'Acanthamoeba' as a model for the investigation of the molecular mechanisms of 'Campylobacter jejuni' pathogenesis and survival in the environment

Campylobacter jejuni is a foodborne pathogen recognised as the leading cause of human bacterial gastroenteritis. Undercooked poultry products and contaminated water are considered as the most important sources of infection. Antimicrobial therapy is warranted only for immunocompromised patients and, although most people recover from this disease, others may develop rare neurodegenerative disorders such as Guillain-Barre Syndrome (GBS). The latter affects the nerves of the body leading to paralysis and requires extensive medical treatment. The wide use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter over the last decade. Investigation of the molecular mechanisms of antibiotic resistance is considered important to control the spreading of resistant bacteria. CmeABC RND-type multidrug efflux (MDR) pump and the tetO gene found on pTet plasmids mediate tetracycline resistance in Campylobacter. CmeABC MDR pump consists of three components: an outer membrane protein CmeA, an inner membrane drug transporter CmeB and a periplasmic protein CmeC. Even though C. jejuni strains G1 and 11168H do not contain the pTet plasmid, the former was shown to be more resistant to tetracycline (Tet). Comparison of the genome of the G1 strain with that of the reference strain, 11168H, revealed a remarkable difference between the nucleotide sequences of their cmeB genes. In addition, it was observed that the transfer of the pTet plasmid from C. jejuni 81-176 to the G1 strain increased the level of Tet resistance above that of the former strain carrying this plasmid. This finding suggests that CmeB of strain G1 has a higher capacity to excrete this drug than its analogue in C. jejuni strains 81-176 and 11168H and thus, the former strain could be considered as an efflux pump variant with increased resistance to antibiotics. In this study we demonstrate that contribution of MDR pumps to antibiotic resistance might be dependent on the sequence variation of CmeB. Although antibiotic resistance is the main function of MDR pumps, these pumps may have other physiological roles, such as in virulence. An important mechanism of bacterial pathogenesis is the survival of Campylobacter inside environmental hosts. As a host of pathogenic microorganisms, the protozoan Acanthamoeba is a good model for the investigation of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The endosymbiotic relationship between this eukaryotic organism and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. Although some studies suggest that Acanthamoeba supports Campylobacter survival in the environment, the type of interaction between these microorganisms needs to be elucidated. Also, the bacterial factors involved in this interaction remain unknown. Using a modified gentamicin protection assay it was found that C. jejuni 81-176 is able to survive and multiply inside this eukaryotic host. Thus, since these microorganisms can co-exist in the same environments (e.g. in poultry farms) the risk of infection with this foodborne pathogen is elevated. It is also reported that the CmeABC MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni within A. polyphaga. However, this MDR pump was found to be dispensable for C. jejuni biofilm formation, motility and oxidative stress. Moreover, it was observed that capsule production is also required for the interaction between C. jejuni and with amoebae. Due to their role in antibiotic resistance and virulence of C. jejuni, MDR pumps could be considered as good targets for the development of antibacterial drugs against this pathogen. During the course of this study, a new chimeric C. jejuni strain was created due to horizontal gene transfer between two different strains, 81-176 and G1, which were growing together. This finding emphasises how easily Campylobacter can exchange its genetic material and thus adapt to the surrounding environment