Campylobacter jejuni: Biofilm formation and oxidative stress

Campylobacter jejuni (C. jejuni ) is a zoonotic pathogen most often associated with poultry and the leading cause of bacterial gastroenteritis in the developed world. It is an obligate microaerobe, requiring low levels of oxygen and increased levels of carbon dioxide for growth. Attempts to understand how this sensitive pathogen survives the variety of environmental challenges it faces have led to the suggestion that it may find protection in biofilms. This study investigates the effect of oxygen concentration on the formation of C. jejuni biofilms and their subsequent resistance to oxidative stress. In order to determine their ability to develop biofilms when exposed to oxidative stress, three strains (NCTC 11168 V1, NCTC 11168 V26 and 16-2R) of C. jejuni biofilm cells were grown on glass fibre filters in different atmospheres: MA -- microaerobic (5 % O2, 10% CO2, 85% N) AIR -- aerobic (20% O2, 0.05% CO2, 78% N) and ACO2 -- aerobic enhanced with 5% CO2 (19% O2, 5% CO 2, 74% N). Biofilm formation was assessed using plate counts, biovolume analysis and scanning electron microscopy (SEM). Tolerance to oxidative stress was determined by exposure of biofilm cells to the exogenous oxidant hydrogen peroxide (H2O2).Biofilm cells grown in ACO2 showed higher tolerance to the exogenous oxidant H2O2 (2.70 log10 colony forming units/g reduction) than those from the MA condition (up to 4.57 log10 CFU/g reduction) for C. jejuni 16-2R. Growth condition appeared to have no effect on subsequent tolerance to oxidative stress for the other 2 strains (NCTC 11168 V1 and NCTC 11168 V26). However, the original clinical isolate, NCTC 11168 V1, exhibited more than twice the tolerance to H 2O2 than the lab -passaged variant, NCTC 11168 V26.C. jejuni formed biofilms equally well in both MA (10% CO2 and 5%O2) and ACO2 (5% CO2 and 19% O 2), but only very poorly in AIR (0.05% CO2 and 20% O 2). This suggests that it is not the concentration of oxygen, but rather the concentration of CO2 which influences the development of biofilm, for the three strains investigated. Furthermore, tolerance to oxidative stress was subject to strain variation.Plate count data indicated no significant difference between biofilm cells for MA (9.6 to 9.8 log10 CFU/g) and ACO2 (9.6 to 9.9 log 10 CFU/g) (p > 0.05). Biofilm cells grown in AIR (4.0 to 4.3 log 10 CFU/g) were significantly lower than MA or ACO2 for all three strains (p<0.001). These results were supported by the SEM images. Biofilm development was more extensive in both MA and ACO2 than in AIR. SEM images indicated some strain variation with respect to biofilm development. Values for biovolumes also supported the trends observed in the plate count data. Total biovolume values for AIR (7.74 to 8.34 log10 mum3/g) were at most 15% of the corresponding values for MA (8.82 to 9.22 log10 mum 3/g). Strains NCTC 11168 V1 and 16-2R showed a slight preference for ACO2 (9.00 and 9.43 log10 mum3/g) over MA (8.82 and 9.22 log10 mum3/g). The NCTC 11168 V26 strain had very similar values for MA and ACO2 (9.15 and 9.16 log10 mum 3/g).