Emergence of Conjugative IncC Type Plasmid Simultaneously Encoding Resistance to Ciprofloxacin, Ceftriaxone, and Azithromycin in Salmonella

LETTER

Nontypical Salmonella is a primary cause of foodborne diseases and is considered a major public health concern worldwide (1, 2). The current choices of treatment for systemic Salmonella infections are ciprofloxacin, ceftriaxone, and azithromycin (3). Clinical Salmonella isolates resistant to these three antibiotics have been detected occasionally in recent years, but cotransfer of such three antimicrobial resistance determinants in one plasmid was rarely reported (4).

In this study, a Salmonella enterica serovar Typhimurium strain, Sa1735, which was isolated from a retail pork sample purchased from a supermarket in Shenzhen, China, was shown to be resistant to most of the antibiotics tested, including ciprofloxacin, azithromycin, and ceftriaxone, and had intermediate resistance to ofloxacin and levofloxacin but remained susceptible to amikacin, nalidixic aid, norfloxacin, kanamycin, and meropenem (Table S1 in the supplemental material). Conjugation experiments showed that phenotypic resistance to multiple antibiotics, including ciprofloxacin, ceftriaxone, and azithromycin, could be successfully cotransferred from S. enterica serovar Typhimurium Sa1735 to the recipient Escherichia coli J53 with conjugation rates of ∼1.2 × 10⁻⁶ per donor. The transconjugant exhibited sharply increased MICs for ciprofloxacin and other fluoroquinolone tested, ceftriaxone, and azithromycin, with up to 64-, 1,024-, and 128-fold increases recorded, respectively (Table S1). S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) indicated that strain Sa1735 and its transconjugant (Sa1735-TC) contained a single plasmid of ∼160 kb (Fig. S1), suggesting that genetic determinants that encode the resistance phenotypes of this strain are in this plasmid, which is designated pSa1735.

The plasmid pSa1735 was sequenced and found to be IncC type, 169,152 bp in size, and it comprised 209 predicted coding sequences (CDS) and exhibited a GC content of 52.5%. Two multiple antibiotic-resistant islands (ARI), designated ARI-A and ARI-B, were observed upstream of the genes rhi1 and parA, respectively (Fig. 1). Using BLAST against the ResFinder (5) and ISfinder (6) database showed that the genetic environments of corresponding resistant profiles were int-aac(6′)lb-cr-qnrVC1-aac(6′)lb-cr-bla_CMY-2-aadA1-dfrA14, IS26-mphA-mrx-mphR-IS6100, and merD-merB-merA-merP-merT-merR-IS4321R, which exhibited resistance to antibiotics such as ciprofloxacin, azithromycin, and heavy metal mercury. Among them, the association of three genes [aac(6′)lb-cr-qnrVC1-aac(6′)lb-cr] encoding in a colinear manner may reduce susceptibility to ciprofloxacin. Similarly, ARI-B of pSa1735 contained a total of 6 antibiotic-resistant determinants, including erm(42), floR, tet(A), strA, strB, and sul2 together in a 10 kb-region, and mediated potential resistance to erythromycin, florfenicol, tetracycline, and streptomycin. In addition, the ISEcp1-bla_CMY-2 association, which is observed in many type 1 IncC plasmids (7), can also be observed at the upstream of traA gene and exhibits resistance to the third-generation cephalosporin antibiotics such as ceftriaxone and cefotaxime.

FIG 1.

Genetic organization of representative IncC type plasmids. Liner alignment of pSa1735 and other IncC type plasmids, including pVb1796 (GenBank accession no. MH113855), pTB221 (GenBank accession no. CP032238), pEC009 (GenBank accession no. MN254970), and pSL131_IncA/C-IncX3 (GenBank accession no. MH105050) using EasyFig. Among them, horizontal arrows indicate the location, size, and orientation of predicted coding sequences (CDS) with different functions by specified colors. Red, antimicrobial resistance-associated genes; pink, genes that involve plasmid maintenance; yellow, genes that encode insertion sequences and integrases; green, genes that denote the transfer protein (tra, pil, and vir locus); blue, black, and purple, replication, regulation, and metabolism genes, respectively; blue flame in each plasmid, class I integron. The position of ARI-A and ARI-B, the ISEcp1-bla_CMY-2 islands, are indicated above. The degree of genetic similarity between the five plasmids is depicted by the shaded area, and a scale indicating the degree of similarity is shown at the bottom right.

