Coexistence of optrA and fexA in Campylobacter

Florfenicol is widely used for the treatment of respiratory infections and as a feed additive in food animal production. As a foodborne pathogen, Campylobacter is constantly exposed to florfenicol, and resistance to this antimicrobial agent has increased in recent years.

IMPORTANCE Florfenicol is widely used for the treatment of respiratory infections and as a feed additive in food animal production. As a foodborne pathogen, Campylobacter is constantly exposed to florfenicol, and resistance to this antimicrobial agent has increased in recent years. Previous studies indicated that Campylobacter has developed several mechanisms that confer resistance to florfenicol. This study describes for the first time the coexistence of the florfenicol exporter FexA and the ribosomal protective protein OptrA in Campylobacter jejuni isolated from pigs. The two genes were located in various multidrug resistance genomic islands within different regions of the Campylobacter genomes. Although phenicols are not commonly used for the treatment of Campylobacter infections, the extensive use of florfenicol in food animals may play a role in the coselection of multidrug resistance genomic island (MDRGI)-carrying Campylobacter isolates which also exhibited resistance to critically important antimicrobial agents (macrolides, aminoglycosides, and tetracyclines) commonly used for the treatment of human campylobacteriosis. KEYWORDS Campylobacter, fexA, optrA, multidrug resistance C ampylobacter is the leading bacterial pathogen that causes diarrheal illness worldwide, with most cases of campylobacteriosis being triggered by Campylobacter jejuni. As a foodborne pathogen, Campylobacter is constantly exposed to multiple antimicrobial agents used during food animal production. Thus, Campylobacter has developed various resistance mechanisms, including the formation of multidrug resistance genomic islands (MDRGIs), for fitness advantage upon exposure to multiple antimicrobial agents (1)(2)(3)(4). Florfenicol is a fluorinated thiamphenicol derivative that was exclusively approved as a broad-spectrum antimicrobial agent for the treatment of animals raised for food (5). To date, several mechanisms of antibiotic resistance to florfenicol have been characterized, including the multidrug resistance protein Cfr(C), the multidrug efflux pump RE-CmeABC, and the recently described florfenicol exporter FexA and the ribosomal protective OptrA (4,(6)(7)(8)(9)(10). cfr(C), RE-cmeABC, and fexA were characterized in both C. jejuni and Campylobacter coli, whereas optrA was identified only in C. coli (4, 6-10).
The phenicol exporter gene fexA is responsible for florfenicol resistance. optrA not only confers resistance to phenicols but also results in elevated MICs of the oxazolidinone linezolid. Although these drugs are not commonly used for the treatment of Campylobacter infections, the extensive use of florfenicol in food animals may play a role in the coselection of MDRGI-carrying Campylobacter isolates, which also exhibit resistance to macrolides, aminoglycosides, and tetracyclines, commonly used for treating human campylobacteriosis (7). Linezolid represents one of the last-resort antimicrobial agents for the treatment of severe infections caused by methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. Thus, the coexistence of these two drug-resistant genes in Campylobacter aggravates the spread of antimicrobial resistance and poses a threat to human health. In this study, the coexistence of optrA and fexA was identified in C. jejuni isolates from pig and C. coli isolates from chicken and duck, and whole-genome sequencing was used to characterize their genetic environment.
To determine the presence of optrA, the primers A-F (59-AGGTGGTCAGCGAACTAA-39) and A-R (59-ATCAACTGTTCCCATTCA-39) (11) were used for PCR analysis of 146 C. coli and 54 C. jejuni strains isolated from poultry and swine farms in Zhejiang and Hunan provinces, China. The optrA sequence was identified in two C. jejuni and five C. coli isolates. Of note, all the strains also contained fexA.
To further characterize these seven optrA 1 fexA 1 isolates and the genetic environment of the genes, a hybrid sequencing strategy using Illumina short-read and MinION long-read technology was used to generate the complete genomes, as previously described (12). Five complete and two draft genome sequences were obtained for further mining (Table 1). In silico multilocus sequence typing of the whole-genome sequencing data showed that these seven isolates belonged to three sequence types (ST), including ST825, ST828, and a new ST (aspA_8, glnA_620, gltA_292, glyA_28, pgm_1072, tkt_668, and uncA_23). Acquired antimicrobial resistance genes can explain the resistance phenotype, including florfenicol resistance, determined by the broth microdilution method (Table 2). Only three and one single nucleotide polymorphisms were detected in the optrA and fexA sequences, respectively, in these seven strains (see Fig. S1 in the supplemental material).
C. coli CC19DZ036 and CC19DZ037 belong to ST828, and the complete genomes were 1,761,335 and 1,761,334 bp in length, with a GC content of 31.37% (Table 1) (accession no. CP068565 and CP068566, respectively). In total, only three gaps difference were found between the two strains. The order of gene content was tet(O)hp-catA9-IS1216E-hp-fexA-hp-optrA-IS1216E-tet(L), which was inserted between nfnB and smc.
This study revealed that the emerging gene optrA is associated with various MDRGIs in C. jejuni. Moreover, the core segment fexA-hp-optrA-IS1216E was identified in both C. jejuni and C. coli, which agrees with previous reports (7). Of note, the optrAcontaining MDRGIs varied from 9,611 to 22,697 bp and were inserted into different regions over the genomes of Campylobacter, all of which contained tet(O) and tet(L) at the two ends. The GC content of these MDRGIs ranged from 34.79% to 38.01%, which is different from the GC content of the Campylobacter genome (;31.0%), suggesting that Campylobacter might have obtained these MDRGIs from other species. There were 12 antimicrobial resistance genes in MDRGI, including aac(69)-aph(299), aph(39)-III, aph (299)-If, ant(6)-Ia, tet(L), tet(O), optrA, fexA, cat, catA9, bla OXA-465 , and erm(B), which were resistant to aminoglycosides, tetracyclines, phenicol, and macrolides. All of these antibiotics were used for the prevention and treatment of infections in farm animals in China. In addition, the GC content within several MDRGIs was not evenly distributed, and the presence of multiple insertion sequences suggested that their integration may have occurred through a multistep process. Therefore, MDRGI in Campylobacter was likely to be the product of multiple-antibiotic coselection. Due to the use of florfenicol in livestock and poultry production, the emergence of fexA and optrA could confer a fitness advantage under selection pressure, which will support the spread of fexA and optrA and their associated MDRGIs through Campylobacter natural transformation.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.