Atomic structure of the Campylobacter jejuni flagellar filament reveals how ε Proteobacteria escaped Toll-like receptor 5 surveillance.

Vertebrates, from zebra fish to humans, have an innate immune recognition of many bacterial flagellins. This involves a conserved eight-amino acid epitope in flagellin recognized by the Toll-like receptor 5 (TLR5). Several important human pathogens, such as Helicobacter pylori and Campylobacter jejuni, have escaped TLR5 activation by mutations in this epitope. When such mutations were introduced into Salmonella flagellin, motility was abolished. It was previously argued, using very low-resolution cryoelectron microscopy (cryo-EM), that C. jejuni accommodated these mutations by forming filaments with 7 protofilaments, rather than the 11 found in other bacteria. We have now determined the atomic structure of the C. jejuni G508A flagellar filament from a 3.5-Å-resolution cryo-EM reconstruction, and show that it has 11 protofilaments. The residues in the C. jejuni TLR5 epitope have reduced contacts with the adjacent subunit compared to other bacterial flagellar filament structures. The weakening of the subunit-subunit interface introduced by the mutations in the TLR5 epitope is compensated for by extensive interactions between the outer domains of the flagellin subunits. In other bacteria, these outer domains can be nearly absent or removed without affecting motility. Furthermore, we provide evidence for the stabilization of these outer domain interactions through glycosylation of key residues. These results explain the essential role of glycosylation in C. jejuni motility, and show how the outer domains have evolved to play a role not previously found in other bacteria.

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.

Association of some Campylobacter jejuni with Pseudomonas aeruginosa biofilms increases attachment under conditions mimicking those in the environment.

Campylobacter jejuni is a microaerophilic bacterial species which is a major food-borne pathogen worldwide. Attachment and biofilm formation have been suggested to contribute to the survival of this fastidious bacteria in the environment. In this study the attachment of three C. jejuni strains (C. jejuni strains 2868 and 2871 isolated from poultry and ATCC 33291) to different abiotic surfaces (stainless steel, glass and polystyrene) alone or with Pseudomonas aeruginosa biofilms on them, in air at 25°C and under static or flow conditions, were investigated using a modified Robbins Device. Bacteria were enumerated and scanning electron microscopy was carried out. The results indicated that both C. jejuni strains isolated from poultry attached better to Pseudomonas aeruginosa biofilms on abiotic surfaces than to the surfaces alone under the different conditions tested. This suggests that biofilms of other bacterial species may passively protect C. jejuni against shear forces and potentially oxygen stress which then contribute to their persistence in environments which are detrimental to them. By contrast the C. jejuni ATCC 33291 strain did not attach differentially to P. aeruginosa biofilms, suggesting that different C. jejuni strains may have alternative strategies for persistence in the environment. This study supports the hypothesis that C. jejuni do not form biofilms per se under conditions they encounter in the environment but simply attach to surfaces or biofilms of other species.

Genomic correlates of extraintestinal infection are linked with changes in cell morphology in Campylobacter jejuni.

Campylobacter jejuni is the most common cause of bacterial diarrheal disease in the world. Clinical outcomes of infection can range from asymptomatic infection to life-threatening extraintestinal infections. This variability in outcomes for infected patients has raised questions as to whether genetic differences between C. jejuni isolates contribute to their likelihood of causing severe disease. In this study, we compare the genomes of ten C. jejuni isolates that were implicated in extraintestinal infections with reference gastrointestinal isolates, in order to identify unusual patterns of sequence variation associated with infection outcome. We identified a collection of genes that display a higher burden of uncommon mutations in invasive isolates compared with gastrointestinal close relatives, including some that have been previously linked to virulence and invasiveness in C. jejuni. Among the top genes identified were mreB and pgp1, which are both involved in determining cell shape. Electron microscopy confirmed morphological differences in isolates carrying unusual sequence variants of these genes, indicating a possible relationship between extraintestinal infection and changes in cell morphology.

High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly.

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

Genomic variations leading to alterations in cell morphology of Campylobacter spp.

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens.

