Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

Abstract

ObjectivesThis study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture.MethodsChloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations.Results C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides.ConclusionsThis study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

“…In our study, resistance to CHL was consistently the most difficult to obtain. Although this finding is in apparent disagreement with the observed 50% of persistent bacteraemia after CHL treatments 21 , it might be related to changes at the fitness level as has been observed in the presence of specific 23S rRNA gene point mutations in other microorganisms 23 24 25 .…”

Chloramphenicol Derivatives as Antibacterial and Anticancer Agents: Historic Problems and Current Solutions

“…More crucial are mutations and/or deletions of ribosomal proteins, which are usually encoded by single gene copies. However, mutations in ribosomal proteins causing CAM-resistance have been identified only in a few cases so far [ 35 , 36 ]. Because interactions of CAM with ribosomal proteins have not been detected by crystallographic analyses [ 18 , 20 ], such resistance is suspected to arise indirectly, probably by induced conformational changes in rRNA.…”

“…It has been found that combination of both ribosomal protein and rRNA mutations lead to increased resistance; e.g. G-to-A nucleotide change at position 2073 (2058, E. coli numbering) in all three copies of the 23S rRNA gene in combination with G14D modification in ribosomal protein L4 confers CAM resistance in Campylobacter jejuni [ 36 ]. Nevertheless, such mutations are often accompanied by fitness cost and consequently are not extensively spread.…”

Integrated Genomic and Proteomic Analyses of High-level Chloramphenicol Resistance in Campylobacter jejuni

“…In our previous work, a point mutation, G2073A, was identified in 23 S rRNA in CAP-selected mutants of C. jejuni 19 . However, further study was needed to investigate whether this point mutation alone was sufficient to confer high-level resistance to CAP in the C. jejuni mutants.…”