−3	2

^aThe mcr-5 genes are either located on Tn6452 or within a putative mobile insertion cassette (pMIC).

^bPlasmid sequence obtained from short-read WGS data.

^cStrains harbor copies of Tn6452 on the respective plasmid and the chromosome.

^dPlasmid sequence obtained from short-read sequencing of plasmid extractions.

FIG 1.

Comparison of mcr-5-harboring plasmid types identified in Salmonella Typhimurium isolates using Easyfig. The nucleotide sequence identities between the plasmid sequences are indicated by color shading according to the scale for nucleotide sequence identity on the right, protein-coding genes are indicated by open arrows, and gene functions are indicated according to the gene function key on the right.

Three Salmonella isolates contained plasmids identical to the initially described ColE-like plasmid pSE13-SA01718 from avian S. Paratyphi B dTA+ (10) (Table 2). Two Salmonella isolates carried nontypeable plasmids (pSE11-03155 and pSE12-02784) which are highly similar (1 base substitution each) to plasmid pEC0674 previously observed in porcine Escherichia coli isolates from Germany (15) (Table 2). Furthermore, three novel mcr-5-harboring plasmids, pSE11-03671 (8,936 bp), pSE12-02284 (13,989 bp), and pSE13-SA02717 (50,928 bp), were observed in the remaining three monophasic S. Typhimurium isolates (Table 2 and Fig. 1).

Plasmid typing revealed that plasmids pSE11-03671 and pSE12-02284 could be typed as ColE-like plasmids, and plasmid pSE13-SA02717 belonged to the incompatibility group X1 (IncX1) and carried the dfrA1-sat2-aadA1 cassette array located in a class 2 integron (Table 2 and Fig. 1). Both Inc groups (ColE-like and IncX) play a major role in AMR-related plasmid families (26).

Filter mating studies revealed that the IncX1 plasmid pSE13-SA02717 is a self-transmissible plasmid (Table 2). Moreover, in contrast to other strains carrying nonmobilizable pSE13-SA01718-like plasmids, successful comobilization was observed for pSE16-SA02404, which was transferred together with the 115-kbp helper plasmid located in 16-SA02404 (Fig. S2 and S3 and Table 2). In general, conjugative transfer of the pSE13-SA01718-like and pEC0674-like plasmids in vivo is highly likely because these plasmids were detected in different serovars and species (10, 15).

Nonconjugative plasmids pSE11-03155, pSE11-03671, and pSE12-02284 could be successfully transferred in chemically competent E. coli DH5α (Table 2).

MIC testing results confirmed that mcr-5 location seems to have a major effect on the MIC value due to copy number effects, as previously hypothesized (10). S. Typhimurium strain 13-SA02717 carrying mcr-5 on a low-copy-number IncX1 plasmid (as indicated by the plasmid-to-chromosome coverage ratio obtained by WGS) showed a decreased colistin MIC value compared to those of the other strains harboring mcr-5 on high-copy-number plasmids (Tables 1 and 2). In filter mating studies, the uptake of the low-copy-number IncX1 plasmid pSE13-SA02717 in E. coli K-12 J53 led to a decreased colistin MIC value compared to that with the uptake of the multicopy ColE-type plasmid pSE16-SA02404 (Table 2). However, not only the mcr-5 location and copy number but also the genetic background of the host strain have an effect on colistin susceptibility, as shown in transformation experiments. The E. coli DH5α transformants generated in this study showed MIC values reduced by half (MIC, 2 mg/liter instead of 4 mg/liter) compared to those with the S. Typhimurium hosts (Tables 1 and 2). On the other side, S. Typhimurium isolates described here showed MIC values reduced by half (MIC, 4 mg/liter instead of 8 mg/liter) compared to the originally described S. Paratyphi B dTa+ isolates harboring 13-SA01718-like plasmids (10).

Analysis of the Tn6452 structure showed that pSE11-03671, like the previously published plasmid pEC0674, carried a 3,803-bp variant of Tn6452 which lacks the transposase genes and harbors imperfect Tn3-like inverted repeats (IRs) (Fig. S4). This structure was previously described as a putative mobile insertion cassette (22). In both plasmid types observed in this study, the 3,803-bp putative mobile insertion cassette is flanked by 5-bp direct repeats. These target site duplications are usually generated during the insertion of Tn3 transposons (Fig. S4). However, the mobility of these elements remains to be explored, as stated previously (22). Interestingly, the frameshift mutation in major facilitator superfamily (MFS)-type transporter gene proP, which led to a premature stop codon and resulted in two open reading frames in the Tn6452 transposon observed in pSE13-SA01718, was not detected in the 3,803-bp putative mobile insertion cassettes.

The plasmid pSE12-02284 carried a transposon nearly identical to the one observed in pSE13-SA01718 (including one base substitution). In pSE13-SA02717, a slightly different version of Tn6452 was observed due to a 208-bp deletion in the proP MFS transporter region, as well as minor base substitutions (Fig. S4).

Interestingly, expanded WGS analysis including long-read MinION data revealed that in the strains 13-SA02717 and 12-02284, an additional copy of the respective Tn6452 transposon has integrated in the bacterial chromosome (Fig. S4).

