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Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions.

ABSTRACT: Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was introduced to calculate false-discovery rates for each response. This novel analytical concept was used to investigate global survival mechanisms in the environment of the major food-borne pathogen C. jejuni. We simulated nongrowth environmental conditions by investigating combinations of the factors temperature (5 and 25 degrees C) and oxygen tension (anaerobic, microaerobic, and aerobic). Data were generated with DNA microarrays for information about gene expression patterns and Fourier transform infrared (FT-IR) spectroscopy to study global macromolecular changes in the cell. Microarray analyses showed that most genes were either unchanged or down regulated compared to the reference (day 0) for the conditions tested and that the 25 degrees C anaerobic condition gave the most distinct expression pattern with the fewest genes expressed. The few up-regulated genes were generally stress related and/or related to the cell envelope. We found, using FT-IR spectroscopy, that the amount of polysaccharides and oligosaccharides increased under the nongrowth survival conditions. Potential mechanisms for survival could be to down regulate most functions to save energy and to produce polysaccharides and oligosaccharides for protection against harsh environments. Basic knowledge about the survival mechanisms is of fundamental importance in preventing transmission of this bacterium through the food chain.

SUBMITTER: Moen B

PROVIDER: S-EPMC1082531 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions.

Moen Birgitte B Oust Astrid A Langsrud Øyvind Ø Dorrell Nick N Marsden Gemma L GL Hinds Jason J Kohler Achim A Wren Brendan W BW Rudi Knut K

Applied and environmental microbiology 20050401 4

Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was intro ...[more]

PMID: 15812042

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Project description:Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonas putida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.

Project description:BackgroundMacrolide is the drug of choice to treat human campylobacteriosis, but Campylobacter resistance to this antibiotic is rising. The mechanisms employed by Campylobacter jejuni to adapt to erythromycin treatment remain unknown and are examined in this study. The transcriptomic response of C. jejuni NCTC 11168 to erythromycin (Ery) treatment was determined by competitive microarray hybridizations. Representative genes identified to be differentially expressed were further characterized by constructing mutants and assessing their involvement in antimicrobial susceptibility, oxidative stress tolerance, and chicken colonization.ResultsFollowing the treatment with an inhibitory dose of Ery, 139 genes were up-regulated and 119 were down-regulated. Many genes associated with flagellar biosynthesis and motility was up-regulated, while many genes involved in tricarboxylic acid cycle, electron transport, and ribonucleotide biosynthesis were down-regulated. Exposure to a sub-inhibitory dose of Ery resulted in differential expression of much fewer genes. Interestingly, two putative drug efflux operons (cj0309c-cj0310c and cj1173-cj1174) were up-regulated. Although mutation of the two operons did not alter the susceptibility of C. jejuni to antimicrobials, it reduced Campylobacter growth under high-level oxygen. Another notable finding is the consistent up-regulation of cj1169c-cj1170c, of which cj1170c encodes a known phosphokinase, an important regulatory protein in C. jejuni. Mutation of the cj1169c-cj1170c rendered C. jejuni less tolerant to atmospheric oxygen and reduced Campylobacter colonization and transmission in chickens.ConclusionsThese findings indicate that Ery treatment elicits a range of changes in C. jejuni transcriptome and affects the expression of genes important for in vitro and in vivo adaptation. Up-regulation of motility and down-regulation of energy metabolism likely facilitate Campylobacter to survive during Ery treatment. These findings provide new insight into Campylobacter adaptive response to antibiotic treatment and may help to understand the mechanisms underlying antibiotic resistance development.

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Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions.

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Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions.

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