Characterization of the genetic environment of the ribosomal RNA methylase gene erm(B) in Campylobacter jejuni

Sir, Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis in humans.1C. jejuni is increasingly resistant to clinically important antibiotics, particularly fluoroquinolones and macrolides.2,3 Because of the lack of ribosomal RNA methylases and the fitness cost of the point mutations associated with resistance, the overall macrolide resistance rate in C. jejuni has been maintained at a relatively low level compared with that of fluoroquinolone resistance.2,4 Our recent study identified a ribosomal RNA methylase gene, erm(B), conferring macrolide resistance in Campylobacter.5 Subsequent investigation indicated that the majority of Campylobacter strains carrying erm(B) were Campylobacter coli, with only one C. jejuni isolate identified (C179b).6 Investigation of the genetic environment of erm(B) in C. coli indicated that all erm(B) genes are located within multidrug resistance genomic islands (MDRGIs).6 Generally, these MDRGIs are inserted into either the region between cadF and CCO1582 or into aac located between nfsB and cinA.6 Our earlier study failed to discern the flanking regions of erm(B) in C. jejuni C179b using a primer-walking strategy. Additionally, long-range PCR could not amplify any MDRGI in C. jejuni C179b the same as those MDRGIs carrying erm(B) in C. coli in our previous publication.6 Here, we use whole genome sequencing to characterize the genetic environment of erm(B) in C. jejuni C179b. We also determined the transferability and stability of erm(B) in this C. jejuni isolate. Genetic environment of erm(B) in C. jejuni C179b and within its MDRGI in natural transformants C179b_11168 and C179b_81-176 in comparison with their homologues in the donor and recipient. The locations of primers cj0168c-F and sodB-R as well as cpp50-F and cpp7-R, used to detect MDRGI, are indicated. To confirm the transferability of the erm(B)-carrying MDRGI in C. jejuni, the genomic DNA of C179b was introduced into macrolide-susceptible C. jejuni strains NCTC 11168 and 81-176 by natural transformation.8 The transformants, designated C179b_11168 (NCTC 11168 as recipient) and C179b_81-176 (81-176 as recipient), exhibited ≥128-, ≥2048- and ≥8-fold increases in the MICs of macrolides (erythromycin, azithromycin, tylosin and spiramycin), clindamycin and streptomycin, respectively, compared with NCTC 11168 and 81-176 (Supplementary Data), indicating that this MDRGI was transferred to the recipient strains. To confirm the presence of type VII MDRGI in the transformants, a long-range PCR assay was conducted using the primers cj0168c-F (5′-AGCTGGTGTTTGCTCTTTTGT-3′) and sodB-R (5′-TGCCTTCTTTTAAAGCCCATTC-3′), which are located in the conserved regions of cj0168c and sodB, respectively (Figure 1). A 11 035 bp amplicon containing the intact type VII MDRGI was obtained from both C179b and its transformant C179b_11168, but not from C179b_81-176. Considering the high level of similarity between the genes in the type VII MDRGI and the pTet plasmid in 81-176, there is a strong possibility that the MDRGI inserted into pTet instead of the chromosome (Figure 1). We performed several analyses to locate the exact insertion site of the MDRGI in 81-176. S1-nuclease PFGE and Southern blotting indicated that erm(B) and the tetracycline resistance gene tet(O) were located on a ∼50 kb plasmid (Supplementary Data). It should be noted that this original tet(O) gene in pTet showed 94.6% nucleotide identity to that in the type VII MDRGI. A modified random primer-walking strategy revealed that the MDRGI was linked to the genes on the pTet plasmid. Further sequence analysis indicated that the pTet plasmid kept its original features [tet(O), cpp2, cpp3, cpp4 and repa] and 8 out of 13 ORFs from the MDRGI were incorporated between cpp2 and cpp3 (Figure 1). Finally, long-range PCR using the primers cpp50-F (5′-CCACTTGGCTTTATCTTTTGC-3′) and cpp7-R (5′-GCAATGTCTTTCTCATATTCTGC-3′) (Figure 1), which are located in cpp50 and cpp7 on pTet, confirmed the presence of the MDRGI in C179b_81-176. These results showed that type VII MDRGI could be transferred amongst different C. jejuni strains and could be integrated into both plasmid and chromosome. Based on these and previous findings,5,6 we concluded that the characterized MDRGIs could be integrated into at least four regions in Campylobacter and the plasticity of the insertion regions enhances the possible dissemination of MDRGIs among Campylobacter. To determine the stability of erm(B) and its associated type VII MDRGI in transformants, two transformants (C179b_11168 and C179b_81-176) were subcultured daily in antibiotic-free Mueller–Hinton (MH) broth for 14 days. Cultures were diluted and then plated onto MH agar plates to distinguish single colonies. PCR screening of 20 colonies using the primers described above revealed that all of the colonies maintained the acquired MDRGI, confirming that the two transformants remained stable in the absence of antibiotics for ≥14 passages (Supplementary Data). These findings suggest that natural transformation might contribute to the dissemination of high-level resistance to macrolides amongst Campylobacter species. In conclusion, this study characterized the erm(B)-associated MDRGI in C. jejuni. Together with our previous study, these findings indicated that MDRGIs carrying erm(B) could be disseminated amongst C. jejuni isolates and be transferred from C. coli to C. jejuni in multiple regions. Even though the isolation rate of C. jejuni carrying erm(B) is relatively low at this stage, C. jejuni accounts for ∼90% of campylobacteriosis in humans. Thus, it is worthwhile to monitor the emergence and spread of erm(B) and its associated MDRGI in C. jejuni. The study was funded by grants from the National Basic Research Program of China and the National Natural Science Foundation of China (2013CB127200 and U1031004). None to declare.