Association with HeLa cells of Campylobacter jejuni and Campylobacter coli isolated from human feces.

We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples.

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

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.     

Polynucleotide sequence relationships among flagellin genes of Campylobacter jejuni and Campylobacter coli.

DNA probes that encode a complete flagellin gene and various internal regions of the Campylobacter coli VC167 flagellin genes were hybridized to 30 strains of C. coli or C. jejuni from 20 different Lior serogroups. The results indicated a high overall degree of homology among all of the strains examined. Although the most variable regions occurred within the middle of the gene, significant DNA homology was observed among many serogroups in this region of the molecule.     

Molecular characterization and genotypic antimicrobial resistance analysis of Campylobacter jejuni and Campylobacter coli isolated from broiler flocks in northern Italy

Genetic variability and genotypic antimicrobial resistance (AMR) of Campylobacter jejuni and Campylobacter coli from commercial broiler farms were investigated in this study. Campylobacter isolates were genetically characterized by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and flaA-SVR and flaB-SVR sequence-based typing. Eight RAPD types were identified in C. jejuni and three in C. coli, while 16 fla profiles were detected among all isolates. Further, 13 flaA-SVR and 13 flaB-SVR alleles were identified. Both typing methods detected a high level of genetic diversity, but fla-SVR typing showed a higher discriminatory power. Indeed, Simpson's index of fla typing (D=0.920) was higher than that of RAPD typing (D=0.814). Moreover, the association of flaA-SVR and flaB-SVR sequence analysis showed a higher discriminatory power compared with the sequence analysis of single loci. Isolates were also analysed by the mismatch amplification mutation assay PCR test and the detection of cmeB gene to determine the occurrence of genetic determinants of AMR to macrolides and fluoroquinolones and multidrug resistance. The A2074C and A2075G mutations in the 23S rRNA gene, the C257T mutation in the gyrA gene, and the cmeB gene were higher in C. coli (19.0%, 67.0%, 100.0% and 100.0%, respectively) than in C. jejuni (0.0%, 3.1%, 48.3% and 48.3%, respectively). This study showed a high degree of genetic diversity and a high prevalence of genetic determinants of macrolide resistance, fluoroquinolone resistance and multidrug resistance among C. jejuni and C. coli isolates from Italian commercial broiler farms.     

Sequence variation in the outer membrane protein-encoding gene cmeC, conferring multidrug resistance among Campylobacter jejuni and Campylobacter coli strains isolated from different hosts

A novel PCR primer pair was used to detect the presence of cmeC in 131 Campylobacter jejuni and C. coli strains isolated from various hosts (cattle, turkeys, humans, and pigs). DNA sequence analysis revealed a high degree of genetic variation between the two species, while extremely limited genetic variation among isolates of the same species was detected.     

Distribution of sero-biotypes of Campylobacter jejuni and C. coli isolated from paediatric patients

During a one-year period, 258 isolates of Campylobacter jejuni and C. coli were obtained from children with gastroenteritis or bacteraemia at the Red Cross Children's Hospital, Cape Town, South Africa. These isolates were biotyped by hippurate hydrolysis, H2S production and tolerance to 2,3,5-triphenyltetrazolium chloride (TTC). Our study indicated that 95.4% of the isolates were C. jejuni biotype 1, 1.5% were C. jejuni biotype 2 and 3.1% were C. coli; 70% of the isolates were resistant to TTC. Serotyping on the basis of soluble, thermostable antigens detected by a passive-haemagglutination technique revealed that 79% of the Cape Town isolates were typable and that the most common serotypes, in order, were: 4, 2, 12, 23/36 and 19, together comprising 25% of the isolates. About 37% of the typable isolates belonged to nine serotypes. The finding that 21% of the isolates were non-typable suggests the existence of antigenic specificities different from those defined by the 60 antisera in current use.     

Pathogenicity of Campylobacter jejuni isolates from animals and humans.

Fourteen isolates of Campylobacter jejuni of different serotypes and one Campylobacter coli isolate, from various human and animal sources, were tested for potential pathogenic mechanisms. Enterotoxin production was not detected in the infant mouse test or by calf and piglet ligated intestinal loop studies. Isolates were not invasive by the Sereny test. All isolates associated with and penetrated HeLa cells, although both actions occurred generally in a minor way under the conditions of our study. The C. coli isolate showed extensive HeLa cell association, but three other C. coli isolated tested did not. None of the 15 isolates produced diarrhea or death in 3-day-old chickens inoculated orally and observed for 3 days, nor did they consistently produce diarrhea and death in 9- to 10-day-old infant mice over a 3-day period after oral inoculation. Diarrheal disease and mortality were not observed when 3-day-old gnotobiotic chickens were infected with one of five isolates and observed over a 2-week period.     

