Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms

Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ≥ 3) led to 1 to 3 log₁₀ CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log₁₀ CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.     

Characterization of Campylobacter jejuni biofilms under defined growth conditions

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.     

Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O₂) than under microaerobic conditions (5% O₂, 10% CO₂), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.Infection with Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world and is often associated with the consumption of undercooked poultry products (19). The United Kingdom Health Protection Agency reported more than 45,000 laboratory-confirmed cases for England and Wales in 2006 alone, although this is thought to be a 5- to 10-fold underestimation of the total number of community incidents (20, 43). The symptoms associated with C. jejuni infection usually last between 2 and 5 days and include diarrhea, vomiting, and stomach pains. Sequelae of C. jejuni infection include more-serious autoimmune diseases, such as Guillain-Barré syndrome, Miller-Fisher syndrome (18), and reactive arthritis (15).Poultry represents a major natural reservoir for C. jejuni, since the organism is usually considered to be a commensal and can reach densities as high as 1 × 10⁸ CFU g of cecal contents⁻¹ (35). As a result, large numbers of bacteria are shed via feces into the environment, and consequently, C. jejuni can spread rapidly through a flock of birds in a broiler house (1). While well adapted to life in the avian host, C. jejuni must survive during transit between hosts and on food products under stressful storage conditions, including high and low temperatures and atmospheric oxygen levels. The organism must therefore have mechanisms to protect itself from unfavorable conditions.Biofilm formation is a well-characterized bacterial mode of growth and survival, where the surface-attached and matrix-encased bacteria are protected from stressful environmental conditions, such as UV radiation, predation, and desiccation (7, 8, 28). Bacteria in biofilms are also known to be >1,000-fold more resistant to disinfectants and antimicrobials than their planktonic counterparts (11). Several reports have now shown that Campylobacter species are capable of forming a monospecies biofilm (21, 22) and can colonize a preexisting biofilm (14). Biofilm formation can be demonstrated under laboratory conditions, and environmental biofilms, from poultry-rearing facilities, have been shown to contain Campylobacter (5, 32, 44). Campylobacter biofilms allow the organism to survive up to twice as long under atmospheric conditions (2, 21) and in water systems (27).Molecular understanding of biofilm formation by Campylobacter is still in its infancy, although there is evidence for the role of flagella and gene regulation in biofilm formation. Indeed, a flaAB mutant shows reduced biofilm formation (34); mutants defective in flagellar modification (cj1337) and assembly (fliS) are defective in adhering to glass surfaces (21); and a proteomic study of biofilm-grown cells shows increased levels of motility-associated proteins, including FlaA, FlaB, FliD, FlgG, and FlgG2 (22). Flagella are also implicated in adhesion and in biofilm formation and development in other bacterial species, including Aeromonas, Vibrio, Yersinia, and Pseudomonas species (3, 23, 24, 31, 42).Previous studies of Campylobacter biofilms have focused mostly on biofilm formation under standard microaerobic laboratory conditions. In this work we have examined the formation of biofilms by motile and nonmotile C. jejuni strains under atmospheric conditions that are relevant to the survival of this organism in a commercial context of environmental and food-based transmission.     

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.     

Energy Taxis Drives Campylobacter jejuni toward the Most Favorable Conditions for Growth