BLASTn analysis was performed on pSa1735, with results showing that this plasmid shared 98.9% identity and 90% coverage to plasmid pVb1796 (GenBank accession no. MH113855), which was obtained from a Vibrio strain and contained an mphA-bearing transposable unit and plasmid-mediated quinolone resistance (PMQR) determinants similar to those harbored by pSa1735, but it was lacking the ISEcp1-bla_CMY-2 genetic elements. However, pVb1796 was a novel IncC plasmid with characteristics of both type 1 and type 2 plasmids (8), suggesting that these two plasmids were not likely to be shared from a common ancestor. On the other hand, we found that pSa1735 exhibited much higher homology (99.9% similarity, 91% to 96% coverage) to plasmids originated from E. coli and Salmonella, such as pTB221 (GenBank accession no. CP032238), pEC009 (GenBank accession no. MN254970), and pSL131_IncA/C-IncX3 (GenBank accession no. MH105050) (Fig. 1; Fig. S2). Among these plasmids, the backbone sequences were extremely conserved after removing the combinations of resistance profiles in the ARI-A island, indicating that it was more likely that pSa1735, together with pTB221, pEC009, and pSL131_IncA/C-IncX3, was derived from a shared ancestral plasmid. The main possible formation process of pSa1735 was an evolution event resulting from acquisition of antibiotic-resistant determinants in class 1 integron, which may also play a significant role in plasmid evolution and contribute to increased adaptation of the new bacterial host to the ever-changing environment (Fig. S3).

In conclusion, we reported the discovery of a novel type of conjugative plasmid that encodes resistance to azithromycin, ciprofloxacin, and ceftriaxone, the three primary antimicrobial agents used for treatment of salmonellosis. New antimicrobial agents need to be developed to prevent worldwide dissemination of these multidrug resistance-encoding plasmids among Salmonella and other Enterobacteriaceae species.

Salmonella strain Sa1735 was recovered from a retail pork sample collected from a supermarket in Shenzhen, China, in 2017. The species identification was verified by screening of Salmonella-specific gene invA and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker, Germany). Serotyping was then conducted according to the Kauffmann-White scheme by using a commercial antiserum (Difco, Detroit, MI).

The MICs of different antibiotics were ascertained by the broth dilution method. The breakpoints for each antibiotic were set as recommended by the Clinical and Laboratory Standards (CLSI; M100-S27) (9). Escherichia coli ATCC 25922 and S. aureus ATCC 29213 were used as the quality control strains.

The transmission of multidrug resistance of Salmonella isolate Sa1735 was assessed by performing the conjugation experiments according to filter mating experiment described previously (10). Transconjugants were selected on eosin-methylene blue agar (EMB) plates supplemented with azithromycin (16 mg liter⁻¹) and sodium azide (100 mg liter⁻¹), ciprofloxacin (0.5 mg liter⁻¹) and sodium azide (100 mg liter⁻¹), or ceftriaxone (2 mg l⁻¹) and sodium azide (100 mg l⁻¹), respectively. The transconjugants were confirmed by antimicrobial susceptibility testing and S1-PFGE as described previously. At least four transconjugant colonies were confirmed for each conjugation assay. The filter assay was repeated triple times, and the transfer efficiencies were counted as the number of transconjugants per donor.

The total genomic DNA of transconjugant Sa1735-TC was extracted using the Qiagen Plasmid Midi kit (Qiagen, Valencia, CA). The Illumina paired-end libraries were constructed with the NEBNext Ultra DNA library prep kit for Illumina (NEB). Whole-genome sequencing was performed on Illumina HiSeq 500 and Nanopore MinION long-read sequencing platform. Genome sequence was assembled with SPAdes 3.12.1 (11). Long contigs assembled from Nanopore were used to align and join contigs obtained from Illumina assembly with the CLC Genomics Workbench v10 (CLC bio, Denmark). The complete genome sequence was annotated using the Prokka server (12). EasyFig and BLAST Ring Image Generator (BRIG) were used to compare circular plasmid maps.

Data availability.

The plasmid sequencing data of pSa1735 have been deposited into GenBank under the accession number MT859309.