Genetic characterization and antibiotic resistance of Campylobacter jejuni isolated from meats, water, and humans in Sweden

The incidence of Campylobacter jejuni has increased during the last decade, and today it is the leading cause of bacterial enteritis in most developed countries. Still, there is a lack of knowledge about infection routes and to what extent identified sources are responsible for spreading the bacterium to humans. The major objective of this work was to explore the genetic similarity between C. jejuni isolated from different sources. C. jejuni isolated from patients (n = 95), five types of meat (n = 71), and raw water (n = 11) during the year 2000 were subtyped by pulsed-field gel electrophoresis (PFGE). The pulsotypes obtained after digestion with SmaI revealed not only that C. jejuni is genetically diverse but also that specific pulsotypes occur frequently. Five clusters comprising 88 of the 162 SmaI-digested isolates were obtained. After digestion with KpnI most isolates in four of the five clusters were still indistinguishable, while the fifth cluster was strongly dissolved. The clusters comprised high frequencies of human and meat isolates, while only one of nine water isolates belonged to a cluster. The largest cluster comprised 21 human isolates, one raw water isolate, and seven chicken meat isolates, originating from at least six different broiler flocks. Low frequencies of antibiotic resistance were revealed when the meat and water isolates were tested for sensitivity to six antibiotics. Interestingly, the five isolates resistant to quinolones displayed similar or identical pulsotypes. The results showed that PFGE has proved useful in identifying clones and will be used in future work focusing on identification and eradication of the major reservoirs for common clones.     

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.     

Enterotoxin production and serogroups of Campylobacter jejuni and Campylobacter coli from patients with diarrhea and from healthy laying hens.

Enterotoxin production, a possible virulence factor, was determined in Campylobacter jejuni and Campylobacter coli by two different techniques, the CHO cell test and the GM1 enzyme-linked immunosorbent assay. The frequency of enterotoxigenic Campylobacter strains was 32% in strains from both humans with acute enteritis and healthy laying hens, as measured by the CHO cell test. The CHO cell test was significantly more sensitive than the GM1 enzyme-linked immunosorbent assay in the detection of enterotoxigenic strains. Enterotoxin production was compared with the presence of heat-stable and heat-labile antigens. There was no significant correlation between enterotoxin production and serogroups for C. jejuni or C. coli. The difference in enterotoxigenicity between C. jejuni (34.1%) and C. coli (21.9%) was not significant.     

Serotype distribution of Campylobacter jejuni and Campylobacter coli isolated from hospitalized patients with diarrhea in central Australia.

Campylobacter jejuni and/or Campylobacter coli was cultured from 218 of 1,078 patients of all age groups admitted to Alice Springs Hospital, Alice Springs, central Australia, between July 1988 and June 1989 for treatment of diarrhea. One hundred sixty-six Campylobacter colonies from 127 patients were subjected to O serotyping by using the Penner typing scheme. All except 29 colonies could be serotyped. A total of 46 serotypes were identified, and the predominant serotypes were O:8, 17, O:22, O:1,44, and O:19. A large proportion of colonies reacted with more than one antiserum, and nine serotypes had antigenic compositions not observed previously. Several patients had multiple infections with more than one serotype, and some patients were shown for the first time to be infected with up to three different serotypes. Repeated reinfections with different serotypes were seen in some patients. In some patients, provided it was not due to reinfection with the same serotype, long-term excretion of the same serotype was seen, and for the first time, one patient showed evidence of excretion of the same serotype for up to 73 days.     

Occurrence of plasmid DNA in serologically defined strains of Campylobacter jejuni and Campylobacter coli

Forty Campylobacter jejuni and 17 Campylobacter coli strains that constitute the set of reference strains for our serotyping scheme were each examined for the presence of plasmid DNA. Agarose gel electrophoresis of alkaline-extracted DNA showed the occurrence of 29 bands in 11 C. jejuni strains and 40 bands in C. coli strains. Plasmids ranged in size from 1.6 to 70 megadaltons. Most strains that carried plasmids had between 2 and 6 of them; however, one strain had 14 plasmids, and two strains contained only 1 plasmid each. Repeated electrophoresis demonstrated that all plasmid profiles were stable. A different plasmid profile was seen for each of the 19 plasmid-carrying strains, but it was clear that plasmids of the same or similar molecular weight could be found in different strains. On the basis of these findings, we are persuaded that plasmid profiles determined by a rapid procedure for DNA extraction will play a significant role in resolving complexities among strains that are difficult to serotype and could be useful in epidemiological studies in which the implicated isolates are plasmid bearers.     