Campylobacter jejuni is a serious food-borne bacterial pathogen in the developed world. Poultry is a major reservoir, and C. jejuni appears highly adapted to the gastrointestinal tract of birds. Several factors are important for chicken colonization and virulence, including a taxis mechanism for environmental navigation. To explore the mechanism of chemotaxis in C. jejuni, we constructed mutants with deletions of five putative mcp (methyl-accepting chemotaxis protein) genes (tlp1, tlp2, tlp3, docB, and docC). Surprisingly, the deletions did not affect the chemotactic behavior of the mutants compared to that of the parental strain. However, the tlp1, tlp3, docB, and docC mutant strains displayed a 10-fold decrease in the ability to invade human epithelial and chicken embryo cells, hence demonstrating that the corresponding proteins affect the host interaction. l-Asparagine, formate, d-lactate, and chicken mucus were identified as new attractants of C. jejuni, and we observed that chemical substances promoting tactic attraction are all known to support the growth of this organism. The attractants could be categorized as carbon sources and electron donors and acceptors, and we furthermore observed a correlation between an attractant''s potency and its efficiency as an energy source. The tactic attraction was inhibited by the respiratory inhibitors HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) and sodium azide, which significantly reduce energy production by oxidative phosphorylation. These findings strongly indicate that energy taxis is the primary force in environmental navigation by C. jejuni and that this mechanism drives the organism toward the optimal chemical conditions for energy generation and colonization.The food-borne pathogen Campylobacter jejuni is highly adapted to the environment of the avian gut, where the mucus-filled crypts of the lower gastrointestinal tract are the primary site of colonization (6). It has been speculated that C. jejuni bacteria apply chemotaxis to reach this particular milieu (10, 42). Chemotaxis allows motile bacteria to navigate according to the extracellular chemical composition. The bacteria are either attracted or repelled by chemicals sensed by trans-membrane methyl-accepting chemotaxis proteins (MCP), and the information is transmitted to the flagellum motor via the histidine kinase CheA and the response regulator CheY. In contrast to the classical, metabolism-independent chemotaxis, some bacteria, such as Azospirillum brasilense and Rhodobacter sphaeroides, display metabolism-dependent “energy taxis” (or redox taxis) in which the signal for navigation originates within the electron transport system (1, 37).C. jejuni NCTC11168 encodes 10 MCP-like proteins, termed Tlp (transducer-like proteins), and two proteins with homology to aerotaxis receptors (Aer) (31). A taxis mechanism is essential for C. jejuni colonization, since strains with mutations of the central histidine kinase, cheA, or the response regulator, cheY, are unable to establish colonization in mice, chickens, and ferrets (10, 20, 51). Furthermore, C. jejuni strains with mutations in docB (tlp10) and docC (tlp4) are severely impaired in establishing colonization in chickens, whereas none of the other putative receptors are required for colonization (however, tlp5 could not be mutated) (20). Mutants of aer2 (cetB) and tlp9 (cetA) displayed reduced migration in minimal medium supplemented with pyruvate or fumarate, which leads to the speculation that CetA and CetB mediate an energy taxis response of C. jejuni (19).C. jejuni is attracted to amino acids, organic acids, or mucus components, while it is repelled by bile components (23). However, specific Tlp proteins have not been matched to any of these substances. It is speculated that the attraction toward chicken mucus directs and retains C. jejuni in the optimal environment of the avian intestinal lumen and thus prevents direct interaction with epithelial cells. This notion is based on in vitro observations where chicken mucus inhibited C. jejuni invasion of primary human epithelial cells, while increased invasion was observed for mutants carrying deletions of either cheA or cheY (9, 44, 51).To explore the mechanism of C. jejuni chemotaxis and analyze the biological functions of individual MCP-like proteins, we have analyzed five mutants with deletions of tlp genes (tlp1, tlp2, tlp3, docB, and docC). Furthermore, we have explored whether C. jejuni is primarily driven by chemotaxis or energy taxis.     

Characterization of cross-reacting serotypes of Campylobacter jejuni

Some strains of Campylobacter jejuni react with more than one reference antiserum from the serotyping scheme based on heat-stable lipopolysaccharide antigens. To investigate the molecular basis of these cross-reactions, lipopolysaccharides from the reference strains for serotypes 4, 13, 16, 43, and 50 and isolates recovered during two different outbreaks of C. jejuni enteritis were analyzed by passive haemagglutination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. The results showed that lipopolysaccharides from the reference strains and the isolates reacted with antisera prepared against heterologous strains in various combinations and that both silver-stainable, low Mr and non-silver-stainable, high Mr lipopolysaccharide components provided the antigenic determinants associated with the cross-reactions. There were strain-to-strain differences in the structural and antigenic properties of these macromolecules and shared antigenic determinants were not always provided by a common structure. Analysis of the silver-stained lipopolysaccharide profiles, outer membrane protein patterns, and chromosomal DNA restriction patterns indicated that strains with the same lipopolysaccharide profile could have the same outer membrane protein pattern and the same DNA restriction pattern. These results provided evidence for the presence of clones within this antigenic complex and implicated antigenic variation in some strains as the phenomenon responsible for the multiplicity of cross-reactions.     

Genomic Characterization of Campylobacter jejuni Strain M1

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1.     

