Diverse and Flexible Transmission of fosA3 Associated with Heterogeneous Multidrug Resistance Regions in Salmonella enterica Serovar Typhimurium and Indiana Isolates

TEXT

Bacterial resistance to antibiotics is a global problem, and the increase in multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacterial strains places an enormous burden on clinicians treating these infections. Fosfomycin, a decades-old antibiotic, has recently been reintroduced into clinical use because of its perceived activity against MDR and XDR Gram-negative pathogens, including extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae (1). Mutations in chromosomal genes are the main mechanism for fosfomycin resistance in Enterobacteriaceae (2). However, the most prevalent plasmid-borne fosfomycin resistance gene, fosA3, was implicated in the recent increased occurrence of fosfomycin resistance among Enterobacteriaceae, especially Escherichia coli isolates from diverse origins that included animals, retail meat, and people in East Asia (3). The high prevalence of fosA3 in E. coli strains from these sources did not exclude its absence from major foodborne pathogens, such as Salmonella spp. (4–6). However, there is little information regarding the transmission of fosA3 in the latter group (7–9). In the present study, we investigated the prevalence of fosA3 among Salmonella isolates from food animals and characterized the plasmids and the chromosomal genomes containing fosA3.

A total of 310 Salmonella enterica strains, including serotypes Typhimurium (n = 100), Indiana (n = 28), and others (n = 182), were obtained from food animals from livestock farms and animal clinics in China between 2016 and 2017, as previously described (10). The MICs of fosfomycin were determined by the agar dilution method on Mueller-Hinton agar containing 25 μg/ml glucose 6-phosphate, and the results (resistant breakpoint, ≥256 μg/ml) were interpreted according to guideline M100-S25 of the Clinical and Laboratory Standards Institute (11). Fosfomycin-resistant isolates (>512 μg/ml) were screened for the fosA3, fosA, and fosC2 genes by PCR, as previously described (4). Eleven isolates (3.5%) were fosfomycin resistant (>512 μg/ml), and 8 (2.6%) were positive for fosA3. None of these isolates contained fosC2 or fosA (Table 1). This detection rate was consistent with the sporadic occurrence of fosA3 in Salmonella isolates (7).

Pulsed-field gel electrophoresis (PFGE) (12) was successfully performed on the 8 FosA3 producers, and 7 distinct Xba I-PFGE patterns were observed (see Fig. S1 in the supplemental material). MIC values for the 8 fosA3-positive isolates indicated that all were resistant to ampicillin and cefotaxime, and 7 were resistant to florfenicol, olaquindox, tetracycline, and sulfamethoxazole-trimethoprim (see Table S1 in the supplemental material). In addition, some of these isolates were resistant to colistin (n = 5), ciprofloxacin (n = 5), gentamicin (n = 2), and amikacin (n = 1). We then screened the isolates for the presence of bla_CTX-M-1G, bla_CTX-M-9G, rmtB, oqxA, oqxB, aac-(6′)-Ib-cr, floR, and mcr-1 by PCR amplification and sequencing as previously described (13, 14). Five S. Typhimurium isolates harbored fosA3, bla_CTX-M-14, mcr-1, oqxAB, and floR, and 1 harbored fosA3 and bla_CTX-M-55. The fosA3 genes were cotransferred with bla_CTX-M-14-mcr-1-oqxAB/bla_CTX-M-55 among the 6 fosA3-positive S. Typhimurium isolates through conjugation or transformation assays (4, 10). Each transconjugant/transformant carried a single fosA3-positive plasmid, and these plasmids were assigned to ST3-IncHI2 (∼250 kb) (n = 5) and F33:A−:B− (∼90 kb) (n = 1) by plasmid replicon typing (15–17), S1-PFGE, and Southern blot analysis (18) (see Fig. S3 in the supplemental material and Table 1). The S. Indiana strain FJC33 harbored diverse antibiotic resistance genes (ARGs), including fosA3, rmtB, bla_CTX-M-55, oqxAB, aac-(6′)-Ib-cr, and floR, as previously described (10); whereas S. Indiana strain GDD125 carried fosA3, oqxAB, and floR. The fosA3 genes in these 2 S. Indiana isolates could not be transferred by conjugation or transformation in spite of repeated attempts. A chromosomal location of fosA3 for strain FJC33 and an ∼216-kb plasmid location for strain GDD125 were confirmed using S1-PFGE and Southern blot analysis (18) (see Fig. S2 in the supplemental material).

