The effect of campylobacter lipopolysaccharide on fetal development in the mouse

Purified lipopolysaccharide (LPS) obtained from isolates of Campylobacter fetus ss. fetus and Campylobacter jejuni impaired fetal development when administered to mice on day 13 of pregnancy. Strikingly more fetal resorption was produced by C. jejuni LPS than by similar amounts of C. fetus ss. fetus LPS. Three of the four Campylobacter strains examined produced LPS that had no effect on maternal health, but LPS from one C. jejuni strain killed all of the mice to which it was administered.     

Hippurate hydrolysis by and triphenyltetrazolium tolerance of Campylobacter fetus.

A rapid test of hippurate hydrolysis and a test of tolerance to triphenyltetrazolium chloride (TTC) were studied in 315 strains of Campylobacter fetus subsp. jejuni to determine their usefulness for biotyping this organism and for distinguishing it from C. fetus subsp. intestinalis. Of the 315 strains tested, 84% hydrolyzed hippurate and 97% were resistant to TTC. Ability to hydrolyze hippurate was seen in 99% of 155 human isolates, 75% of 60 avian isolates, 100% of 41 cattle and dog isolates, 84% of 31 zoo mammal isolates, and none of 28 hog isolates. Resistance to 400 micrograms of TTC per ml was seen in 97% of the human isolates, 95% of the avian isolates, and 100% of the mammalian isolates (other than human). In no case did any of the 315 isolates of C. fetus subsp. jejuni show both lack of ability to hydrolyze hippurate and sensitivity to TTC. In contrast, all 18 strains of C. fetus subsp. intestinalis failed to hydrolyze hippurate and were sensitive to TTC. These two tests may be useful to distinguish between C. fetus subsp. jejuni and subsp. intestinalis and also to biotype strains of C. fetus subsp. jejuni.     

Campylobacters and impaired fetal development in mice

Intravenous injection of eight human strains of Campylobacter fetus ss fetus and Campylobacter jejuni into mice at various stages of pregnancy demonstrated significant strain differences in ability to affect implantation of the fertilised ovum and to cause resorption of the mouse fetus. Implantation was significantly impaired when C. fetus ss fetus was injected intravenously on day 2 of pregnancy, but no effect was observed in mice receiving C. jejuni. On day 6 of pregnancy, before the development of placental circulation, both C. fetus ss fetus and C. jejuni impaired fetal growth; one strain of C. jejuni had a greater effect than others of the same species. In animals inoculated on day 13 of pregnancy, after the development of placental circulation, six of the eight campylobacter strains caused resorption of the mouse embryos. A similar effect on the embryos was observed after injection of heat-killed organisms, and endotoxin-like substances may have been responsible. It is also suggested that factors other than endotoxin-like substances have a deleterious effect on embryonic growth.     

Restriction endonuclease analysis of Campylobacter strains with particular reference to Campylobacter fetus ss. fetus

Forty-three strains of Campylobacter fetus ss. fetus isolated from sheep abortions in New Zealand, and reference strains of C. fetus ss. fetus (four), C. fetus ss. venerealis (two), C. jejuni (one) and C. coli (one) were examined by restriction endonuclease analysis with the enzymes BstE II and Xho I. DNA fragment patterns of C. fetus, C. jejuni and C. coli differed strikingly from each other, but there were many similarities in the patterns obtained for all strains of C. fetus ss. fetus (47) and C. fetus ss. venerealis (three). The 43 local strains of C. fetus ss. fetus were clearly divided into four types. Two of these types produced patterns nearly identical with those of overseas reference strains.     

Molecular evidence for zoonotic transmission of an emergent, highly pathogenic Campylobacter jejuni clone in the United States

Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.     