BACKGROUND: Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens. RESULTS: Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. The minimal inhibitory concentrations of MgO nanoparticles to 10(4) colony-forming unit/ml (CFU/ml) of Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella Enteritidis were determined to be 0.5, 1 and 1 mg/ml, respectively. To completely inactivate 10(8-9) CFU/ml bacterial cells in 4 h, a minimal concentration of 2 mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8 mg/ml nanoparticles. Scanning electron microscopy examination revealed clear morphological changes and membrane structural damage in the cells treated with MgO nanoparticles. A quantitative real-time PCR combined with ethidium monoazide pretreatment confirmed cell membrane permeability was increased after exposure to the nanoparticles. In a cell free assay, a low level (1.1 µM) of H2O2 was detected in the nanoparticle suspensions. Consistently, MgO nanoparticles greatly induced the gene expression of KatA, a sole catalase in C. jejuni for breaking down H2O2 to H2O and O2. CONCLUSIONS: MgO nanoparticles have strong antibacterial activity against three important foodborne pathogens. The interaction of nanoparticles with bacterial cells causes cell membrane leakage, induces oxidative stress, and ultimately leads to cell death.

Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging.

Flagella-mediated adhesion and extracellular DNA release contribute to biofilm formation and stress tolerance of Campylobacter jejuni.

Campylobacter jejuni is a leading cause of foodbourne gastroenteritis, despite fragile behaviour under standard laboratory conditions. In the environment, C. jejuni may survive within biofilms, which can impart resident bacteria with enhanced stress tolerance compared to their planktonic counterparts. While C. jejuni forms biofilms in vitro and in the wild, it had not been confirmed that this lifestyle confers stress tolerance. Moreover, little is understood about molecular mechanisms of biofilm formation in this pathogen. We previously found that a ΔcprS mutant, which carries a deletion in the sensor kinase of the CprRS two-component system, forms enhanced biofilms. Biofilms were also enhanced by the bile salt deoxycholate and contained extracellular DNA. Through more in-depth analysis of ΔcprS and WT under conditions that promote or inhibit biofilms, we sought to further define this lifestyle for C. jejuni. Epistasis experiments with ΔcprS and flagellar mutations (ΔflhA, ΔpflA) suggested that initiation is mediated by flagellum-mediated adherence, a process which was kinetically enhanced by motility. Lysis was also observed, especially under biofilm-enhancing conditions. Microscopy suggested adherence was followed by release of eDNA, which was required for biofilm maturation. Importantly, inhibiting biofilm formation by removal of eDNA with DNase decreased stress tolerance. This work suggests the biofilm lifestyle provides C. jejuni with resilience that has not been apparent from observation of planktonic bacteria during routine laboratory culture, and provides a framework for subsequent molecular studies of C. jejuni biofilms.

Ultrastructure and complex polar architecture of the human pathogen Campylobacter jejuni.

Campylobacter jejuni is one of the most successful food-borne human pathogens. Here we use electron cryotomography to explore the ultrastructure of C. jejuni cells in logarithmically growing cultures. This provides the first look at this pathogen in a near-native state at macromolecular resolution (~5 nm). We find a surprisingly complex polar architecture that includes ribosome exclusion zones, polyphosphate storage granules, extensive collar-shaped chemoreceptor arrays, and elaborate flagellar motors.

Bacteriophage receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.

In vivo modulation of Campylobacter jejuni virulence in response to environmental stress.

Campylobacters have developed a number of mechanisms for responding to environmental conditions, although the different virulence properties of these cells following exposure to stress are still poorly understood. We analyzed in vitro stress responses and the consequent in vivo modulation of Campylobacter jejuni pathogenicity in BALB/c mice, as a result of the exposure of the C. jejuni to environmental stress (starvation, oxidative stress, heat shock). In vitro, the influence of starvation and oxidative stress was milder than that of heat shock, although the majority of the stress conditions influenced the survival of C. jejuni. During starvation, C. jejuni viability was maintained longer than its culturability. Additionally, starvation elicited transformation of stressed bacteria to coccoid forms. In contrast, bacteria exposed to oxygen remained culturable, but their viability decreased. Pre-starvation did not contribute to improved survival of C. jejuni cells during oxygen exposure. Changes in bacteria numbers and the levels of several cytokines (interleukins 6 and 10, tumor necrosis factor-α, interferon-γ) were followed in vivo, in liver homogenates from the mice intravenously infected with either control (untreated) or stressed C. jejuni. The systemic infection with the control or stressed C. jejuni occurred with different production dynamics of the cytokines investigated. Starvation was the most powerful stress factor, which significantly decreased infectious potential of C. jejuni during the first 3 days postinfection. The most pronounced differences in cytokine production were found in interferon-γ and interleukin-10 production, which indicates that these have roles in the immune response to C. jejuni infection. These in vivo studies of environmental impact on bacterial virulence reveal that microbial adaptation during stress challenge is crucial not just for pathogen survival out of the host, but also during host-pathogen interactions, and thus for the bacterial pathogenicity.

