Key Role of Mfd in the Development of Fluoroquinolone Resistance in Campylobacter jejuni

Abstract

Campylobacter jejuni is a major food-borne pathogen and a common causative agent of human enterocolitis. Fluoroquinolones are a key class of antibiotics prescribed for clinical treatment of enteric infections including campylobacteriosis, but fluoroquinolone-resistant Campylobacter readily emerges under the antibiotic selection pressure. To understand the mechanisms involved in the development of fluoroquinolone-resistant Campylobacter, we compared the gene expression profiles of C. jejuni in the presence and absence of ciprofloxacin using DNA microarray. Our analysis revealed that multiple genes showed significant changes in expression in the presence of a suprainhibitory concentration of ciprofloxacin. Most importantly, ciprofloxacin induced the expression of mfd, which encodes a transcription-repair coupling factor involved in strand-specific DNA repair. Mutation of the mfd gene resulted in an approximately 100-fold reduction in the rate of spontaneous mutation to ciprofloxacin resistance, while overexpression of mfd elevated the mutation frequency. In addition, loss of mfd in C. jejuni significantly reduced the development of fluoroquinolone-resistant Campylobacter in culture media or chickens treated with fluoroquinolones. These findings indicate that Mfd is important for the development of fluoroquinolone resistance in Campylobacter, reveal a previously unrecognized function of Mfd in promoting mutation frequencies, and identify a potential molecular target for reducing the emergence of fluoroquinolone-resistant Campylobacter.

Effect of ciprofloxacin exposure on DNA repair mechanisms in Campylobacter jejuni

“…When we compared ciprofloxacin induced gene expression in C. jejuni strain 81-176 with another C. jejuni strain NCTC 11168 (Han et al, 2008), we found that only a set of genes in 81-176 showed a similar response to ciprofloxacin as NCTC 11168 (Table 1). For example, we did not find any response in the transcriptional-repair coupling gene mdf as found in the studies of Han et al (2008).…”

“…These transcriptome studies have shown that responses to ciprofloxacin exposure involve DNA-damage repair mechanisms through the SOS response (Kuzminov, 1999). However, C. jejuni lacks LexA (Han et al, 2008), which is important in SOSmediated DNA repair and thus a typical SOS response system is not functional in C. jejuni. It is not known which molecular mechanisms underlying responses to ciprofloxacin cause direct DNA damage, like DNA breaks and fragmentation.…”

“…It is not known which molecular mechanisms underlying responses to ciprofloxacin cause direct DNA damage, like DNA breaks and fragmentation. Based on a gene expression study of C. jejuni NCTC 11168, ciprofloxacin exposure can cause various responses in general cellular processes, but only two factors were directly associated with DNA metabolism (Han et al, 2008): downregulation of the DNA polymerase subunit dnaE and upregulation of the mdf gene. The transcriptional-repair coupling factor Mdf functions in strand-specific DNA repair and it was suggested to contribute to the emergence of ciprofloxacin-resistant mutants (Han et al, 2008).…”

“…Based on a gene expression study of C. jejuni NCTC 11168, ciprofloxacin exposure can cause various responses in general cellular processes, but only two factors were directly associated with DNA metabolism (Han et al, 2008): downregulation of the DNA polymerase subunit dnaE and upregulation of the mdf gene. The transcriptional-repair coupling factor Mdf functions in strand-specific DNA repair and it was suggested to contribute to the emergence of ciprofloxacin-resistant mutants (Han et al, 2008). Another recent study by Gaasbeek et al (2009) that employed insertional mutants of uvrB, ung, recA, and mutS proved that nucleotide excision repair (NER) and RecA-dependent recombinational repair were functional DNA repair systems in C. jejuni (Gaasbeek et al, 2009;Guerry et al, 1994).…”

“…RT-qPCRs were performed with gene specific primers (Table S1) in triplicate for three independent samples from each time point, using a previously published amplification protocol (Hyytiäinen et al, 2012). Fold changes of genes were calculated with the method described by Pfaffl (Pfaffl, 2001) using rrs (16S rRNA) as a normalization reference (Han et al, 2008;Hyytiäinen et al, 2012). Statistical differences in relative gene expression were calculated with one-sample t-tests using SPSS for Windows, Release 15.0.1 software (SPSS).…”