Differentiation of the virulence potential of Campylobacter jejuni strains by use of gene transcription analysis and a Caco-2 assay

Abstract

Campylobacter jejuni is the leading cause of bacterial diarrheal disease in humans, and contaminated poultry and poultry products are recognized as the main vehicle of infection. Despite the significance of C. jejuni as a foodborne pathogen, little is known about its response to stress, and, especially, how its virulence is modulated under such conditions. The aim of this study was to assess the effect of temperature shift in a broth model system on virulence expression and cell survival of three different Campylobacter jejuni strains: two clinical (TB1048 and NCTC11168) and one chicken isolate (DFVF1099). Firstly, cells were transferred from 42 to 4°C to investigate the effect of low temperature storage for short (30min) and long (24h) periods of time. A shift in temperature from 4 to 37°C for 30min was performed to investigate the effect of a momentary increase in temperature. Virulence properties were evaluated by analyzing transcriptions of the virulence genes cdtB, ciaB, cadF and the stress associated genes clpP, htrB using reverse transcription quantitative PCR (RT-qPCR) and by the ability of the C. jejuni strains to adhere to and invade Caco-2 cells. Similar cell survival and no growth was seen for all strains at 4°C and after transfer to 37°C for 30min. Interstrain variation was observed as transcription levels of cdtB, cadF and clpP were upregulated in NCTC11168 but not in DFVF1099 after 24h at 4°C. Bioinformatic analysis of invasion associated genes, showed differences in one gene, cipA between DFVF1099 and NCTC11168 resulting in a 14 amino acid deletion and 28 amino acid addition at the N and C terminal ends respectively of the CipA protein of DFVF1099. In contrast to DFVF1099, strains NCTC1168 and TB1048 were able to invade Caco-2 cells. Invasion ability was not affected by temperature shifts, as was also displayed by RT-qPCR analysis of another invasion associated gene, ciaB. The adhesion capacity was increased only for the TB1048 strain with incubation time. In conclusion, this study showed that low storage temperature is not enough to control the survival and virulence expression of C. jejuni. The clinical strains appeared to be more virulent than the chicken isolate as measured by the Caco-2 invasion assay which could be due to differences in CipA functionality. The RT-qPCR analysis and Caco-2 assay showed to be useful tools for differentiating virulence potentials of three C. jejuni strains under growth conditions where cell survival rates were similar.