Based on the results of plasmid analysis, 4 fosA3-bearing plasmids (pGDD27-24, pGDP37-4, pGDP25-25, and pJXP9) were randomly selected from the corresponding transconjugants/transformants and subjected to complete sequencing using the Illumina MiSeq platform as described previously (19). After filtering E. coli J53 chromosomal DNA data, the remaining reads were assembled by Velvet into contigs, and the gaps were closed by PCR mapping using the corresponding reference sequences pHN7A8 (GenBank accession no. JN232517) and pHNSHP45-2 (GenBank accession no. KU341381). Gene prediction and annotation were performed using RAST and BLAST tools. The ARGs and plasmid replicon types were analyzed using the CGE server (https://cge.cbs.dtu.dk/services/). The sequence comparison and map generation were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and Easyfig (version 2.1).

Plasmid pGDD27-24 belonged to the F33:A−:B− type and contained 121 coding sequences (CDS) with a size of 97,614 bp. Sequence comparison analysis showed that the backbone sequence of pGDD27-24 was almost identical to those of the fosA3-bearing F33:A−:B− plasmids pHN7A8, pHNEC55 (GenBank accession no. KT879914), and pHNFP460-1 (GenBank accession no. KJ020575) from animal E. coli isolates from China (Fig. 1a). These four F33:A−:B− plasmids contained ∼60-kb conserved backbone regions, and the primary differences were within the regions located between pemKI and ycdA. Plasmid pGDD27-24 identified in this study possessed an ∼38.1-kb insertion between these two genes, which contained an important genetic structure, i.e., IS26-tetR-orf2-ofr1-fosA3-IS26-bla_TEM-1-orf477-bla_CTX-M-55-ΔISEcp1 (∼7.7 kb).

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FIG 1

Characteristics of the complete nucleotide sequences of four fosA3-carrying plasmids identified in this study. (a) Linear comparison of F33:A−:B− plasmid pGDD27-24 with another three fosA3-carrying plasmids from the GenBank database. (b) Comparison with three ST3-IncHI2 plasmids bearing fosA3 identified in this study with another two fosA3-carrying IncHI2 plasmids pHNSHP45-2 (KU341381) and pA3T (KX421096) from the GenBank database.

Plasmids pGDP37-4, pGDP25-25, and pJXP9 belonged to the ST3-IncHI2 type and contained 230 to 240 CDSs with sizes of 249,627, 257,318, and 244,799 bp, respectively. All of these plasmids showed extremely high similarity to fosA3-carrying ST3-IncHI2 plasmids pHNSHP45-2 (E. coli, pig, China) and pA3T (GenBank accession no. KX421096, S. Indiana, chicken, China) and possessed typical and conserved IncHI2 backbones (Fig. 1b). The primary differences of the 5 plasmids were located between the umuC and dcm genes that contained a large multidrug resistance region (MRR). The 5 MRRs also showed high similarity to each other, and important structural units were frequently present, including IS26-bla_CTX-M-14-fosA3-IS26 (ΔIS26) (n = 4), IS26-oqxAB-IS26 (n = 5), and rcr2-floR-virD2 (n = 5) (see Fig. S4 in the supplemental material). In addition to the MRR, the structural unit ISApl1-mcr-1-pap2 was inserted downstream of terF (n = 4) (Fig. 1b). Taken together, these findings indicated that fosA3-carrying ST3-IncHI2 and F33:A−:B− plasmids were a cluster of plasmids with similar backbones but various MRRs that disseminated fosA3 in E. coli and Salmonella isolates in China.

The S. Indiana strain FJC33 carrying the chromosomal fosA3 was sequenced using a combined MiSeq/PacBio RSII single-molecule real-time (SMRT) sequencing platform. Genome assembly was performed with Unicycler version 0.4.1 (20) using a combination of short and long reads, followed by error correction by Pilon version 1.12 (21). Gene prediction and annotation and ARG analysis were carried out as described above. The complete nucleotide sequence of FJC33 revealed that it contained one 4.941-Mb chromosome and lacked any plasmid contigs. This was consistent with the lack of plasmids according to our S1-PFGE analysis (see Fig. S2). Strain FJC33 harbored 22 ARGs, including fosA3-rmtB-bla_CTX-M-55-oqxAB-aac-(6′)-Ib-cr-floR described above (see Table S2 in the supplemental material). Sequence analysis of S. Indiana strain FJC33 (ST17) showed high similarity to the XDR ST17 S. Indiana strain C629 (GenBank accession no. CP015724) from a chicken carcass in China (68% query coverage, 99% overall nucleotide identity), and it differed by containing 3 insertions of ∼60 kb each (Fig. 2a). In particular, 21 of the 22 ARGs present in FJC33 were contained in insertion regions I and II.