Campylobacter jejuni and Campylobacter coli serotypes isolated from chickens, cattle, and pigs

A total of 191 Campylobacter jejuni and 125 Campylobacter coli were isolated from the intestinal content of 398 chickens, 421 cattle, and 203 pigs. All 108 chicken isolates and 73 of 80 cattle isolates were C. jejuni, but 115 of the 118 pig isolates were C. coli. A total of 84% of the C. jejuni and 64% of the C. coli isolates were typed on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes in Campylobacter enteritis in man (15 C. jejuni, 6 C. coli serotypes). A total of 96% of the chicken isolates and 67% of the cattle isolates belonged to 11 C. jejuni serotypes that occur most frequently in human cases of enteritis (serotypes 1, 2, 3, 4, 5, 13/16, 18, 21, 23, 31, and 36). Serotype 8, a relatively common human isolate, was not recovered. The C. coli isolates from pigs belonged to serotypes uncommon among human isolates.     

Emergence of a tetracycline-resistant Campylobacter jejuni clone associated with outbreaks of ovine abortion in the United States

Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.     

The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.     

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.     

Detection of pathogenic Campylobacter species in blood culture systems.

Because differences in recognition of Campylobacter fetus and Campylobacter jejuni in systemic infections may be due partially to differences in the ability to cultivate these organisms, we studied their growth characteristics in two widely used blood culture systems. In the Roche Septi-Chek system (Hoffman-La Roche, Inc., Nutley, N.J.), over a broad range of inocula all strains were detected in broth within 2 days and on paddles within 3 days. In the BACTEC 6B aerobic bottles (Johnston Laboratories, Inc., Towson, Md.), C. jejuni and C. fetus took a median of 5 and 3 days, respectively, to reach the growth index threshold. However, in the BACTEC 7D anaerobic bottles, C. fetus required a median of 2 days to reach the growth index threshold, whereas for C. jejuni the median was greater than 10 days. The poor performance of C. jejuni in both BACTEC systems may have been due to unfavorable incubation atmospheres and may partially explain why C. jejuni bacteremia is so infrequently detected. Overall, the Roche Septi-Chek system was excellent for detecting Campylobacter strains in blood cultures.     

Uneven distribution of the luxS gene within the genus Campylobacter

Polymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacter species using a degenerate primer pair designed in silico to generate a product of the luxS gene or its homologue from Campylobacter organisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuni and C. coli and some of C. upsaliensis and C. fetus, it failed to amplify fragments with all four isolates of C. lari (two urease-negative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxS gene fragments prepared from the C. jejuni, C. coli, C. upsaliensis and C. fetus strains as a probe, all C. jejuni, C. coli, C. upsaliensis and C. fetus isolates gave positive signals, but no positive signal was detected with any C. lari isolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensis and C. fetus carry the luxS gene or its homologue. However, no luxS gene or its homologue was identified to occur in the C. lari genome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensis and C. fetus isolates, it was negative with all the C. lari isolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. lari species does not appear to correlate with the occurrence of the luxS gene because biofilm formation occurred among some isolates of C. lari.     

Thermo-tolerant Campylobacter fetus bacteraemia identified by 16S ribosomal RNA gene sequencing: an emerging pathogen in immunocompromised patients

Eight Campylobacter isolates that were able to grow at 25 degrees C and 42 degrees C and had the same biochemical profile were isolated from the blood of eight immunocompromised patients. Conventional biochemical tests were unable to determine whether they were isolates of thermo-tolerant C. fetus, H2S-negative C hyointestinalis, or a new Campylobacter species. Sequencing of the 16S ribosomal RNA genes showed that all eight isolates had the same nucleotide sequence and this was identical to that of C. fetus (GenBank accession no. AF219233). All eight patients had underlying disease and two died despite antibiotic treatment. Because of the ability of C fetus to grow over a wide range of temperatures and a higher incidence of bacteraemia by this organism than C. jejuni in the past 5 years in Hong Kong, thermo-tolerant C fetus may be an emerging pathogen in immunocompromised patients in the years to come.     

Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains

The cytolethal distending toxin (cdt) gene clusters of Campylobacter coli strain Co1-243 and C. fetus strain Col-187 were cloned and sequenced to understand the importance of Cdt as a virulence factor. The cdt genes of C. coli and C. fetus consist of three closely linked genes termed cdtA, cdtB, cdtC whose sizes are 774, 801, and 570 bp, and 702, 798, and 546 bp, respectively. The homologies of each subunit of cdt genes between C. jejuni and C. coli, C. jejuni and C. fetus, or C. coli and C. fetus are 59.6%, 40.3%, or 46.5% for cdtA, 70.2%, 62.4%, or 61.3% for cdtB, 61.3%, 52.3%, or 50.1% for cdtC, respectively. Colony hybridization assay revealed that the genes homologous to the cdtABC gene were distributed in all 27, 19, 20 strains of C. jejuni, C. coli, and C. fetus, respectively, isolated from patients and animals in species-specific manner. Furthermore, nucleotide sequence of the cdt operon, including flanking region, of 10 strains of each species indicated that though the size of the cdtB gene was conserved in each species, those of cdtA and cdtC genes varied particularly among C. coli strains. Amino acid residues demonstrated to be important for toxin activity in CdtB, corresponding to H152, D185, D222, D258, H259 in Cj-CdtB, were also conserved in Cc-CdtB and Cf-CdtB. The cdt gene cluster was located in different sites among different species but in the same site of genomes of the same species. Cdt activity produced by C. jejuni and C. fetus varied among strains, however, any C. coli strains exhibited Cdt activity on HeLa cells. These data indicate that the cdt gene may have a potential for virulence factor at least in C. jejuni and C. fetus.     

Uncommon Campylobacter species in infant Macaca nemestrina monkeys housed in a nursery.

Studies were conducted to characterize 18 isolates of Campylobacter spp. that could not be identified as either Campylobacter jejuni or C. coli. The isolates were cultured from specimens from 13 of 18 infant nonhuman primates during a prospective epidemiologic study reported previously. Phenotypic tests, DNA hybridization, and analysis of DNA coding for rRNA identified the isolates as C. butzleri (seven isolates), C. hyointestinalis (seven isolates), and C. fetus subsp. fetus or C. fetus subsp. fetus-like organisms (four isolates). Ribotype and polyacrylamide gel electrophoresis patterns indicated that there was heterogeneity among the isolates of C. butzleri and C. fetus subsp. fetus-like organisms.     

Cellular fatty acid composition of Campylobacter fetus.

The cellular fatty acid composition of 29 human and animal isolates of Campylobacter fetus was determined with gas-liquid chromatography. All strains contained small amounts of 3-hydroxytetradecanoic acid, suggesting the presence of lipid A. Each of 13 different blood or stool isolates of C. fetus subsp. jejuni obtained from humans or fowl contained a 19-carbon cyclopropane acid which was not present in C. fetus subsp. fetus (6 strains from cattle) or C. fetus subsp. intestinalis (10 strains from humans and cattle). These findings indicate that C. fetus subsp. jejuni can be rapidly differentiated from other C. fetus by gas-liquid chromatography analysis of cellular fatty acids.     

Campylobacter jejuni in black-headed gulls (Larus ridibundus): prevalence,genotypes, and influence on C. jejuni epidemiology