We report three novel mcr-5-carrying plasmids, the reoccurrence of two already-described plasmid types (10, 15) in S. Typhimurium, the detection of a conjugative mcr-5 plasmid, and the additional integration of Tn6452 in two bacterial chromosomes. Generally, the high variation of mcr-5-carrying plasmids in only eight analyzed Salmonella isolates as well as the presence of Tn6452 in two of the host genomes indicate a high mcr-5 mobility, an independent acquisition of mcr-5-harboring plasmids by individual strains, and a successful integration in and adaptation to different genomic environments. This is supported by the increasing number of publications reporting the presence of mcr-5 in various bacterial species worldwide (10, 15–23). However, mcr-5 is only reported sporadically, and so far, no links can be made between isolates from the environment, livestock, food, and humans. Therefore, extended mcr-5 screening in pathogens and commensal bacteria is necessary to understand the dissemination of mcr-5.

Strain selection.

This study focused on 315 colistin-resistant (MIC, >2 mg/liter) Salmonella enterica isolates from the collection of the German National Reference Laboratory for Salmonella (with exception of the previously analyzed S. Paratyphi B dTa+ isolates [10] and Salmonella spp. of serogroup D) originating from food-producing animals and food products and received between 2011 and 2017 in the framework of routine diagnostic, monitoring, and control programs.

PCR screening for mcr-5.

PCR screening on the presence of mcr-5 was performed in 25-μl PCRs containing 12.5 μl DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Carlsbad, CA, USA), 2.5 μl of each 10 μM primer dilution (MCR5_fw and MCR5_rev [10]), 5.5 μl of sterile water, and 2 μl of template DNA using thermal cell lysis preparation and PCR protocols previously described (10). Positive Salmonella isolates showing a PCR product of 1,644 bp were subjected to S1-PFGE analysis and WGS.

Characterization of mcr-5-harboring Salmonella strains.

The mcr-5-positive strains were characterized by antimicrobial susceptibility testing, phage typing, S1-PFGE, Southern blotting, and DNA-DNA hybridization experiments, as previously described (10, 24).

Whole-genome DNA preparations, plasmid extractions, library preparation, sequencing, and data assembly were performed as previously described (10). Assembly of WGS data led to draft genomes with an average coverage of 32.4 to 79.6×. Plasmid sequences were either directly derived from de novo-assembled WGS data or from data obtained by sequencing of plasmid extractions (Table 2), as previously described (10). The obtained plasmid sizes were compared with the S1-PFGE results.

Draft genomes and plasmid sequences were analyzed using the following Web tools hosted by the Center for Genomic Epidemiology (CGE) (http://www.genomicepidemiology.org): MLST 2.0 (https://cge.cbs.dtu.dk/services/MLST/), SeqSero 1.2 (https://cge.cbs.dtu.dk/services/SeqSero/), ResFinder 3.1 (https://cge.cbs.dtu.dk/services/ResFinder/), and PlasmidFinder 2.0 (https://cge.cbs.dtu.dk/services/PlasmidFinder/). PlasmidFinder results were confirmed by in silico PCR using previously described primers (10, 27, 28). Annotation of plasmids was performed using RASTtk (29). Plasmid comparison studies were carried out using Easyfig (30). Whole-genome SNP analysis and clustering of isolates were performed as previously described (10) using the chromosome of S. Typhimurium isolate SO4698-09 (GenBank accession no. LN999997.1) as a reference. Selected strains were also sequenced using Oxford Nanopore MinION technology. MinION libraries were prepared from a genomic DNA extraction using the Rapid barcoding kit (Oxford Nanopore Technologies, Oxford, UK), following the manufacturer’s instructions, and sequenced for approximately 16 h using a FLO-MIN106 R9 flow cell. For genome assembly, the hybrid assembly software Unicycler (version 0.4.4) was used (31, 32). Alignment of mcr-5 genomic environments was carried out using CLC Genomics Workbench 9.5.2 (Qiagen, Hilden, Germany).

Filter mating conjugation and transformation experiments.

Selected mcr-5-carrying strains were used as a donor for filter mating conjugation studies using sodium azide-resistant E. coli K-12 J53 recipient cells. Filter mating conjugation was performed as previously described (33). The reaction mixtures were plated on transconjugant selective plates containing 1 mg/liter colistin sulfate and 100 mg/liter sodium azide (Sigma-Aldrich, Darmstadt, Germany) and incubated for approximately 42 h at 37°C. The average conjugation frequency was determined as the average number of transconjugants obtained per donor cell in two individual experiments, each performed in triplicate. Transformation experiments were performed as previously described (10), and transformants were selected on LB agar plates containing 1 mg/liter colistin sulfate. Selected transformants and transconjugants were confirmed by mcr-5 PCR and S1-PFGE and subjected to antimicrobial susceptibility testing.

Data availability.

The NCBI GenBank accession numbers of the novel plasmids are listed in Table 2. Raw sequence data can be found in the NCBI BioProject database (accession no. PRJEB30493).