Abstract
Many epidemiological studies provide us with the evidence of horizontal gene transfer (HGT) contributing to the bacterial genomic diversity that benefits the bacterial populations with increased ability to adapt to the dynamic environments. Campylobacter jejuni, a major cause of acute enteritis in the U.S., often linked with severe post-infection neuropathies, has been reported to exhibit a non-clonal population structure and comparatively higher strain-level genetic variation. In this study, we provide evidence of the HGT of chromosomally encoded genetic markers between C. jejuni cells in the biphasic MH medium. We used two C. jejuni NCTC-11168 mutants harbouring distinct antibiotic-resistance genes [chloramphenicol (Cm) and kanamycin (Km)] present at two different neutral genomic loci. Cultures of both marker strains were mixed together and incubated for 5 hrs, then plated on MH agar plates supplemented with both antibiotics. The recombinant cells with double antibiotic markers were generated at the frequency of 0.02811 ± 0.0035% of the parental strains. PCR assays using locus-specific primers confirmed that transfer of the antibiotic-resistance genes was through homologous recombination. Also, the addition of chicken cecal content increased the recombination efficiency approximately up to 10-fold as compared to the biphasic MH medium (control) at P < 0.05. Furthermore, treating the co-culture with DNase I decreased the available DNA, which in turn significantly reduced recombination efficiency by 99.92% (P < 0.05). We used the cell-free supernatant of 16 hrs-culture of Wild-type C. jejuni as a template for PCR and found DNA sequences from six different genomic regions were easily amplified, indicating the presence of released chromosomal DNA in the culture supernatant. Our findings suggest that HGT in C. jejuni is facilitated in the chicken gut environment contributing to in vivo genomic diversity. Additionally, C. jejuni might have an active mechanism to release its chromosomal DNA into the extracellular environment, further expediting HGT in C. jejuni populations.
Highlights
Campylobacter jejuni (C. jejuni) infections cause significant impacts on food safety and public health worldwide
The Chloramphenicol resistant (CmR) and Kanamycin resistant (KmR) markers were introduced into distinct genomic loci of C. jejuni strains NCTC 11168, and these marker strains were used for the recombination assay unless described otherwise
To observe the horizontal gene transfer (HGT) in an experimental setting, it was important to use the marker strains with distinct genomic tags inserted at neutral genomic loci. hipO gene locus was used previously by de Boer et al [34] to construct a marker strain, which was used to study HGT in C. jejuni strains
Summary
Campylobacter jejuni (C. jejuni) infections cause significant impacts on food safety and public health worldwide. Many studies point towards genetic diversity within C. jejuni populations and among different species of genus Campylobacter [9,10,11,12,13]. Genetic diversity explains the greater ability of a bacterial population to survive in a hostile environment and its better adaptation in host gut colonization. 1,600 genes are present in a C. jejuni genome [17], but the number of genes estimated in the pan-genome of C. jejuni is about 2,600 [12, 21]. Multiple epidemiological reports indicate towards a complex strain diversity of C. jejuni [23, 24] and a similar observation was made in C. jejuni strains isolated from an infected chicken flock [19]. The analysis of infecting C. jejuni strains using strain-typing methods in a chicken flock is typically complicated as a chicken flock is often infected by different genotypes over time [26]
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