Campylobacter jejuni--microtubule-dependent invasion

Campylobacter jejuni is the leading bacterial cause of food-borne illness worldwide and a major cause of Guillain-Barré paralysis. Recent molecular and cellular studies of one well-characterized C. jejuni strain have begun to unravel the details of an unusual microtubule-dependent (actin-filament-independent) gut-invasion mechanism, through which at least some C. jejuni initiate disease. Although responsible for causing a human dysenteric syndrome remarkably similar to that triggered by Shigella spp., current evidence suggests that C. jejuni use some markedly different molecular mechanisms of pathogenesis compared with shigellae.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni.

Campylobacter jejuni and Campylobacter coli are important causes of human enteric infections. Several determinants of pathogenicity have been proposed based on the clinical features of diarrheal disease and on the phenotypic properties of Campylobacter strains. To facilitate an understanding of the genetic determinants of Campylobacter virulence, we have developed a method for constructing C. jejuni mutants by shuttle mutagenesis. In the example described here, a kanamycin resistance gene was inserted into Campylobacter DNA fragments encoding 16S rRNA cloned in Escherichia coli. These disrupted, modified sequences were returned to C. jejuni via conjugation. Through the apparent process of homologous recombination, the kanamycin resistance-encoding sequences were rescued by chromosomal integration, resulting in the simultaneous gene replacement of one of the 16S sequences of C. jejuni and the loss of the vector. We propose that Campylobacter isogenic mutants could be developed by using this system of shuttle mutagenesis.     

Campylobacter Enteritis on Hopi and Navajo Indian Reservations—Clinical and Epidemiologic Features

From June 22 through September 30, 1981, stool specimens from 522 Hopi and Navajo outpatients were cultured because of diarrheal illnesses at the Keams Canyon Indian Health Service Hospital, Arizona. Campylobacter jejuni was isolated from the specimens of 26 (5%) of the patients. This pathogen was found as frequently as Shigella in patients younger than 2 years or older than 20 years, but was significantly less common in the 2 to 20-year age group (P<.000001). Campylobacter enteritis was indistinguishable clinically from shigellosis in adult patients, but in children younger than 5 years, a rectal temperature higher than 38°C (100.5°F) was significantly more common with Shigella than with Campylobacter infection (P=.003). In a field study of 20 families, we found that households with a case of Campylobacter enteritis were more likely than age- and community-matched controls to own farm animals (P=.05), but were not more likely to own household pets. C jejuni is less common than Shigella as a cause of summer seasonal diarrhea and dysentery among the Hopi and Navajos; the striking differences in the age-specific rates of these two infections suggest different routes of transmission.     

Evaluation of cytotoxic activity in fecal filtrates from patients with Campylobacter jejuni or Campylobacter coli enteritis

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis.     

Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.     

Characterization of Campylobacter jejuni strains occurring in the USSR and the Czech Republic

In 1986-1989 the microbiological laboratory in Prague obtained 100 Campylobacter jejuni strains while its counterpart in Moscow gained 120 such strains. The strains were primarily isolated from humans with diarrheal disease, from domestic and wild animals and from the environment. Most C. jejuni strains were successfully specified and classified using a Czechoslovak serotyping scheme proposed by Kahlich. Serotypes 1, 2, 7, 15, 22 and 24 occurred most frequently in the Czech Republic whereas in the Soviet Union the most common serotypes were 7, 16, 5, 2, 1. The proportion of strains which could not be identified in the serotyping scheme was about 10% in the USSR and 20% in the Czech Republic. Our findings suggest differences between the USSR and the Czech Republic in the prevalence and incidence particular serotypes of C. jejuni strains.     

Guillain-Barré syndrome and Campylobacter jejuni: a serological study.

The association between Campylobacter jejuni infection and Guillain-Barré syndrome was investigated serologically in a retrospective study of 56 patients admitted to this hospital over four years. Evidence of preceding C jejuni infection was found in 21 (38%) of these patients, indicating that C jejuni was the most common single identifiable pathogen precipitating the disease. Among those patients who had presented with preceding diarrhoea the serum antibody response was similar to that in uncomplicated C jejuni enteritis. Patients with serological evidence of preceding C jejuni infection manifested a significantly more severe form of the disease. In cerebrospinal fluid the predominant specific antibody class was IgG, and this was closely related to the serum titres of specific IgG. IgA and IgM specific antibodies were found only in the cerebrospinal fluid of patients with recent C jejuni infection. These findings support the possibility that humoral immune factors are responsible for the neural damage and demyelination seen in Guillain-Barré syndrome.     

Campylobacter jejuni/coli in commercially reared beagles: prevalence and serotypes

A 5 year longitudinal study involving 187 commercially reared beagles from three suppliers was undertaken to determine prevalence and serotypes of Campylobacter jejuni and C. coli. Campylobacter jejuni or C. coli was isolated from the feces in 62 of 177 asymptomatic beagles and 8 of 10 dogs with diarrhea for an overall prevalence of 37%. A total of 36 isolates were serotyped on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes isolated from humans with campylobacter associated enteritis (15 C. jejuni, 5 C. coli serotypes). Of these isolates, 17 (47%) serotyped with antisera to 7 C. jejuni serotypes frequently isolated in human cases of enteric campylobacteriosis (serotypes 1, 4, 10, 16, 18, 19, 37). One C. coli reacted to antisera 24, 34, 37, one strain of C. coli to antisera type 37, and another C. coli to antisera type 34. All three C. coli belonged to serotypes frequently encountered in diarrheic human patients.     

Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5'-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Chronic effects of Campylobacter infection

Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis and chronic sequelae, such as reactive arthritis and Guillain-Barré syndrome (GBS), are known to follow uncomplicated infections. While little is known about reactive arthritis following Campylobacter infection, our knowledge on the pathogenesis of Campylobacter-induced GBS is expanding rapidly and is summarized in this review.     

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Genotypic and phenotypic properties of cattle-associated Campylobacter and their implications to public health in the USA

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P<0.05) higher in fecal samples collected from the South (32.8%) as compared to those from the North (14.8%), Midwest (15.83%), and East (12%). Pulsed Field Gel Electrophoresis (PFGE) analysis showed that C. jejuni and C. coli isolates were genotypically diverse and certain genotypes were shared across two or more of the geographic locations. In addition, 13 new C. jejuni and two C. coli sequence types (STs) were detected by Multi Locus Sequence Typing (MLST). C. jejuni associated with clinically human health important sequence type, ST-61 which was not previously reported in the USA, was identified in the present study. Most frequently observed clonal complexes (CC) were CC ST-21, CC ST-42, and CC ST-61, which are also common in humans. Further, the cattle associated C. jejuni strains showed varying invasion and intracellular survival capacity; however, C. coli strains showed a lower invasion and intracellular survival potential compared to C. jejuni strains. Furthermore, many cattle associated Campylobacter isolates showed resistance to several antimicrobials including ciprofloxacin, erythromycin, and gentamicin. Taken together, our results highlight the importance of cattle as a potential reservoir for clinically important Campylobacter.     

Biochemical and serologic characteristics of Campylobacter jejuni/coli strains causing diarrhea in children

One hundred strains of Campylobacter jejuni/coli isolated from faeces of children with diarrhoea were characterised. Frequency of isolation of these microorganisms from faeces of children with enterocolitis symptoms was evaluated. In this group Campylobacter jejuni/coli constituted 11.4% of all isolates, being the dominant etiologic agent of these infections. Biotype pattern of 100 Campylobacter jejuni/coli strains was determined. Biotype I C.jejuni prevailed and C. coli constituted as much as 35% of all isolated strains. All isolated strains were characterised serologically according to typing scheme of Lior. Seventy four strains were typed and 22 were untypable, out of which four were rough. Two new serotypes were isolated: LIO 71 and LIO 72, LIO 4 and LIO 72 serotypes were the most frequently isolated. Frequency of isolation of Campylobacter jejuni/coli strains were also determined in the period from january 1985 to august 1987.     

Campylobacteriosis--a "new" disease in laboratory animals

Campylobacter fetus ss jejuni is now recognized as a leading cause of diarrhea in humans. With the development of proper culture techniques, C jejuni has been isolated from humans with diarrhea as frequently as Salmonella or Shigella species. Laboratory animal models are being developed to study the pathogenesis of campylobacter-induced diarrhea; also, identification of C jejuni is occurring more frequently in a variety of laboratory animals, whether asymptomatic carriers or those with clinical diarrhea. Animals shedding C jejuni also have been incriminated as a source of campylobacter diarrhea in humans.     

Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle

AIMS: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. METHODS AND RESULTS: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51.2% positive samples) with 94 operations positive (97.9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47.4%), nalidixic acid (4.0%) and ciprofloxacin (2.5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3.6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20.3% of the C. coli strains were multiresistant. CONCLUSIONS: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.     

Determination of toxicity of Campylobacter jejuni isolated from humans and from poultry carcasses acquired at various stages of production

AIM: The research focused on the determination of the toxicity variation associated with Campylobacter jejuni isolated from humans and chickens. METHODS AND RESULTS: Campylobacter jejuni isolates were obtained from chicken carcasses and from humans exhibiting symptoms of campylobacteriosis. Using HeLa cells as the in vitro model, toxicity was determined for each isolate. The mean toxicity level of the chicken isolates was significantly lower than that of the human isolates (P < 0.001). There was a wide range of toxicity in C. jejuni isolated from chickens and the percentage of isolates exhibiting low toxicity remaining relatively constant. All C. jejuni isolates from humans possessed either medium or high levels of toxicity. CONCLUSIONS: All wildtype C. jejuni isolates obtained from poultry carcasses may not be equally important as a human foodborne pathogen. SIGNIFICANCE AND IMPACT OF STUDY: Campylobacter jejuni remains a primary foodborne pathogen and increased efforts are needed to determine the impact of wildtype isolates in causing human illness. The present research indicates that all isolates may not be equally important in regards to disease potential. The information found should be included in efforts to develop C. jejuni detection, control and infection modelling.     

Characterization of Campylobacter jejuni biofilms under defined growth conditions

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.     

Proteomic analyses of a robust versus a poor chicken gastrointestinal colonizing isolate of Campylobacter jejuni

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.