Fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni

The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.     

Critical role of LuxS in the virulence of Campylobacter jejuni in a guinea pig model of abortion

Previous studies on Campylobacter jejuni have demonstrated the role of LuxS in motility, cytolethal distending toxin production, agglutination, and intestinal colonization; however, its direct involvement in virulence has not been reported. In this study, we demonstrate a direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxS complement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902 luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxS gene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivo model of natural disease.     

Fitness cost of fluoroquinolone resistance in Campylobacter coli and Campylobacter jejuni

In this study, the fitness cost of fluoroquinolone resistance was evaluated in vitro, on food matrices, and in vivo, using Campylobacter coli and Campylobacter jejuni in vitro selected mutants. In vitro, the growth rate of the susceptible (wild type) and resistant (mutant) strains did not differ when cultured separately. However, by conducting sequential passages of mixed cultures, the ratio of the resistant mutant to the susceptible strain decreased for C. coli but not for C. jejuni. When the wild type and the mutant were co-inoculated on food matrices, mutants were no longer detectable 3 to 5 days after artificial contamination, but the wild-type strains remained detectable for over 13 days. In mono-inoculated animals, no difference was observed between wild-type and mutant fecal titers. When co-inoculated into chickens, the susceptible strain outcompeted the resistant mutant for C. coli and for C. jejuni. However, for C. coli, if the resistant strain was already present in animals, it could persist at high titers in the digestive tract even in the presence of the wild-type strain. Together, these findings suggest that, depending on strain and study conditions, fluoroquinolone resistance can impose a fitness cost on Campylobacter.     

DNA rearrangements in the flagellin locus of an flaA mutant of Campylobacter jejuni during colonization of chicken ceca

Campylobacter jejuni is an enteropathogen for humans but commensal for chickens. In both hosts, the flagella and motility are important colonization factors. The flagellin gene is duplicated in Campylobacter, but only one flagellin gene, flaA, is sufficient for motility. We found that, during colonization of the chicken intestine, a nonmotile flaA mutant of C. jejuni underwent rearrangements within its flagellin locus, thereby regaining its motility and colonization capacity. In contrast, in vitro motile revertants isolated from liquid culture showed different flagellin DNA rearrangements than after reversion in the chicken.     

Adaptation of Campylobacter jejuni NCTC11168 to high-level colonization of the avian gastrointestinal tract

The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148:473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5, flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.     

Cj1136 is required for lipooligosaccharide biosynthesis, hyperinvasion, and chick colonization by Campylobacter jejuni

Campylobacter jejuni is a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasive C. jejuni strains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue of cj1136, which encodes a putative galactosyltransferase according to the annotation of the C. jejuni NCTC11168 genome. In the current study, we investigated the role of cj1136 in C. jejuni virulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. The cj1136 mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation of cj1136 resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. The cj1136 mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded by cj1136 is involved in LOS biosynthesis and is important for C. jejuni virulence, as disruption of this gene and the resultant truncation of LOS affect both colonization in vivo and invasiveness in vitro.     

Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.     

Isolation of nonchemotactic mutants of Campylobacter jejuni and their colonization of the mouse intestinal tract.

Three nonchemotactic mutants (D54, Y14, and N74) of Campylobacter jejuni were isolated from wild-type strain FUM158432 by either the negative swarming or liquid gradient method with brucella broth as the attractive substance. Strains D54 and Y14 were isolated after mutagenesis with methyl methanesulfonate, and N74 was isolated from a nonmutagenized culture. These mutants all failed to swarm on a semisolid medium and did not show any chemotactic behavior in the hard-agar plus assay method for any of the chemicals which act as attractants for the wild-type strain. They had intact flagella and were actively motile. Swimming behavior examined by a video tracking technique showed that the mutants swim only straight, without any tumbling. When suckling mice were challenged orally with approximately 10(5) CFU of these mutant strains, all of the mutants were cleared from the intestinal tract by 48 h. In contrast, the wild-type strain colonized the intestinal tracts of all mice challenged with 10(2) CFU. We concluded that chemotactic movement is important for colonization of the intestinal tract of suckling mice by C. jejuni.     