Development of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli adapted to biocides.

The potential for adaptive resistance of Campylobacter jejuni and Campylobacter coli after step-wise exposure to increasing sub-inhibitory concentrations of five biocides as triclosan, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate and trisodium phosphate, was investigated, to identify the mechanisms underlying resistance. The biocide resistance and cross-resistance to the antimicrobials erythromycin and ciprofloxacin, and to sodium dodecyl sulphate, were examined according to the broth microdilution method. The presence of active efflux was studied on the basis of restored sensitivity in the presence of the efflux pump inhibitors phenylalanine-arginine beta-naphthylamide, 1-(1-naphthylmethyl)-piperazine, cyanide 3-chlorophenylhydrazone, verapamil and reserpine. Changes in the outer membrane protein profiles and morphological changes in adapted strains were studied, as compared with the parent strains. Repeated exposure of C. jejuni and C. coli to biocides resulted in partial increases in tolerance to biocides itself, to other biocides and antimicrobial compounds. The developed resistance was stable for up to 10 passages in biocide-free medium. More than one type of active efflux was identified in adapted strains. These adapted strains showed different alterations to their outer membrane protein profiles, along with morphological changes. The data presented here suggest that different mechanisms are involved in adaptation to biocides and that this adaptation is unique to each strain of Campylobacter and does not result from a single species-specific mechanism.

Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen.

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.

Investigating antibacterial effects of garlic (Allium sativum) concentrate and garlic-derived organosulfur compounds on Campylobacter jejuni by using Fourier transform infrared spectroscopy, Raman spectroscopy, and electron microscopy.

Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were used to study the cell injury and inactivation of Campylobacter jejuni from exposure to antioxidants from garlic. C. jejuni was treated with various concentrations of garlic concentrate and garlic-derived organosulfur compounds in growth media and saline at 4, 22, and 35°C. The antimicrobial activities of the diallyl sulfides increased with the number of sulfur atoms (diallyl sulfide < diallyl disulfide < diallyl trisulfide). FT-IR spectroscopy confirmed that organosulfur compounds are responsible for the substantial antimicrobial activity of garlic, much greater than those of garlic phenolic compounds, as indicated by changes in the spectral features of proteins, lipids, and polysaccharides in the bacterial cell membranes. Confocal Raman microscopy (532-nm-gold-particle substrate) and Raman mapping of a single bacterium confirmed the intracellular uptake of sulfur and phenolic components. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to verify cell damage. Principal-component analysis (PCA), discriminant function analysis (DFA), and soft independent modeling of class analogs (SIMCA) were performed, and results were cross validated to differentiate bacteria based upon the degree of cell injury. Partial least-squares regression (PLSR) was employed to quantify and predict actual numbers of healthy and injured bacterial cells remaining following treatment. PLSR-based loading plots were investigated to further verify the changes in the cell membrane of C. jejuni treated with organosulfur compounds. We demonstrated that bacterial injury and inactivation could be accurately investigated by complementary infrared and Raman spectroscopies using a chemical-based, "whole-organism fingerprint" with the aid of chemometrics and electron microscopy.

Major host factors involved in epithelial cell invasion of Campylobacter jejuni: role of fibronectin, integrin beta1, FAK, Tiam-1, and DOCK180 in activating Rho GTPase Rac1.

Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin(-/-), integrin beta1(-/-), and focal adhesion kinase (FAK)(-/-) deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin(-/-), integrin beta1(-/-), and FAK(-/-) knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells.

Susceptibility of Campylobacter jejuni to organic acids and monoacylglycerols.

