Abstract
Campylobacter jejuni has long been recognized as a cause of bacterial food-borne illnesses, and today it is the most prevalent bacterial food-borne pathogen in the industrial world. Many bacteria have evolved mechanisms that enable them to survive in different environments. Oxidative stress remains an important challenge to bacteria. It results from the formation of highly reactive oxygen species as a result of incomplete reduction of oxygen during respiration and is also produced by the host immune system to remove or to kill invading pathogenic microorganisms. The reactive oxygen species formed can cause deleterious effects on bacteria as these can cause damage to proteins, nucleic acids and membranes. However, aerobic bacteria and microaerophilic bacteria (Campylobacter species) have developed several enzymes such as superoxide dismutase, to detoxify these very active compounds. Manganese has recently been shown to protect some pathogenic bacteria against oxidative stress, most notably in Neisseria gonorrhoeae. Given this, the aim of this study was to investigate for the first time, the response of different strains of C. jejuni and Campylobacter coli to oxidative stress, and exposure to air, in the absence or presence of manganese. Growth was assessed at various concentrations of MnSO4, and under microaerobic conditions, concentrations of 100 μM and 250 μM enhanced growth slightly whereas 50 μM did not. At higher concentrations, 500 μM and lmM, manganese was bactericidal and its presence resulted in a decrease in viability of over 6-log cycles over the duration of various experiments. However, at concentrations of 100 μM and 250 μM , manganese had a marked effect on the survival of Campylobacters exposed to aerobic conditions and here its presence not only enhanced survival by 0. 5-1 log-cycle depending on the strain being studied, but it also prolonged survival. For example, in the absence of added manganese counts of C. jejuni NCTC 11168 fell to undetectable levels in 110 hours whilst in its presence cells took over 140 hours to reach undetectable levels. The same concentrations of manganese, also increased the resistance of Campylobacters to superoxide radicals and hydrogen peroxide when it was measured using a disc diffusion assay. This study has shown, for the first time that manganese influences the growth and survival of Campylobacters, particularly under aerobic conditions, and during oxidative stress. Mn+2 transporters have been shown to play important roles in the growth and virulence of bacteria. For example, simultaneous mutation in both the ABC-permease and Nramp transporters of Salmonella enterica serovar Typhimurium has been shown to attenuate virulence in this well-studied pathogen. To ascertain, whether manganese transport was necessary for its proective effect, the genome sequence of C. jejuni NCTC11168 was screened for the presence of putative manganese transporter. It was shown to possess Cj0141c possess, a gene encoding a putative permease protein for a MntB-like, ATP binding cassette transporter for manganese. A knock-out mutant, deficient in Cj0141c was generated by allelic exchange, and its survival in air was not shown to be enhanced by the presence of manganese, and it was more resistant to the higher concentrations of this metal, than the corresponding wild-type. Taken together, these results suggest a role of Cj0141c in manganese transport, and in facilitating its protective effect against oxidative stress in Campylobacters.