During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

Campylobacter jejuni is the leading cause of bacterial enteritis in Europe. Human campylobacteriosis cases are frequently associated to the consumption of contaminated poultry meat. To survive under environmental conditions encountered along the food chain, i.e., from poultry digestive tract its natural reservoir to the consumer’s plate, this pathogen has developed adaptation mechanisms. Among those, biofilm lifestyle has been suggested as a strategy to survive in the food environment and under atmospheric conditions. Recently, the clinical isolate C. jejuni Bf has been shown to survive and grow under aerobic conditions, a property that may help this strain to better survive along the food chain. The aim of this study was to evaluate the adhesion capacity of C. jejuni Bf and its ability to develop a biofilm. C. jejuni Bf can adhere to abiotic surfaces and to human epithelial cells, and can develop biofilm under both microaerobiosis and aerobiosis. These two conditions have no influence on this strain, unlike results obtained with the reference strain C. jejuni 81-176, which harbors only planktonic cells under aerobic conditions. Compared to 81-176, the biofilm of C. jejuni Bf is more homogenous and cell motility at the bottom of biofilm was not modified whatever the atmosphere used. C. jejuni Bf whole genome sequence did not reveal any gene unique to this strain, suggesting that its unusual property does not result from acquisition of new genetic material. Nevertheless some genetic particularities seem to be shared only between Bf and few others strains. Among the main features of C. jejuni Bf genome we noticed (i) a complete type VI secretion system important in pathogenicity and environmental adaptation; (ii) a mutation in the oorD gene involved in oxygen metabolism; and (iii) the presence of an uncommon insertion of a 72 amino acid coding sequence upstream from dnaK, which is involved in stress resistance. Therefore, the atypical behavior of this strain under aerobic atmosphere may result from the combination of insertions and mutations. In addition, the comparison of mRNA transcript levels of several genes targeted through genome analysis suggests the modification of regulatory processes in this strain.

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.

Campylobacter jejuni has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO2, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As C. jejuni is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in C. jejuni and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of C. jejuni was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among C. jejuni strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.

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Purkrtová S., Turoňová H., Pilchová T., Demnerová K., Pazlarová J. (2010): Resistance of Listeria monocytogenes biofilms to disinfectants. Czech J. Food Sci., 28: 326-332.We studied the optimal conditions for the biofilm development by Listeria monocytogenes on a model system represented by microtiter plates, and also for determined some effective disinfectant agents. Listeria monocytogenes ATCC 13932 and an industrial isolate of Listeria monocytogenes Lm-24 were compared as to their abilities to form biofilms. The starting concentration of the cells leading to the most reproducible results was 0.5 McFarland. The temperatures tested ranged between 8°C to 37°C, the optimal values to form biofilm in buffered peptone water (BPW) with 0.05% glucose were 25°C and 30°C. Under comparable conditions the persistent strain L. monocytogenes Lm-24 constituted more massive biofilm than did the reference strain. The following disinfectants were applied: Savo, Merades Alco, benzalalkonium chloride. A persistent industry in isolate Listeria monocytogenes Lm-24 was used as the model organism for these tests. Benzalalkonium chloride treatment was found to be the most efficient way to damage the biofilm. One minute treatment with 500 mg/l was lethal for the biofilm cells, and that with 125 mg/l for planctonic cells. Savo suppresed the viability of the biofilm cells only by about 20% on average while being lethal for planctonic cells. Merades Alco exhibited only a weak effect on both the biofilm and planctonic cells.