A tolerogenic mucosal immune response leads to persistent Campylobacter jejuni colonization in the chicken gut

Campylobacter enteritis is the most reported zoonotic disease in many developed countries where it imposes a serious health burden. Campylobacter transmission to humans occurs primarily through the chicken vector. Chicks are regarded as a natural host for Campylobacter species and are colonized with C. jejuni in particular. But despite carrying a very high bacterial load in their gastrointestinal tract, these birds, in contrast to humans, do not develop pathological signs. It seems that in chickens C. jejuni principally harbors in the cecal mucosal crypts, where an inefficient inflammatory response fails to clear the bacterium from the gut. Recent intensive research resulted in an increased insight into the cross talk between C. jejuni and its avian host. This review discusses the chicken intestinal mucosal immune response upon C. jejuni entrance, leading to tolerance and persistent cecal colonization. It might in addition provide a solid base for further research regarding this topic aiming to fully understand the host-bacterium dynamics of C. jejuni in chicks and to develop effective control measures to clear this zoonotic pathogen from poultry lines.     

Experimental infection of chickens with Campylobacter jejuni: strains differ in their capacity to colonize the intestine.

Groups of broiler and layer type chickens (25 to 63 d.o.) were inoculated per os with separate isolates of 10 strains of Campylobacter jejuni. Nine of the 10 strains were originally isolated from chickens and one from a dog. The dog strain and five of the chicken isolates could be isolated after inoculation, but four strains were not recovered from cloacal swabs for up to 4 to 16 days after inoculation. However, it was possible to isolate C. jejuni from these birds, from cloacal swabs, when they were inoculated with organisms which had been previously shown to colonize other birds.     

Chemotactic behavior of Campylobacter jejuni.

The chemotactic behavior of Campylobacter jejuni was determined in the presence of different amino acids, carbohydrates, organic acids, and preparations and constituents of mucin and bile. L-Fucose was the only carbohydrate and L-aspartate, L-cysteine, L-glutamate, and L-serine were the only amino acids producing a chemotactic (positive) response. Several salts of organic acids, including pyruvate, succinate, fumarate, citrate, malate, and alpha-ketoglutarate, were also chemoattractants, as were bile (beef, chicken, and oxgall) and mucin (bovine gallbladder and hog gastric). Most constituents of bile tested individually were chemorepellents, but the mucin component was chemoattractant. The chemotactic behavior of C. jejuni toward L-fucose, a constituent of both bile and mucin, may be an important factor in the affinity of the organism for the gallbladder and intestinal tract.     

Campylobacter jejuni diarrhea model in infant chickens.

To study the pathogenic mechanisms of Campylobacter jejuni infection, 36- to 72-h-old chickens were fed 10(3) to 10(6) live cells, using strains isolated from 40 patients with watery diarrhea and 6 with bloody mucoid diarrhea from whom no other known enteropathogen was detected. Chickens of Starbro strain were more likely to develop C. jejuni-induced diarrhea than were White Leghorn chickens. Diarrhea was defined on the basis of amounts of gut fluid in 288 chicks fed with live C. jejuni versus 183 saline-fed control as an accumulation greater than or equal to 0.4 ml of fluid in the guts (excluding ceca) of chickens. Twenty-five percent of the chickens developed diarrhea on day 2, 49% on day 4, and 81% on day 5. The intestines, including ceca, were distended with watery fluid. The majority of the strains, irrespective of whether they were isolated from watery or bloody mucoid enteritis patients, caused watery diarrhea in chickens, and a few caused mucoid diarrhea. No correlation was observed between the source of a strain and the outcome in the experimental model. Bloody diarrhea was never observed in chickens. The peak incidence of diarrhea on day 5 coincided with the mean of maximum fluid accumulation. The organisms multiplied by 3 to 4 logs in all parts of the intestine, with a steady increase in number until day 5. Systemic invasion occurred frequently: C. jejuni could be recovered from the spleen in 47% of the chickens on day 5, in 25% from the liver on day 6, and in 11% from heart blood on day 4. Histopathological examination of gut tissue of the chickens having watery diarrhea did not reveal any abnormality except slight submucosal edema. However, in chickens with mucoid diarrhea, the organisms were found to adhere to brush borders and penetrate into the epithelial cells with formation of a breach in continuity of the brush border lining. The electrolyte composition of the intestinal fluid from chickens infected with C. jejuni and from saline-fed controls did not show significant differences, except for depletion of K+ in the test group. The results obtained in this highly reproducible chicken diarrhea model indicate that (i) most chickens develop nonexudative watery diarrhea 2 to 5 days after oral feeding of 10(3) to 10(6) live cells of C. jejuni; (ii) the organism multiples in all parts of a chicken intestine, (iii) systemic invasion is common, and (iv) local invasion is sometimes observed.     