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FIG 2

Characteristics of the chromosomal genome sequence of S. Indiana FJC33 carrying chromosomally encoded fosA3 identified in this study. (a) Comparison with the whole chromosomal genome sequence of S. Indiana FJC33 carrying chromosomally encoded fosA3 identified in this study with that of S. Indiana C629 (accession no. CP015724) from the GenBank database. The figure was drawn using Mauve software. (b) Comparison of the ∼293.6-kb chromosomal region containing insertion regions I and II from S. Indiana FJC33 with the corresponding chromosomal region from S. Indiana C629 (accession no. CP015724). The two integrase genes are in dark red. (c) Comparison of the large chromosomal fosA3-containing-MDR region from S. Indiana FJC33 with the corresponding regions from three IncHI2 plasmids from the GenBank database.

Insertion region I possessed two distinct modules that were identified by comparison with related segments (Fig. 2b). Module II (∼17.1 kb) contained 14 open reading frames (ORFs) and was almost identical to the corresponding chromosomal region from a duck pathogenic E. coli O157:H16 isolate from China (GenBank accession no. CP007592). In contrast, module I (∼47.7 kb) contained a large fosA3-rmtB-oqxAB-containing MRR composed of seven structural units, and each was bracketed by two intact IS26 elements. The latter included IS26-oqxAB-IS26, IS26-tetR-orf2-ofr1-fosA3-IS26, and IS26-tnpR-bla_TEM-1-rmtB-IS26 (Fig. 2c). Interestingly, the former two units have frequently been reported on IncHI2 plasmids (6, 22, 23). Module I resembled an analogous region on the IncHI2 plasmid pC629 (GenBank accession no. CP015724) (24), although it contained inversions, deletions, and rearrangements. In addition, partial fragments of module I were found on other IncHI2 plasmids, such as 12051p1 (GenBank accession no. CP012141) and pP2-3T (GenBank accession no. MG012722) (Fig. 2c). Actually, module I mimicked the multidrug resistance clusters on IncHI2 plasmids, in which IS26 played a central role in shaping their MRRs (23, 25). Together these results indicated that the chromosomal fosA3-rmtB-oqxAB-containing MRR identified in S. Indiana strain FJC33 probably resulted from the acquisition of genetic structures that originated from plasmids, especially MDR IncHI2 type. We also found that an integrase gene was present at the left end of the chromosomal fosA3-rmtB-oqxAB-containing MRR that was also present downstream of an ∼22.8-kb phage-related element-containing region, including genes for phage head and tail assembly. Furthermore, an ∼70.5-kb region containing the ∼22.8-kb region and a large partial region of the fosA3-rmtB-oqxAB-containing MRR was identified as an intact prophage (http://phast.wishartlab.com) (Fig. 2b). Thus, the large fosA3-rmtB-oqxAB-containing MRR was probably integrated into the chromosome of the S. Indiana FJC33 by prophages integration.

Insertion region II (∼63.7 kb) was almost identical to the corresponding region on the chromosome of E. coli strain H9 isolated from a mink farmer in China (GenBank accession no. CP029180; 98% query coverage, 100% overall nucleotide identity). Unexpectedly, several acquired ARGs were embedded into the ∼63.7-kb region, and these contained the genetic structure ISEcp1-bla_CTX-M-55 (∼2.3 kb) and an MRR (∼8.4 kb) composed of five ARGs (Fig. 2b). Coincidentally, the other phage integrase gene was found at the right end of insertion region II. Prophage-mediated chromosomal integration of this region from E. coli into S. Indiana is a distinct possibility for this transfer. Indeed, prophages may play crucial roles in transferring chromosomal ARGs, along with long surrounding DNA, between bacteria without direct cell-cell interaction (26). This contributes to the emergence and spread of antibiotic-resistant bacteria.

In conclusion, this study revealed that the fosA3 gene was cospread with diverse important ARGs, including mcr-1, bla_CTX-M-14/55, oqxAB, and rmtB mediated by ST3-IncHI2 and F33:A−:B− plasmids and the chromosome, each of which possessed a heterogeneous fosA3-containing MRR, in S. Typhimurium and S. Indiana isolates from food animals in Guangdong province, China. These findings indicate a high flexibility of fosA3 cotransmission with multiple important ARGs among Salmonella strains. To the best of our knowledge, this is the first report of the complete sequence of a Salmonella chromosome simultaneously containing fosA3-rmtB-oqxAB embedded in a novel MRR. Great concern should be taken for cospread of fosA3 with multiple ARGs in Salmonella spp.