Monosaccharide composition of lipopolysaccharides from Campylobacter jejuni and Campylobacter coli

The monosaccharide composition of the LPS from 5 Campylobacter jejuni strains and 7 Campylobacter coli strains has been studied. All LPS's contained KDO, heptose, glucosamine, glucose, and (with one exception) galactose. All C. jejuni and 3 C. coli LPS's contained greater than 1% galactosamine. 3-Amino-3.6-dideoxyglucose was present in all but one C. coli LPS and in only one C. jejuni LPS.     

Conjugative transfer of chromosomally encoded antibiotic resistance from Helicobacter pylori to Campylobacter jejuni

Many strains of Helicobacter pylori are naturally competent for transformation and able to transfer chromosomal DNA among different isolates using a conjugation-like mechanism. In this study, we sought to determine whether H. pylori can transfer DNA into Campylobacter jejuni, a closely related species of the Campylobacterales group. To monitor the transfer, a chromosomally encoded streptomycin resistance cassette prearranged by a specific mutation in the rpsL gene of H. pylori was used. Mating of the bacteria on plates or in liquid broth medium produced C. jejuni progeny containing the streptomycin marker. DNA transfer was unidirectional, from H. pylori to C. jejuni, and the progeny were genetically identical to C. jejuni recipient strains. DNase I treatment reduced but did not eliminate transfer, and DNase I-treated cell supernatants did not transform, ruling out phage transduction. Recombinants also did not occur when the mating bacteria were separated by a membrane, suggesting that DNA transfer requires cell-to-cell contact. Transfer of the streptomycin marker was independent of the H. pylori comB transformation system, the cag pathogenicity island, and another type IV secretion system called tfs3. These findings indicated that a DNase I-resistant, conjugation-like mechanism may contribute to horizontal DNA transfer between different members of the Campylobacteriales group. The significance of this DNA uptake by C. jejuni in the context of acquiring antibiotic resistance is discussed.     

Distribution and serotypes of Campylobacter jejuni and Campylobacter coli in enteric Campylobacter strains isolated from children in the Central African Republic.

One hundred eighty-five enteric Campylobacter strains isolated from diarrheic or healthy children in Bangui (Central African Republic) were studied to determine their species and serotypes. C. coli was identified in 38.9% of all strains and in 43.9% of strains from diarrheic children. By the hemagglutination technique for heat-stable antigens, 73.5% of the strains could be serotyped. Of the typeable strains, 75% were distributed among 13 more frequent serotypes. C. coli serotype Pen 37,56 was the most common serotype from diarrheic children.     

Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from raw camel, beef, and water buffalo meat in Iran

This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from retail raw meats in Iran. From August 2009 to August 2010, a total of 379 raw meat samples from camel (n?=?130), beef (n?=?207), and water buffalo (n?=?42) were purchased from randomly selected retail outlets in Chaharmahal va Bakhtiari and Khuzestan provinces in Iran. The samples were evaluated for the presence of Campylobacter using traditional bacteriological tests and a nested polymerase chain reaction. Overall, 31 of 379 meat samples (8.2%) were contaminated with Campylobacter. The highest prevalence of Campylobacter spp. was found in water buffalo meat (21.4%), followed by beef (9.2%), and camel (2.3%) meat. The most prevalent Campylobacter species isolated from meat samples was Campylobacter jejuni (77.4%); the remaining isolates were Campylobacter coli (22.6%). Susceptibilities of 31 Campylobacter isolates were determined for ten antimicrobial drugs using the disk diffusion assay. Of 31 Campylobacter isolates, 27 (87.1%) were resistant to one or more antimicrobial agents. Nine strains (29.0%) were resistant to one single antimicrobial agent, and eight strains (25.8%) showed resistance to two antimicrobial agents. Multidrug resistance was found in 32.3% of Campylobacter strains. Resistance to tetracycline was the most common finding (67.7%), followed by resistance to ciprofloxacin (32.7%), and nalidixic acid (32.7%). To the authors' knowledge, the present study is the first report of the isolation of Campylobacter spp. from raw water buffalo meat in Iran.     

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.     

Improved biotyping schemes for Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.