Impact Of Macrolide Resistance On The Virulence Properties And Fitness In Campylobacter Jejuni

The relationship between macrolide resistance and fitness or virulence in Campylobacter jejuni is one of the most concerns in worldwide. The mechanism of macrolide resistance in C. jejuni includes the mutation in 23S rRNA, the modification in ribosome L4 and L22, the expression of efflux pump and other unknown mechanism. Previous studies evaluated the biological cost imposed by 23 S rRNA gene mutations in macrolide-resistant Campylobacter jejuni. However whether acquisition of macrolide resistance through modifications in the ribosomal proteins or through combined mutations (in 23S rRNA gene and ribosomal protein) confers biological cost similar to that of the 23S rRNA gene mutations is unknown. Furthermore the epidemiological studies of macrolide resistance in C. jejuni demonstrated that infections with macrolide-resistant C. jejuni could be associated with an increased risk of adverse events, development of invasive illness or death compared to to macrolide-susceptible isolates. However all these studies were dependent upon epidemiological studies and no scientific evidence that clearly illustrated if macrolide resistance in C. jejuni could be associated with increased virulence of the resistant strain over the susceptible strain. In addition to that the phenotypic alterations in the context of antibiotic resistance have been extensively studied in some bacterial species. However there are no enough data addressing these alterations due to macrolide resistance in Campylobacter jejuni.In this work, an in vitro induction experiment was conducted using susceptible C. jejuni strain and different macrolide drugs as selecting agents to obtain macrolide-resistant mutants with different type of mutations in the macrolide action sites. The relative fitness, growth carves and heat tolerance assay of the macrolide-reistant mutants were compared to that of the susceptible parent strains as well as animal colonization model between a mutant with 23S rRNA gene mutation (A2074C) and the susceptible parent strain. Furthermore the changes in the virulence characteristics between a mutant with 23S rRNA gene mutation (A2074C) and the susceptible parent strain were studied as well as the gene expression profile using DNA microarray. In addition to that, there are no data that examined the effect of exposure to macrolide on the virulence properties such as motility in C. jejuni. The effect of exposure to macrolide through induction with or through exposure to sub-inhibitory concentration of macrolide on the motility and flagellar filaments formation is also studied.Sequence analysis of the macrolide action sites in the resistant mutants demonstrated that macrolide-resistant mutants revealed different type of mutations. Macrolide-resistant mutant with only modifications in the ribosomal proteins L4 (G170A) and L22 (G257A), macrolide-resistant mutants with only 23S rRNA gene mutations (A2074C or A2075G) and macrolide-resistant mutant beside 23S rRNA gene mutation (A2074C) harbored another mutation in the rplV gene (insertion of AGTCGT at position 265) were obtained. The comparison of the growth rate and the relative fitness of the strains that characterized by different type of mutations revealed different level of defects in their growth rates and the different mutations imposed different fitness cost depending on the type and/or the site of the mutation when competed against the susceptible parent strain. However all the strains, including the susceptible parent strain, demonstrated drastic exponential drop in the number of the recovered bacteria in the heat tolerance assay but without statistical significant difference observed.Animal colonization assay between the susceptible parent strain and a mutant with 23S rRNA gene mutation (A2074C) showed that when the two strains inoculated separately in the mice model they showed approximately the same colonization level. Nevertheless, co-inoculation of the two strains in the mice model demonstrated that the resistant strain colonized only for 7 days compared to 21 days for the susceptible strain. In addition to that the changes in the virulence characteristics between the susceptible parent strain and the mutant with 23S rRNA gene mutation (A2074C) were examined. The resistant-mutant strain demonstrated slightly more resistance to bile in the bile tolerance assay compared to the susceptible strain but without statistical significant difference. However the resistant-mutant strain apparently demonstrated reduced adhesion and invasion characteristics to intestinal epithelial cells, murine macrophage and short time intracellular survivability within macrophage compared to the susceptible strain.DNA microarray showed that the majority of the genes upregulated in the resistant strain (A2074C) were genes related to protein metabolism, amino acid transporter and energy production and conversion. While the majority of the downregulated genes in the resistant strain were involved in cell motility and secretion, translation, post-translation modifications, protein turnover and chaperones. Furthermore macrolide through induction or exposure to sub-MIC concentrations impaired the motility of C. jejuni in 0.4% MH agar plates. Electron microscope analysis revealed the absence of flagellar filaments from strains exposed to macrolide. SDS-PAGE demonstrated that macrolide have no effect on protein synthesis and immunoblotting analysis further confirmed that flagellin was fully synthesized within C. jejuni strains exposed to macrolide. Nevertheless C. jejuni strains exposed to macrolide demonstrated defect in their excreted flagellin into the supernatant compared to strains not exposed to macrolide. Accordingly we speculated that macrolide inhibited flagellar filaments formation in the strains exposed to macrolides via affecting the secretion of flagellin without affecting the amount synthesized within the bacteria.Taken together these findings revealed that macrolide resistance through modification in the ribosomal proteins L4 and L22 incurred a fitness cost in C. jejuni. The combined mutations, despite they conferred high resistance level to macrolides, were more detrimental for the bacterial physiology. Moreover these findings demonstrated the increment of the virulence characteristics of Ery-susceptible strain rather than Ery-resistant strain. The adverse events previously observed in the epidemiological studies for macrolide-resistant strains infection, we suggested this maybe attributed to the resistivity of the resistant strains to the treatment and consequently prolonged the symptoms and compromised the disease in patients. Finally we demonstrated that exposure to macrolides through induction or sub-inhibitory concentrations resulted in phenotypic alterations such as inhibition of flagellar filaments formation and loss of motility in C. jejuni.