Campylobacteriosis is a zoonotic disease in which birds have been suggested to play an important role as a reservoir. We investigated the prevalence of Campylobacter jejuni subsp. jejuni in black-headed gulls (Larus ridibundus) in southern Sweden with the aim of examining the nature of C. jejuni infection in this bird species. Birds were sampled in four sampling series each year during 1999 (n = 419) and 2000 (n = 365). Longitudinally sampled C. jejuni isolates from individual gulls were subjected to macrorestriction profiling (MRP) by pulsed-field gel electrophoresis to investigate the genotypical stability during the natural course of infection. Furthermore, a subset (n = 76) of black-headed gull isolates was compared to isolates from broiler chickens (n = 38) and humans (n = 56) originating from the same geographic area. We found a pronounced seasonal variation in C. jejuni carriage, with the highest rates found in late autumn. MRP similarities were higher between isolates of human and broiler chicken origin, than between those of wild bird origin and either of the other two hosts. However, identical MRPs were found in two gull isolates and one human isolate after digestion with two restriction enzymes, strongly indicating that they may have been colonized by the same clone of C. jejuni. The MRPs most prevalent in gull isolates did not occur among isolates from humans and broiler chickens, suggesting the existence of a subpopulation of C. jejuni adapted to species-specific colonization or environmental survival.     

Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa

In the opportunistic pathogen Pseudomonas aeruginosa, the production of several virulence factors such as elastase, rhamnolipids and pyocyanin depends on cell-to-cell signaling or quorum sensing (QS) involving N-acylhomoserine lactone (AHL) signal molecules. In vitro studies with laboratory strains and virulence studies in animals with these same strains have demonstrated the contribution of QS to the pathogenesis of P. aeruginosa. However, the importance of P. aeruginosa QS systems in the development of human infections is not clearly known. In order to determine if deficiency within the QS system compromises the ability of P. aeruginosa to cause infections in humans, we collected 50 P. aeruginosa clinical isolates. Phenotypic characterization showed that isolates I-457, I-458, I-459 and I-461 were defective in the production of N-butanoyl-l-homoserine lactone (C4-HSL) signaling molecule and virulence factors elastase, protease, pyocyanin and rhamnolipids. Analysis of the sequences of the lasR, lasI, rhlR and rhlI genes of these four isolates showed that two of the four isolates had mutational defects in both rhlR and rhlI genes while other two isolates were only mutated in the rhlI gene. The combination of rhlR and rhlI mutations or only rhlI mutation probably explains their C4-HSL and virulence factors deficiencies. These observations suggest that QS deficient P. aeruginosa clinical isolates are able to cause infections and that in addition to known virulence factors, factors yet unidentified may contribute to the pathogenesis of P. aeruginosa.     

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.     

Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France

Five hundred eighty-two Campylobacter isolates (177 from humans, 319 from poultry and 86 from pig) collected in Brittany, France, in 2003 and 2004 were typed by pulsed-field gel electrophoresis. The number of human cases increased during the hot season, particularly for C. jejuni. Twelve genetic groups out of 27 contained human isolates collected over the two years. These groups had 21.3 and 17.0% of the isolates obtained in 2003 and 2004, respectively. In four cases, isolates from 2003 have the same Pulsed-field gel electrophoresis (PFGE) profile as isolates from 2004. Six PFGE profiles common to poultry and human isolates were identified. Poultry isolates were found in 47 clusters containing human isolates. Caeca from farms and slaughterhouses accounted for 66% of these isolates, with chicken legs obtained from supermarkets accounting for the other 34%. Pig isolates never clustered with poultry and human isolates. In conclusion, the analysis of the genetic profiles of Campylobacter resulting from human cases showed that there were few identical or genetically close isolates between the human cases declared in 2003 and those declared in 2004. This highlighted a great genetic diversity in the isolates and indicated that it should be difficult to bind the human infections with groups of Campylobacter isolates presenting particular genetic profiles. The Campylobacter isolates obtained from the two animal production systems had different genotypes, and isolates from pigs differed genetically from isolates obtained from humans. We found that 44.6% of human Campylobacter isolates were genetically related to genotypes found in poultry and a part of these campylobacteriosis are due to contact with poultry. This is not particularly surprising in Brittany, a farming area with many animal-rearing farms and slaughterhouses. This work highlights the implication of the poultry in the French human cases and that handling of poultry is also an important risk for Campylobacter infection in humans.     

Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis.

The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.