Systematic investigation of enrichment media for wild-type Campylobacter jejuni strains

Of the media examined, thioglycolate broth supplemented with 5% lysed sheep blood, Butzler antibiotic mixture, and 0.1% lauryl sulfate was the most sensitive enrichment medium for recovery of wild-type strains of Campylobacter jejuni from cecal contents of chickens and chicken livers. It allowed the retrieval of 1 CFU as did solid media but permitted the screening of 50-times larger volumes. Double-strength enrichment medium required 5 to 10 CFU for growth. Omission of lauryl sulfate reduced the sensitivity. Replacement of Butzler antibiotic mixture with Blaser antibiotic mixture increased overgrowth and, therefore, decreased retrieval of C. jejuni.     

The role of Klebsiella pneumoniae urease in intestinal colonization and resistance to gastrointestinal stress

The first step in nosocomial infections due to Klebsiella pneumoniae is colonization of the patient's gastrointestinal (GI) tract. In a previous work, signature-tagged mutagenesis was used in a murine model to identify 13 genes required for efficient colonization, two of which were involved in urea metabolism. The role of urease was further investigated by the construction and analysis of an isogenic urease-deficient mutant. The behavior of both the wild-type strain and the urease-deficient mutant was tested under hostile conditions, reproducing stresses encountered in the GI environment. The wild-type strain had an acid tolerance response (ATR) to inorganic acid, was resistant to organic acids (38.5% survival) and was able to survive concentrations of bile encountered in vivo. The absence of urease did not affect the resistance of K. pneumoniae to acid and bile stresses, but the enhanced adhesion response to Int-407 cells after exposure to bile observed with the wild-type strain was no longer detected with the urease mutant. When tested in the murine intestinal colonization model, both strains were mainly recovered in the large intestine parts, and the mutant was impaired in its colonization capacities, but only when tested in competition with the wild-type strain. These findings emphasize the prominent role played by metabolic function in the colonization process of such a complex ecosystem as the host GI tract.     

Campylobacter jejuni colonization of mice with limited enteric flora

We have developed experimental murine Campylobacter infection models which demonstrate efficient establishment and reproducible, high-level colonization. Following oral inoculation, wild-type C3H mice with normal enteric flora were colonized inconsistently and inefficiently by C. jejuni strain 81-176. However, C3H mice with a limited gut flora (LF) were efficiently colonized at high levels (10(8) CFU/g of stool or large intestine tissue) followed by clearance after several weeks. Large intestine tissue showed minimal to mild inflammation at days 7 and 28 postinoculation. In striking contrast, C3H SCID mice with the same limited flora remained persistently colonized at a consistently high level until they were euthanized 8 months postinoculation. Lower gastrointestinal tract tissue from LF-SCID mice showed marked to severe inflammation in the colon and cecum at days 7 and 28 and intense inflammation of the stomach at day 28. These findings indicate that although the innate response alone cannot block colonization persistence, it is sufficient to orchestrate marked gut inflammation. Moreover, the adaptive immune response is critical to mediate C. jejuni clearance from the colonized gut. To validate our LF murine model, we verified that motility and chemotaxis are critical for colonization. Insertion-deletion mutations were generated in motB and fliI, which encode products essential for motility and flagellar assembly, and in the presumptive chemotaxis gene cheA (histidine kinase). All mutants failed to establish colonization in LF mice. Our limited flora murine colonization models serve as tractable, reproducible tools to define host responses to C. jejuni infection and to identify and characterize virulence determinants required for colonization.     

Role of catalase in Campylobacter jejuni intracellular survival

The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.     

The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.