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The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

The Gram-negative bacterial pathogen Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide. Rapid detection and identification of C. jejuni informs timely prescription of appropriate therapeutics and epidemiological investigations. Here, for the first time, we report the applicability of Raman spectroscopy, surface-enhanced Raman scattering (SERS) and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF-MS) combined with chemometrics, for rapid differentiation and characterisation of mutants of a single isogenic C. jejuni strain that disrupt the production of prominent surface features (capsule, flagella and glycoproteins) of the bacterium. Multivariate analysis of the spectral data obtained from these different physicochemical tools revealed distinctive biochemical differences which consistently discriminated between these mutants. In order to generate biochemical and phenotypic information from different locations in the cell-cell wall versus cytoplasm - we developed two different in situ methods for silver nanoparticle (AgNP) production, and compared this with simple mixing of bacteria with pre-synthesised AgNPs. This SERS trilogy (simple mixing with premade AgNPs and two in situ AgNP production methods) presents an integrated platform with potential for rapid, accurate and confirmatory detection of pathogenic bacteria based on cell envelope or intracellular molecular dynamics. Our spectral findings demonstrate that Raman, SERS and MALDI-TOF-MS are powerful metabolic fingerprinting techniques capable of discriminating clinically relevant cell wall mutants of a single isogenic bacterial strain.

The high prevalence of Campylobacter spp. in retail liver products was previously reported and has been linked to several outbreaks of campylobacteriosis. The main objective of this study was to investigate the influence of retail liver juices on the survivability of several strains of C. jejuni and C. coli, which were previously isolated from various retail meats at 4 °C. All tested Campylobacter strains showed higher survival in beef liver juice (BLJ) and chicken liver juice (CLJ) as compared to beef and chicken juices (BJ and CJ) or Mueller Hinton broth (MHB) at 4 °C. Overall, C. jejuni strains showed greater survival in retail liver and meat juices as compared to C. coli. CLJ enhanced biofilm formation of most C. coli strains and supported growth in favorable conditions. When diluted, retail liver and meat juices enhanced survival of Campylobacter strains at low temperatures and increased aerotolerance. In conclusion, beef and chicken liver juices enhanced the survival of C. jejuni and C. coli strains at low temperatures, which helps explain the high prevalence of Campylobacter spp. in retail liver products.

Campylobacter jejuni is the leading cause of bacterial diarrhoeal disease in many areas of the world. The high incidence of sporadic cases of disease in humans is largely due to its prevalence as a zoonotic agent in animals, both in agriculture and in the wild. Compared with many other enteric bacterial pathogens, C. jejuni has strict growth and nutritional requirements and lacks many virulence and colonization determinants that are typically used by bacterial pathogens to infect hosts. Instead, C. jejuni has a different collection of factors and pathways not typically associated together in enteric pathogens to establish commensalism in many animal hosts and to promote diarrhoeal disease in the human population. In this Review, we discuss the cellular architecture and structure of C. jejuni, intraspecies genotypic variation, the multiple roles of the flagellum, specific nutritional and environmental growth requirements and how these factors contribute to in vivo growth in human and avian hosts, persistent colonization and pathogenesis of diarrhoeal disease.

Campylobacter jejuni cells have bipolar flagella. Both flagella have similar lengths of about one helical turn, or 3.53±0.52 µm. The flagellar filament is composed of two homologous flagellins: FlaA and FlaB. Mutant strains that express either FlaA or FlaB alone produce filaments that are shorter than those of the wild-type. It is reported that the flaG gene could affect filament length in some species of bacteria, but its function remains unknown. We introduced a flaG-deletion mutation into the C. jejuni wild-type strain and flaA- or flaB-deletion mutant strains, and observed their flagella by microscopy. The ΔflaG mutant cells produced long filaments of two helical turns in the wild-type background. The ΔflaAG double mutant cells produced very short FlaB filaments. On the other hand, ΔflaBG double mutant cells produced long FlaA filaments and their morphology was not helical but straight. Furthermore, FlaG was secreted, and a pulldown assay showed that sigma factor 28 was co-precipitated with purified polyhistidine-tagged FlaG. We conclude that FlaG controls flagella length by negatively regulating FlaA filament assembly and discuss the role of FlaA and FlaB flagellins in C. jejuni flagella formation.

Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81-176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation. Whole genome sequencing of CFW-passaged strains showed no specific changes correlating to CFW exposure. The cjj81176_1279 (recR; recombinational DNA repair) and cjj81176_1449 (unknown function) genes were highly variable in all 81-176 strains sequenced. A frameshift mutation in pgp1 of our laboratory isolate of the straight genome sequenced variant of 11168 (11168-GS) was also identified. The PG muropeptide profile of 11168-GS was identical to that of Δpgp1 in the original minimally passaged 11168 strain (11168-O). Introduction of wild type pgp1 into 11168-GS did not restore helical morphology. The recR gene was also highly variable in 11168 strains. Microbial cell-to-cell heterogeneity is proposed as a mechanism of ensuring bacterial survival in sub-optimal conditions. In certain environments, changes in C. jejuni morphology due to genetic heterogeneity may promote C. jejuni survival., (© 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped 'wild type' C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

1. The aim was to determine the importance of a contaminated diet as a possible cause of Campylobacter jejuni infection in broilers. 2. This study evaluated the viability of C. jejuni in both starter and finisher diets and the interference from other mesophilic bacteria in this viability. 3. Starter and finisher samples of broiler diet were deliberately contaminated with 3 or 5 log CFU·g -1 of C. jejuni (NCTC 11351) and then maintained at two different storage temperatures (25°C or 37°C) for 3 or 5 d. 4. C. jejuni survived during this period and, when inoculated at 10 3 CFU·g -1 , multiplied with greater proliferation at a storage temperature of 37°C. There was no relationship between the amount of mesophilic bacteria and C. jejuni viability. 5. This study highlights the importance of the diet in the epidemiology of C. jejuni in broilers.

Campylobacter jejuni is a helix-shaped enteric bacterial pathogen and a common cause of gastroenteritis. We recently developed a mouse model for this human pathogen utilizing the SIGIRR-deficient mouse strain, which exhibits significant intestinal inflammation in response to intestinal C. jejuni infection. In the current study, this mouse model was used to define whether C. jejuni's characteristic helical shape plays a role in its ability to colonize and elicit inflammation in the mouse intestine. Mice were infected with the previously characterized straight-rod Δpgp1 and Δpgp2 mutant strains, along with a newly characterized curved-rod Δ1228 mutant strain. We also compared the resultant infections and pathology to those elicited by the helix-shaped wild-type C. jejuni and complemented strains. Despite displaying wild-type colonization of the intestinal lumen, the straight-rod Δpgp1 and Δpgp2 mutants were essentially nonpathogenic, while all strains with a curved or helical shape retained their expected virulence. Furthermore, analysis of C. jejuni localization within the ceca of infected mice determined that the primary difference between the rod-shaped, nonpathogenic mutants and the helix-shaped, pathogenic strains was the ability to colonize intestinal crypts. Rod-shaped mutants appeared unable to colonize intestinal crypts due to an inability to pass through the intestinal mucus layer to directly contact the epithelium. Together, these results support a critical role for C. jejuni's helical morphology in enabling it to traverse and colonize the mucus-filled intestinal crypts of their host, a necessary step required to trigger intestinal inflammation in response to C. jejuni., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Studies have collectively shown the wide impact that luxS mutation has on the expression and function of various aspects of Campylobacter jejuni virulence. Previous work from our group demonstrated that LuxS mutagenesis negatively impacts colonization of the gastrointestinal tract of several host species. To determine what is responsible for the colonization defect, we used a mechanistic approach to understand how the luxS mutation affects the expression of key physiologic factors important to the colonization ability of C. jejuni. This included expression of genes from the CmeABC efflux system, cell morphology, and motility through mucin substrate between wildtype, luxS mutant, and luxS complement of the C. jejuni strains 11168 and/or IA3902. We also measured and compared the activated methyl cycle (AMC) metabolite levels of the IA3902 luxS mutant to wildtype. Results showed that mutagenesis of the luxS gene completely disrupted the AMC with altered concentrations of AMC metabolites both upstream and downstream of LuxS. Multidrug efflux pump genes cmeABC and cmeR showed no significant changes in expression levels within the luxS mutant. Though motility through mucin was not completely unaffected by the luxS mutation, the lack of differences in cell morphology between wildtype and luxS mutant suggest that morphology is not responsible for the slight changes in mucin penetration observed in one of our luxS mutants. Though additional studies are warranted, these findings suggest that the CmeABC multi-drug efflux pump, cell morphology and mucin penetration are not major mechanisms responsible for the luxS mutant's colonization defect in its host., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.

Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.

Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of the Campylobacter jejuni Tlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix. In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm.

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with Bac Light biovolume analysis and conventional plate counting for the enumeration of C. jejuni-removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or Bac Light biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the Bac Light assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

Previous studies have identified a specific modification of the capsular polysaccharide as receptor for phages that infect Campylobacter jejuni. Using acapsular kpsM mutants of C. jejuni strains NCTC11168 and NCTC12658, we found that bacteriophage F341 infects C. jejuni independently of the capsule. In contrast, phage F341 does not infect C. jejuni NCTC11168 mutants that either lack the flagellar filaments (ΔflaAB) or that have paralyzed, i.e., nonrotating, flagella (ΔmotA and ΔflgP). Complementing flgP confirmed that phage F341 requires rotating flagella for successful infection. Furthermore, adsorption assays demonstrated that phage F341 does not adsorb to these nonmotile C. jejuni NCTC11168 mutants. Taken together, we propose that phage F341 uses the flagellum as a receptor. Phage-host interactions were investigated using fluorescence confocal and transmission electron microscopy. These data demonstrate that F341 binds to the flagellum by perpendicular attachment with visible phage tail fibers interacting directly with the flagellum. Our data are consistent with the movement of the C. jejuni flagellum being required for F341 to travel along the filament to reach the basal body of the bacterium. The initial binding to the flagellum may cause a conformational change of the phage tail that enables DNA injection after binding to a secondary receptor., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

Vitamin B6 (pyridoxal-5'-phosphate, PLP) is linked to a variety of biological functions in prokaryotes. Here, we report that the pdxA (putative 4-hydroxy-L-threonine phosphate dehydrogenase) gene plays a pivotal role in the PLP-dependent regulation of flagellar motility, thereby altering host colonization in a leading foodborne pathogen, Campylobacter jejuni. A C. jejuni pdxA mutant failed to produce PLP and exhibited a coincident loss of flagellar motility. Mass spectrometric analyses showed a 3-fold reduction in the main flagellar glycan pseudaminic acid (Pse) associated with the disruption of pdxA. The pdxA mutant also exhibited reduced growth rates compared with the WT strain. Comparative metabolomic analyses revealed differences in respiratory/energy metabolism between WT C. jejuni and the pdxA mutant, providing a possible explanation for the differential growth fitness between the two strains. Consistent with the lack of flagellar motility, the pdxA mutant showed impaired motility-mediated responses (bacterial adhesion, ERK1/2 activation, and IL-8 production) in INT407 cells and reduced colonization of chickens compared with the WT strain. Overall, this study demonstrated that the pdxA gene affects the PLP-mediated flagellar motility function, mainly through alteration of Pse modification, and the disruption of this gene also alters the respiratory/energy metabolisms to potentially affect host colonization. Our data therefore present novel implications regarding the utility of PLP and its dependent enzymes as potent target(s) for the control of this pathogen in the poultry host.

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific "successful alleles" at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a "successful"Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the "unsuccessful"Cj0045 11G allele experienced phase variation to "successful" 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the "successful" 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tetrathionate (S4 O6 (2-) ) is used by some bacteria as an electron acceptor and can be produced in the vertebrate intestinal mucosa from the oxidation of thiosulphate (S2 O3 (2-) ) by reactive oxygen species during inflammation. Surprisingly, growth of the microaerophilic mucosal pathogen Campylobacter jejuni under oxygen-limited conditions was stimulated by tetrathionate, although it does not possess any known type of tetrathionate reductase. Here, we identify a dihaem cytochrome c (C8j_0815; TsdA) as the enzyme responsible. Kinetic studies with purified recombinant C. jejuni TsdA showed it to be a bifunctional tetrathionate reductase/thiosulphate dehydrogenase with a high affinity for tetrathionate. A tsdA null mutant still slowly reduced, but could not grow on, tetrathionate under oxygen limitation, lacked thiosulphate-dependent respiration and failed to convert thiosulphate to tetrathionate microaerobically. A TsdA paralogue (C8j_0040), lacking the unusual His-Cys haem ligation of TsdA, had low thiosulphate dehydrogenase and tetrathionate reductase activities. Our data highlight a hitherto unrecognized capacity of C. jejuni to use tetrathionate and thiosulphate in its energy metabolism, which may promote growth in the host. Moreover, as TsdA represents a new class of tetrathionate reductase that is widely distributed among bacteria, we predict that energy conserving tetrathionate respiration is far more common than currently appreciated., (© 2013 Blackwell Publishing Ltd.)

Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.

Background: Inorganic polyphosphate (poly P) plays an important role in stress tolerance and virulence in many bacteria. PPK1 is the principal enzyme involved in poly P synthesis, while PPK2 uses poly P to generate GTP, a signaling molecule that serves as an alternative energy source and a precursor for various physiological processes. Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans, possesses homologs of both ppk1 and ppk2. ppk1 has been previously shown to impact the pathobiology of C. jejuni., Methodology/principal Findings: Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The Deltappk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Deltappk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Deltappk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization., Conclusions/significance: Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. PPK2 is absent in humans and animals; therefore, can serve as a novel target for therapeutic intervention of C. jejuni infections.

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Deltappk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Deltappk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Deltappk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Deltappk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.

Campylobacter spp. are one of the leading bacterial etiologic agents of acute human gastroenteritis among industrialized countries. Poultry are implicated as a major source of the organism for human illness; however, the factors involved with colonization of poultry gastrointestinal systems remain unclear. Genomics and proteomics analyses were used to identify differences between poor- versus robust-colonizing Campylobacter jejuni isolates, 11168(GS) and A74/C, respectively. Sequence analyses of subtracted DNA resulted in A74/C-specifc genes similar to a dimethyl sulfoxide reductase, a serine protease, polysaccharide modification proteins, and restriction modification proteins. DNA microarray analyses were performed for comparison of A74/C to the complete genome sequences published for two C. jejuni. A total of 114 genes (7.1%) were determined absent from A74/C relative to those genomes. Additionally, proteomics was completed on both soluble and membrane protein extracts from 11168(GS) and A74/C. Variation in protein expression and physical characteristics such as pI was detected between the two isolates that included the major outer membrane protein, flagella, and aconitate hydratase. Several proteins including cysteine synthase and a Ni/Fe hydrogenase were determined to be differentially present between the two isolates. Finally, DNA hybridization analyses of 19 C. jejuni isolates recovered from chickens and humans worldwide over the past 20 years were performed to determine the distribution of a subset of differentially identified gene sequences.

Under conditions of stress, cells of Campylobacter assume a coccoid shape that may be an evolutionary strategy evolved by the organism to enable survival between hosts. However, the physiology of Campylobacter as it devolves from spiral to coccoid-shaped morphology is poorly understood. In this study, conditions influencing the survival of Campylobacter jejuni ATCC 35921 in broth were determined. Cells in late log phase were resuspended in broth at 4 or 60 degrees C. The culturability of these cold- or heat-stressed cell suspensions was determined by spread plate counts and the activity of cells by the direct viable count technique and 5-cyano-2,3-ditolyltetrazolium chloride staining. C. jejuni changed form completely from culturable to viable but nonculturable cells (VBNC) within 25 days at 4 degrees C, and 15 min at 60 degrees C. Light microscopy of C. jejuni VBNC cells showed that the spiral-shaped cells became coccoid, and transmission electron microscopy of C. jejuni VBNC cells showed that the outer membrane was lost in aging cell suspensions. Furthermore, a limited proteomic study was carried out to compare C. jejuni proteins that exhibited increased or decreased synthesis on exposure to 60 degrees C.

The PEB1a protein of the gastrointestinal pathogen Campylobacter jejuni mediates interactions with epithelial cells and is an important factor in host colonization. Cell fractionation and immunoblotting showed that PEB1a is most abundant in the periplasm of C. jejuni, and is detectable in the culture supernatant but not in the inner or outer membrane. The protein is homologous with periplasmic-binding proteins associated with ABC transporters and we show by fluorescence spectroscopy that purified recombinant PEB1a binds L-aspartate and L-glutamate with sub microM K(d) values. Binding of L-14C-aspartate or L-14C-glutamate was strongly out-competed by excess unlabelled aspartate or glutamate but only poorly by asparagine and glutamine. A mutant in the Cj0921c gene, encoding PEB1a, was completely unable to transport 5 microM L-14C-glutamate and showed a large reduction (approximately 20-fold) in the rate of L-14C-aspartate transport compared with the wild type. Although microaerobic growth of this mutant was little affected in complex media, growth on aspartate or glutamate in defined media was completely prevented, whereas growth with serine was similar to wild type. 1H-NMR analysis of the culture supernatants of the Cj0921c mutant showed some utilization of aspartate but not glutamate, consistent with the transport data. It is concluded that in addition to the established role of PEB1a as an adhesin, the PEB1 transport system plays a key role in the utilization of aspartate and glutamate, which may be important in vivo carbon sources for this pathogen.

Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc to flagellin. Mutation of Cj1316c or pseA had been shown to result in loss of the acetamidino form of pseudaminic acid (PseAm). Mutation of a second gene also resulted in loss of PseAm, as well as a minor modification that appears to be PseAm extended with N-acetyl-glutamic acid. Previously described mutants in C. jejuni 81-176 and Campylobacter coli VC167 that produced flagella lacking PseAm or PseAc failed to autoagglutinate. This suggests that interactions between modifications on adjacent flagella filaments are required for autoagglutination. Mutants (81-176) defective in autoagglutination showed a modest reduction in adherence and invasion of INT407 cells. However, there was a qualitative difference in binding patterns to INT407 cells using GFP-labelled 81-176 and mutants lacking PseAm. A mutant lacking PseAm was attenuated in the ferret diarrhoeal disease model.

Campylobacter jejuni undergoes a dramatic morphological transformation from a corkscrew-shaped rod to a coccoid form in response to unfavorable conditions. It has been speculated that the coccoid plays an important role in the survival and dissemination of C. jejuni but questions still remain regarding the viability of coccoid cells. Characterization of the genome of coccoid cells found that newly formed coccoid cells (i.e., 1-3 days) had a SmaI-digestion profile identical to that of spiral-shaped cells; however, there was a progressive degradation of the DNA with continued incubation at 37 degrees C. Concomitant with genome degradation was the detection of DNA in supernatants of coccoid cells. In contrast, cells incubated at 4 degrees C retained a spiral shape and their SmaI-digestion profile for 8 weeks and released little DNA into the medium. Thus, low temperature inhibited both coccoid formation and genome degradation. Collectively, these data support the theory that the coccoid form of C. jejuni is a manifestation of cellular degradation and spiral-shaped cells, or possibly coccoid cells formed at low temperature, are the most probable candidates for a viable but nonculturable form of this pathogen.

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.

The expression of a wide variety of physiological functions in many bacterial species is modulated by quorum sensing, a population-dependent signalling mechanism that involves the production and detection of extracellular signalling molecules. The genome sequence of Campylobacter jejuni NCTC 11168 contains a gene encoding an orthologue of LuxS, which is required for autoinducer-2 (AI-2) production in other bacterial species, but does not contain genes predicted to encode any known acyl-homoserine lactone synthetase. This study demonstrates that C. jejuni produces functional AI-2 activity through the ability of cell-free extracts to specifically induce bioluminescence in Vibrio harveyi BB170, a reporter strain for quorum-sensing system 2. Production of this signalling compound was shown to be dependent upon the product of the C. jejuni luxS gene (Cj1198). While the luxS mutant showed comparable growth rate, resistance to oxidative stress and ability to invade Caco-2 cell monolayers to the parental strain, it exhibited decreased motility haloes in semisolid media, suggesting a role for quorum sensing in the regulation of motility.

Three potential regulators of flagellar expression present in the genome sequence of Campylobacter jejuni NCTC 11168, the genes rpoN, flgR, and fliA, which encode the alternative sigma factor sigma(54), the sigma(54)-associated transcriptional activator FlgR, and the flagellar sigma factor sigma(28), respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated in C. jejuni strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that the rpoN and flgR mutants were nonflagellate and that the fliA mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of rpoN, flgR, and fliA in the expression of flagellin.

In 1996, it was reported that the enteric pathogen Campylobacter jejuni produces pilus-like appendages in response to bile salts such as deoxycholate (DOC), and that the formation of these appendages requires the putative peptidase PspA. Pili were known to be important virulence determinants in other pathogenic bacteria but had never before been observed for C. jejuni. We report here that these appendages are not pili, but are instead a bacteria-independent morphological artifact of the growth medium. Furthermore, the pspA gene is not required for their formation. Broth cultures containing a threshold concentration of DOC inoculated with no bacteria produced identical abundant, fibrous, pilus-like structures as those cultures that had been inoculated with C. jejuni. These fibres were also found in growth media from DOC-containing pspA:CmR mutant cultures. These results are consistent with the absence of candidate pilin monomers in protein gel analyses as well as the dearth of pilin-like genes and pilus formation gene clusters in the C. jejuni genome.