Characterization of mono- and mixed-culture Campylobacter jejuni biofilms

Campylobacter jejuni, one of the most common causes of human gastroenteritis, is a thermophilic and microaerophilic bacterium. These characteristics make it a fastidious organism, which limits its ability to survive outside animal hosts. Nevertheless, C. jejuni can be transmitted to both humans and animals via environmental pathways, especially through contaminated water. Biofilms may play a crucial role in the survival of the bacterium under unfavorable environmental conditions. The goal of this study was to investigate survival strategies of C. jejuni in mono- and mixed-culture biofilms. We grew monoculture biofilms of C. jejuni and mixed-culture biofilms of C. jejuni with Pseudomonas aeruginosa. We found that mono- and mixed-culture biofilms had significantly different structures and activities. Monoculture C. jejuni biofilms did not consume a measurable quantity of oxygen. Using a confocal laser scanning microscope (CLSM), we found that cells from monoculture biofilms were alive according to live/dead staining but that these cells were not culturable. In contrast, in mixed-culture biofilms, C. jejuni remained in a culturable physiological state. Monoculture C. jejuni biofilms could persist under lower flow rates (0.75 ml/min) but were unable to persist at higher flow rates (1 to 2.5 ml/min). In sharp contrast, mixed-culture biofilms were more robust and were unaffected by higher flow rates (2.5 ml/min). Our results indicate that biofilms provide an environmental refuge that is conducive to the survival of C. jejuni.     

Explorative Multifactor Approach for Investigating Global Survival Mechanisms of Campylobacter jejuni under Environmental Conditions

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°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°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.     

Campylobacter jejuni

This review describes characteristics of the family Campylobacteraceae and traits of Campylobacter jejuni. The review then focuses on the worldwide problem of C. jejuni antimicrobial resistance and mechanisms of pathogenesis and virulence. Unravelling these areas will help with the development of new therapeutic agents and ultimately decrease illness caused by this important human pathogen.     

Cloning and Characterization of a Campylobacter jejuni Iron-Uptake Operon

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C). Received: 19 June 2000 / Accepted: 30 August 2000     

Characterization of the biochemical properties of Campylobacter jejuni RNase III

Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn²⁺ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn²⁺-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.     

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-d-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.     

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

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.Campylobacter food-borne infections are the most prevalent bacterial enteric infections in humans in industrialized and developing countries (1). It has been shown that most human infections are related to poultry meat and food produced from cattle or sheep (34, 41). Campylobacter jejuni, the species most frequently causing human disease, can be isolated from the animal intestinal tract at levels of up to 10⁹ CFU per gram of feces and can thus be called a well-adapted intestinal microorganism (30, 37). Nevertheless, because it causes human disease as a food-borne pathogen, it has to survive outside the gut. By cross-contamination at the level of the abattoir, Campylobacter bacteria hit the meat surface and have to adapt to different environmental challenges. C. jejuni is a true microaerophilic bacterium; thus, on the one hand it requires oxygen, but on the other hand it cannot grow under normal atmospheric oxygen tension conditions (15). Despite its sensitivity to high oxygen tension in vitro, viable and culturable Campylobacter bacteria can be isolated from nonskinned chicken meat at frequencies of 10⁴ CFU/g (9, 19). Assumptions on the mechanisms by which Campylobacter cells survive on meat surfaces are diverse, for example, by growing in biofilms, entering a “viable but nonculturable state,” or interacting with other microorganisms.For instance, C. jejuni is able to resist protozoa digestion and can parasitize inside protozoa, e.g., Tetrahymena pyriformis (35). This mechanism provides survival in harsh environments and resistance to antimicrobial substances and thus enhances the potential for transmission. But bacterium-bacterium interaction has also been demonstrated to be of a high level of importance for intestinal survival and uptake (20). Accordingly, members of Campylobacter have been identified to initiate cellular uptake of commensal bacteria into enterocytes (14). However, a bacterial community can also mean competition, e.g., bacteriocin production by Lactobacillus salivarius that is effective against Campylobacter colonization (36).Meat surfaces harbor numerous bacterial species (24). Some of these bacteria have adapted to this specific environmental niche and are well-known spoilage bacteria. Most relevant species belong to the family Pseudomonadaceae. But also different members of the Enterobacteriaceae can be found on meat. To date, information regarding the interaction between spoilage bacteria and pathogens is of increasing importance for public health safety measures.Hence, experimental data on the survival of C. jejuni isolates in the presence of selected meat-spoiling bacteria were analyzed and clearly demonstrated a specific interaction with type strains and isolates of Pseudomonas putida, Pseudomonas fragi, and Pseudomonas fluorescens from chicken meat surfaces.     

Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.