Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.     

Survival of Campylobacter jejuni in Waterborne Protozoa

The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.     

Survival of Campylobacter jejuni in waterborne protozoa

The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.     

A Novel Campylobacter jejuni Two-Component Regulatory System Important for Temperature-Dependent Growth and Colonization

Campylobacter jejuni colonizes the intestines of domestic and wild animals and is a common cause of human diarrheal disease. We identified a two-component regulatory system, designated the RacR-RacS (reduced ability to colonize) system, that is involved in a temperature-dependent signalling pathway. A mutation of the response regulator gene racR reduced the organism's ability to colonize the chicken intestinal tract and resulted in temperature-dependent changes in its protein profile and growth characteristics.     

Survival of Campylobacter jejuni in different gas mixtures

Campylobacter jejuni in fresh chilled chicken meat is known to be a major risk factor for human gastrointestinal disease. In the present study, the survival under chilled conditions of different C. jejuni strains exposed to different gas mixtures usually used for gas packaging of food was examined. Bolton broth and fresh, skinless chicken fillets were inoculated with six and four strains, respectively, and exposed to the gas mixtures 70/30% O(2)/CO(2), 70/30% N(2)/CO(2), and 100% N(2) (the latter only investigated in broth) at refrigeration temperature (4-5 degrees C). In broth culture, the strains survived significantly longer when exposed to 100% N(2) and 70/30% N(2)/CO(2) than in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001). For the two anaerobic gas mixtures, the reductions only reached 0.3-0.8 log(10) CFU mL(-1) within the same period. In the presence of oxygen, the numbers of C. jejuni were reduced by a minimum of 4.6 log(10) CFU mL(-1) over 21 days. When inoculated onto chicken fillets, the C. jejuni strains also died significantly faster in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001), reaching reductions of 2.0-2.6 log(10) CFU g(-1) after 8 days. In the gas mixture without oxygen (70/30% N(2)/CO(2)), no reductions were observed.     

Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4',6'-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

Survival of Campylobacter jejuni in foods and comparison with a predictive model

Campylobacter jejuni was inoculated into a range of raw and cooked foods and survival determined during storage at 20°, 10° and 20°C for up to 56 d. To facilitate easy enumeration, two antibiotic-resistant strains of Camp. jejuni , which had similar survival characteristics to the parent strain, were used. Campylobacter jejuni survived for longer at lower temperatures in all foods and inactivation was most rapid in pâté. There was generally good agreement between the survival data and predictions from a Camp. jejuni survival model (Food MicroModel).     

Survival of Campylobacter jejuni in chicken meat at frozen storage temperatures

The aim of this study was to determine the survival of Campylobacter jejuni in chicken meat samples at frozen temperatures and given length of incubation and to determine the impact of aerobic bacteria on the survival of C. jejuni. The chicken meat samples were inoculated with C. jejuni NCTC 11351 suspensions and stored in bags at temperatures of -20°C and -70°C. The mean value of C. jejuni from meat samples decreased from 7.52 log10 CFU/g after 30 minutes of incubation at ambient temperature, to 3.87 log10 CFU/g on the eighth week of incubation at -20°C, and to 3.78 log10 CFU/g at incubation at -70°C after the same incubation period. Both freezing temperatures, -20°C and -70°C, decreased the number of campylobacters. The presence of aerobic mesophilic bacteria did not influence the survival of C. jejuni in chicken meet samples. Keeping poultry meat at freezing temperatures is important for the reduction of C. jejuni, which has a strong influence on the prevention of occurrence of campylobacteriosis in humans.     

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.     

Survival of Escherichia coli and Campylobacter jejuni in untreated and filtered lake water

L.K. KORHONEN AND P.J. MARTIKAINEN. 1991. The survival of Campylobacter jejuni and Escherichia coli in lake water was studied using viable counts. Escherichia coli survived better than C. jejuni in all the test conditions studied. Both the species survived better in filtered than in untreated water. This suggests that predation and/or competition for nutrients affect the survival of both the species in an aquatic environment. Campylobacter jejuni survived less well in filtered autoclaved water and in 0.9% NaC1 than in filtered water without autoclaving. The lack of some essential nutrients, which may be degraded by autoclaving, might explain these results.     

Survival and injury of Escherichia coli, Campylobacter jejuni, and Yersinia enterocolitica in stream water.

Experiments were done to describe the survival and injury of three strains each of enteropathogenic Escherichia coli, Yersinia enterocolitica, and Campylobacter jejuni in agricultural surface water. Bacterial suspensions within membrane diffusion chambers were immersed in two large vessels of stream water that were held at 6 and 16 degrees C and changed daily. The results of daily plate counts, using selective and nonselective media, revealed some variation among strains and genera. Injury increased rapidly in all of the bacteria examined during an initial 4-day period of population stability. Bacterial persistence was generally prolonged at 6 degrees C, while the occurrence of injury was directly related to temperature. However, both survival and injury in C. jejuni were less dependent on temperature, while Y. enterocolitica displayed the greatest survival at both 6 and 16 degrees C. These results suggest that surface water in the temperate zone might serve as a persistent vehicle for the transmission of these enteropathogenic bacteria between animals and humans.     

Persistence of Campylobacter jejuni on surfaces in a processing environment and on cutting boards

AIMS: The objectives of the study were to determine the spread and persistence of Campylobacter in a poultry processing plant and to provide a quantitative estimate of the survival of Campylobacter jejuni on the surface of a cutting board. METHODS AND RESULTS: Several contact surfaces in a poultry processing plant were sampled before the start of processing, after 30 min and after 120 min. Next, the survival of four C. jejuni strains was studied on a beech and polypropylene cutting board during 120 min. CONCLUSIONS: A rapid introduction and spread of Campylobacter in a well cleaned processing plant as well as a significant survival in time on the example of a cutting board is shown. SIGNIFICANCE AND IMPACT OF THE STUDY: The need to prevent cross-contamination in the food processing and preparation area and the importance of an integrated approach throughout the whole food chain to control transmission of Campylobacter is highlighted.     

Survival of Escherichia coli and Campylobacter jejuni in untreated and filtered lake water

The survival of Campylobacter jejuni and Escherichia coli in lake water was studied using viable counts. Escherichia coli survived better than C. jejuni in all the test conditions studied. Both the species survived better in filtered than in untreated water. This suggests that predation and/or competition for nutrients affect the survival of both the species in an aquatic environment. Campylobacter jejuni survived less well in filtered autoclaved water and in 0.9% NaCl than in filtered water without autoclaving. The lack of some essential nutrients, which may be degraded by autoclaving, might explain these results.     

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.     

Survival of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at different temperatures

The survival of a human strain of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at 5°, 10°, 15° and 20°C was studied. No viable units were detected in raw milk after 24 h at 20°C and 48 h at 15°C. None were detected in pasteurized milk after 48 h at 20°C. In all other samples, there was a decline in viable units in the first 24 h but very little decline in the next 24 h period. The organism survived best at 5° and 10° C.     

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase,DsbA

Background

Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.

Methods and Results

Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.

Conclusions

Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.     

Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates,NW and D2600

The Campylobacter jejuni human clinical isolates NW and D2600 colonized C57BL/6 interleukin-10-deficient (IL-10^−/−) mice without inducing a robust inflammatory response (J. A. Bell et al., BMC Microbiol. 9:57, 2009). We announce draft genome sequences of NW and D2600 to facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model.