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.

The incubation of Campylobacter jejuni suspension in aerobic conditions results in the conversion of spiral cells into coccoid form. The present paper shows that such forms introduced into the alimentary tract of two-day-old chicks are not able to convert back into cultivable, infectious spiral forms, as has been suggested by some authors. Technical problems connected with this type of experiment are also discussed.

As part of a study of genes whose transcription is maintained in stationary phase, cloned segments of DNA were selected from a Lambda ZAP II library of Campylobacter jejuni NCTC 11,168. One such clone was found to encode a homologue of the Escherichia coli cell division gene ftsA. Examination of mRNA by transcription mapping revealed that the Campylobacter gene has one major and three minor transcription start sites. There were several significant differences in the structure and organisation of the C. jejuni ftsA promoter compared to that of E. coli.

Detection of the coccoid form of Campylobacter jejuni with the use of the polymerase chain reaction (PCR) was examined. Coccoid cells of this pathogen, formed at different temperatures, showed different detection characteristics in the PCR. For spirals and cocci formed at 4 degrees C and 12 degrees C, the detection limit was about 2 x 10(3) cells/PCR. However, for detection of coccoid cells formed at 25 degrees C and 37 degrees C, at least 2 x 10(4) cells per PCR were needed. PCR was also performed on homogenates in peptone saline solution and enrichment broths of chicken meat and chicken liver that were artificially contaminated with cocci formed at 4 degrees C. PCR-products of these samples could not be demonstrated clearly.

Studies were conducted into the formation and physiological state of coccoid cells of a strain of the human and animal pathogen Campylobacter jejuni. It was found that growth phase and the presence of chloramphenicol did not affect the rate of shape transformation from spiral to coccoid, while nutrient limitation, aeration of the medium and the presence of free-radical scavengers had profound effects. Coccoid cells were found to reduce the tetrazolium salts INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) to their respective formazans and this was linked to cellular respiration. However, respiring coccoid cells could not sustain their existence in prolonged adverse conditions, and it was concluded that they represent a degenerative stage rather than a dormant state of the organism.

Campylobacter jejuni growing in liquid culture was found to exhibit gross morphological changes with time. Exponentially growing cells showed typical short spiral forms. At mid-stationary phase the cells became approximately twice the length of the exponential forms. Late stationary/early decline phase cells were seen to be a mixture of coccal forms and cells which were between 3 and 4 times the length of exponentially growing cells. Continued incubation of cultures eventually resulted in a population largely of coccal forms. These morphological changes have not previously been observed when Camp. jejuni has been grown on agar-based solid medium. It is likely that such changes result from the differential expression of genes that control the timing of cell division.

The virulence of Campylobacter jejuni for 11-day-old chick embryos is associated with the ability to invade the chorio-allantoic membrane, to resist phagocytosis and to survive and proliferate in vivo. The pathogenicity of a well characterised avirulent C. jejuni strain was enhanced by passaging it intravenously and chorio-allantoically through chick embryos. The resulting isogenic variants had greatly increased ability to survive in vivo. In this study, the morphological and cell-surface characteristics of the avirulent parental strain were compared with those of the more virulent variants to determine whether pathogenicity was associated with one or more cell-surface constituents. Changes associated with the increased virulence of the two variants included alterations in cultural and cellular morphology, loss of flagella, expression of a new outer-membrane protein, alterations in cell-surface carbohydrates and decreases in cell-surface hydrophobicity.

The behaviour of Campylobacter jejuni in the environment is poorly documented. Rapid loss of viability on culture media is reported. This phenomenon is associated with the development of so-called coccoid cells. It has been suggested that these cells can be infective to animals and man. Results obtained with ATP-measurements of coccoid cells and Direct Viable Count (DVC) support this hypothesis. Introduction of coccoid cells into simulated gastric, ileal and colon environments did not result in the presence of culturable cells. Oral administration to laboratory animals and volunteers caused no typical symptoms of campylobacteriosis. Until 30 days after uptake of the cells antibodies against C. jejuni could not be detected in the blood, and the presence of this microorganism in stool samples could not be demonstrated.