Organic acids can be used as feed supplements or for treatment of poultry carcasses in processing plants. The antimicrobial activity of nineteen organic acids and two monoacylglycerols in cultures of Campylobacter jejuni CCM 6214(T) (ATCC 33560) was determined using a SYBR Green-based real-time PCR assay. The IC(50) was a concentration at which only 50 % of a bacteria specific DNA sequence was amplified. Caprylic, capric and lauric acids were the most efficient antimicrobials among the compounds tested (IC(50) < or = 0.1 mg/mL). In a weakly acidic environment (pH 5.5), the antimicrobial activity was more pronounced than at pH 6.5. At pH 5.5, oleic and fumaric acid also had clear antimicrobial activity, as did monocaprylin. The antimicrobial activity of acetic, butyric, stearic and succinic acid was low. In cells treated with fumaric acid, the potential of potassium and tetraphenylphosphonium ion-selective electrodes changed, indicating an increase in cytoplasmic and outer membrane permeability, respectively. No changes in membrane permeability were observed in cells treated with capric acid or monocaprin. Transmission electron microscopy revealed separation of the inner and outer membrane in cells treated with capric and fumaric acid, as well as cytoplasmic disorganization in cells exposed to capric acid.

Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length.

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food-borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti-sigma factor proteins: it represses transcription of sigma(28)-dependent genes, forms a complex with sigma factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook-basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the sigma(28)- and the sigma(54)-dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence sigma(28)-dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

In vitro susceptibility to antimicrobial agents and ultrastructural characteristics related to swimming motility and drug action in Campylobacter jejuni and C. coli.

Campylobacter jejuni has recently been noted as the most common cause of bacterial food-borne diseases in Japan. In this study, we examined in vitro susceptibility to 36 antimicrobial agents of 109 strains of C. jejuni and C. coli isolated from chickens and patients with enteritis or Guillain-Barré syndrome from 1996 to 2009. Among these agents, carbapenems (imipenem, meropenem, panipenem, and biapenem) showed the greatest activity [minimal inhibitory concentration (MIC)(90), 0.03-0.125 microg/ml]. This was followed by sitafloxacin (MIC(90), 0.25 microg/ml), furazolidone and azithromycin (MIC(90), 0.5 microg/ml), gentamicin and clindamycin (MIC(90), 1 microg/ml), and clavulanic acid (beta-lactamase inhibitor; MIC(90), 2 microg/ml). All or most strains were resistant to aztreonam, sulfamethoxazole, and trimethoprim. Marked resistance was also observed for levofloxacin and tetracyclines. Resistance was not present for macrolides and rare for clindamycin. C. jejuni (and C. coli) exhibited high swimming motility and possessed a unique end-side (cup-like) structure at both ends, in contrast to Helicobacter pylori and Vibrio cholerae O1 and O139. The morphology of C. jejuni (and C. coli) changed drastically after exposure to imipenem (coccoid formation), meropenem (bulking and slight elongation), and sitafloxacin (marked elongation), and exhibited reduced motility. In the HEp-2 cell adherence model, unusually elongated bacteria were also observed for sitafloxacin. The data suggest that although resistance to antimicrobial agents (e.g., levofloxacin) has continuously been noted, carbapenems, sitafloxacin, and others such as beta-lactamase inhibitors alone showed good in vitro activity and that C. jejuni (and C. coli) demonstrated a unique ultrastructural nature related to high swimming motility and drug action.

The mode of biofilm formation on smooth surfaces by Campylobacter jejuni.

Many microorganisms produce extracellular polymers referred to collectively as "slime" or glycocalyx, and form biofilms on solid surfaces in natural ecosystems. Campylobacter jejuni, one of the most important foodborne pathogens, also has the ability to form biofilm on stainless steel, glass, or polyvinyl chloride in vitro. However, the issue of biofilm formation by Campylobacter species has not been extensively examined. The present study was performed to examine the mode of adhesion of C. jejuni to a smooth surface. When bacterial suspensions in Brucella broth were incubated in microplate wells with a glass coverslip, microcolonies 0.5~2 mm in diameter were formed on the coverslip within 2 hr from the start of incubation. These microcolonies gradually grew and formed a biofilm of net-like connections within 6 hr. Transmission electron microscopy indicated that massive amounts of extracellular material masked the cell surface, and this material bound ruthenium red, suggesting the presence of a polysaccharide moiety. Scanning electron microscopy indicated that the flagella acted as bridges, forming net-like connections between the organisms. To determine the genes associated with biofilm formation, aflagellate (flaA(-)) and flagellate but non-motile (motA(-)) mutants were constructed from strain 81-176 by natural transformation-mediated allelic exchange. The flaA(-) and motA(-) mutants did not form the biofilm exhibited by the wild-type strain. These findings suggest that flagella-mediated motility as well as flagella is required for biofilm formation in vitro.