Emergence of the Phenicol Exporter Gene fexA in Campylobacter coli and Campylobacter jejuni of Animal Origin

LETTER

Bacteria of the genus Campylobacter are major foodborne pathogens that have become increasingly resistant to clinically important antimicrobial agents (1). In China, Campylobacter jejuni is the main cause of human Campylobacter infections, whereas Campylobacter coli isolates exhibit a distinctly higher multidrug resistance rate. Fluoroquinolones and macrolides are the most frequently used antimicrobial agents to treat Campylobacter infections (2). Florfenicol has been widely used in food animals to treat bacterial infections for many years (3). Although it was not the treatment of choice for Campylobacter infections, the resistance rate of florfenicol in Campylobacter spp. of animal origin in China has increased in the past few years (4). The resistance-enhancing efflux pump CmeABC (RE-CmeABC) and the rRNA methyltransferase Cfr(C) were the only two identified transferable resistance proteins that conferred low-level florfenicol resistance (MIC, ≤16 mg/liter) in Campylobacter (5, 6). In this study, we identified for the first time the florfenicol resistance gene fexA in Campylobacter spp. of animal origin and investigated its genetic environment.

We recovered 1,088 Campylobacter isolates (157 C. jejuni and 931 C. coli) from Shandong, Shanghai, and Guangdong provinces in 2014 to 2016 during our annual laboratory antimicrobial resistance surveillance program for Campylobacter from chickens and swine in China (4). The C. coli isolates were obtained from 803 chickens and 128 pigs, and the C. jejuni isolates were isolated from 151 chickens and 6 pigs. Antimicrobial susceptibility testing was conducted as described previously (6). We obtained 238/1,088 (21.9%) florfenicol-resistant Campylobacter, 90 and 148 of which were PCR positive and negative for RE-cmeABC (5), respectively. C. coli strain 16SHKX65C isolates were randomly selected from the 148 RE-cmeABC-negative isolates. This isolate originated from a chicken in Shanghai in 2016 and exhibited resistance to erythromycin (128 mg/liter), ciprofloxacin (256 mg/liter), clindamycin (≥512 mg/liter), gentamicin (≥512 mg/liter), tetracycline (256 mg/liter), chloramphenicol (128 mg/liter), and florfenicol (64 mg/liter). Genomic DNA was subjected to whole-genome sequencing using a combination of the Illumina HiSeq 2000 system (Sinobiocore, Beijing, China) and the PacBio RSII system (Sinobiocore) as previously described (7). The results revealed that the isolate was positive for the phenicol exporter gene fexA, which confers resistance to florfenicol (8). A 1,987-bp segment, including an intact copy of fexA and its putative promoter, was cloned into the vector plasmid pRRK using primers VfexA-F (5′-GGAGCTAAAGAGGTCCCTATCTAGATGGAAACACTACCGGAAAAC-3′) and VfexA-R (5′-TATCAATTTACTATAAATTCTCTAGAGCATATCTGGGTGAGAAAC-3′) at an annealing temperature of 65°C (9). The recombinant plasmid was then transferred into C. jejuni strain NCTC 11168 using natural transformation. The florfenicol MIC values of the C. jejuni transformants indicated that fexA could increase them 16-fold compared with those of the recipient strain (from 1 to 16 mg/liter), although this MIC value was 2 dilution steps lower than that of the wild-type C. coli 16SHKX65C (see Table S1 in the supplemental material). This difference in the florfenicol MIC may suggest that (i) fexA is subject to a differential expression in C. coli and C. jejuni or (ii) an additional, still-unknown florfenicol resistance mechanism is present in C. coli 16SHKX65C isolates.

The size of the genome of C. coli 16SHKX65C was 1,728,473 bp, and this isolate did not harbor plasmids. The fexA gene was located within a 23-kb multidrug resistance genomic island (MDRGI), which was inserted into the intergenic region between cj1528 and moeA2 of C. jejuni strain NCTC 11168 (Fig. 1). The MDRGI contained 17 complete open reading frames (ORFs) and 4 truncated ORFs. The MDRGI could be divided into two parts. The left half exhibited high nucleotide sequence similarity (>99%) to the corresponding region of C. jejuni strain 8C/36C (GenBank accession no. MH257907), which harbored the macrolide-lincosamide-streptogramin B (MLS_B) resistance gene erm(B), the streptomycin resistance gene aadE, and the tetracycline resistance gene tet(O), isolated from Guangdong province, China, in 2016 (10). The right half consisted of the phenicol resistance genes cat_pC194 and fexA and the tetracycline resistance gene tet(L), as well as two intact copies of IS1216E located in the same orientation located up and downstream of the fexA gene (Fig. 1a). This part of the MDRGI did not show striking similarity to any sequences deposited in GenBank. Solely, the insertion sequence IS1216E elements and the fexA gene were indistinguishable from sequences of other bacterial species, such as Enterococcus faecalis plasmid pE508 (GenBank accession no. MK425645) isolated from Henan province, China, in 2015 (Fig. 1a) (11).

FIG 1.

(a) Comparison of the genetic environment of fexA in Campylobacter. Pink, insertion sequences. Antimicrobial resistance genes: red, phenicol resistance; yellow, macrolide resistance; green, aminoglycoside resistance; purple, tetracycline resistance. Gray, genes with predicted functions; white, hypothetical protein genes. Black arrows, genes at both termini of the MDRGIs. Gray shading, regions sharing >98% DNA identity. (b) Prevalence of fexA-positive Campylobacter isolates in 3 successive years, 2014 to 2016.

A 3,724-bp amplicon was generated by PCR (primer-L, 5′-CCATTCCGACACCAACCT-3′; primer-R, 5′-CCATTCCGACACCAACCT-3′; annealing temperature, 54°C), and sequence analysis revealed that it included the fexA gene, two genes for hypothetical proteins, and one copy of IS1216E (IS1216E-hp-hp-fexA). The circular intermediate (also known as translocatable unit) is likely to result from the recombination of the two IS1216E copies. This observation suggested that IS1216E may play an important role in the transmission of fexA (Fig. 1a).

The screening of all 1,088 Campylobacter isolates collected from 2014 to 2016 for the presence of fexA was conducted using PCR (8). A total of 45 (4.1%) of these isolates were positive for fexA, including 6 C. jejuni and 39 C. coli isolates (Fig. 1b). Among them, 42 isolates mediated resistance to florfenicol (MIC, ≥8 mg/liter), of which 38 were negative for RE-cmeABC and cfr(C). There was no significant difference in the fexA carriage rate between C. jejuni and C. coli isolates in the 3 successive years, whereas a significant increase was found between 2015 and 2016 (P < 0.001, χ² test).

In conclusion, we report the emergence of the florfenicol exporter gene fexA in C. coli and C. jejuni isolates. The fexA gene was located in the MDRGI together with the MLS_B resistance gene erm(B), streptomycin resistance gene aadE, chloramphenicol resistance gene cat_pC194, and tetracycline resistance genes tet(O) and tet(L). Both macrolides and aminoglycosides represent extensively used antibiotics in the treatment of human campylobacteriosis. Therefore, the widespread use of florfenicol in food animals may play a role in the coselection of resistance to critically important antibiotics, such as macrolides and aminoglycosides, used in human medicine and therefore pose a threat to public health. Enhanced surveillance is needed to monitor the dissemination of fexA and other florfenicol resistance genes in members of the genus Campylobacter.

Accession number(s).

The sequence of C. coli 16SHKX65C has been deposited in GenBank under accession number SAMN11316573.