Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

AIMS: To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. METHODS AND RESULTS: In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. CONCLUSIONS: All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).     

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.     

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.     

Systemic spread of Campylobacter jejuni after intravenous infections

Abstract Mice were infected intravenously with Campylobacter jejuni in order to study systemic translocation of this vibrio, as well as the interactions between bacteria and the host's defense mechanisms. It was found that granulocytes phagocyte C. jejuni in the bloodstream and that phagocytosis could be stimulated with LPS-pretreatment or, less effectively, opsonizing antibodies. It could also be demonstrated that these circulating 'infected' granulocytes are eliminated from the bloodstream mostly by the hepatic Kupffer's cells and that virulent strains of C. jejuni persist in the liver up to thirty days. It has to be concluded that phagocytosis by granulocytes and clearance of C. jejuni from the bloodstream by the liver represent important defense mechanisms in systemic Campylobacter infections.     

空肠弯曲菌感染动物模型的研究进展

空肠弯曲菌是一种全球关注的人兽共患病原菌,感染后可引起人和动物多种疾病。动物模型是开展致病机理、疫苗评价和药物开发等研究的基础。空肠弯曲菌由于培养条件苛刻以及感染实验动物的疾病相似性、经济性和重复性等因素,仍缺乏良好的感染动物模型,其致病机理迄今尚不清楚。本文对已报道的空肠弯曲菌感染实验动物模型进行综述。     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

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.     

Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni

Campylobacter spp. continue to be the greatest cause of bacterial gastrointestinal infections in humans worldwide. They encounter many stresses in the host intestinal tract, on foods and in the environment. However, in common with other enteric bacteria, they have developed survival mechanisms to overcome these stresses. Many of the survival mechanisms used by Campylobacter spp. differ from those used by other bacteria, such as Escherichia coli and Salmonella spp. This review summarizes the mechanisms by which Campylobacter spp. adapt to stress conditions and thereby increase their ability to survive on food and in the environment.     

Isolation and characterization of bacteriophages specific for Campylobacter jejuni

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni -specific bacteriophages (CPS1–6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni , but not other Gram–negative bacteria, including C. coli , Escherichia coli , Salmonella spp., and Gram–positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni .     

Campylobacter jejuni response to ox-bile stress

Campylobacter jejuni is a pathogen that colonizes the intestinal tract of humans and some animals. The in vitro responses of the bacterium to ox-bile were studied using proteomics to understand the molecular mechanisms employed by C. jejuni to survive bile stress. Its in vitro tolerance to bile was determined by growing the bacterium for 18 h in liquid cultures containing different bile concentrations. Significant growth inhibition was observed in the presence of 2.5% bile, and a decrease of 1.12 log units was measured at a bile concentration of 5%. Protein expression profiles of bacteria grown with and without bile were compared using two-dimensional polyacrylamide gel electrophoresis. Proteins with differential intensities greater than two-fold were identified using tandem mass spectrometry. Nuclear magnetic resonance spectroscopy and spectrophotometry were employed to measure enzyme activities in cell extracts from bacteria grown with and without bile. Together with proteins known to be involved in C. jejuni bile tolerance, the presence of bile modulated the expression of proteins such as elongation factors, ferritin, chaperones, ATP synthase and others, previously unknown to be implicated in the response of the bacterium to bile.     

The structure of the lipid anchor of Campylobacter jejuni polysaccharide

Campylobacter jejuni is a leading cause of gastroenteritis in humans. Campylobacter jejuni produces extracellular polysaccharides that have been characterized structurally and shown to be independent of lipopolysaccharides. Furthermore, it has been suggested that these C. jejuni polysaccharides are capsular in nature, although their lipid anchor has not been identified. In this report, the occurrence of a lipid-linked capsular-like polysaccharide in C. jejuni is conclusively shown, and the lipid anchor identified as dipalmitoyl-glycerophosphate.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Profiles of enterotoxin and cytotoxin production in Campylobacter jejuni and C. coli

Enterotoxin and cytotoxin production of 10 strains of Campylobacter spp. were examined by ELISA and HeLa cells assay, respectively. Both toxins were produced in high concentrations by strains freshly isolated from patients. The maximum enterotoxin activity was found to be at 24 h after incubation, at which time cell growth reached the stationary phase. On the other hand, production of cytotoxin increased after the logarithmic phase of the growth.     

Survival of Campylobacter jejuni in different gas mixtures

Campylobacter jejuni in fresh chilled chicken meat is known to be a major risk factor for human gastrointestinal disease. In the present study, the survival under chilled conditions of different C. jejuni strains exposed to different gas mixtures usually used for gas packaging of food was examined. Bolton broth and fresh, skinless chicken fillets were inoculated with six and four strains, respectively, and exposed to the gas mixtures 70/30% O(2)/CO(2), 70/30% N(2)/CO(2), and 100% N(2) (the latter only investigated in broth) at refrigeration temperature (4-5 degrees C). In broth culture, the strains survived significantly longer when exposed to 100% N(2) and 70/30% N(2)/CO(2) than in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001). For the two anaerobic gas mixtures, the reductions only reached 0.3-0.8 log(10) CFU mL(-1) within the same period. In the presence of oxygen, the numbers of C. jejuni were reduced by a minimum of 4.6 log(10) CFU mL(-1) over 21 days. When inoculated onto chicken fillets, the C. jejuni strains also died significantly faster in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001), reaching reductions of 2.0-2.6 log(10) CFU g(-1) after 8 days. In the gas mixture without oxygen (70/30% N(2)/CO(2)), no reductions were observed.     

The Campylobacter jejuni glycome

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.     

肉及肉制品中空肠弯曲菌的污染情况调查

我们对辽宁地区肉及肉制品进行弯曲杆菌属检查,发现12.9%(23/177)的样品为弯曲杆菌属阳性.经API Campy鉴定系统鉴定,其中26.1%(6/23)为空肠弯曲菌(C.jejuni)阳性,同时发现1株大肠弯曲菌.该调查证实,辽宁地区进出口的肉及肉制品中,存在空肠弯曲菌的污染,如果销售不当或再加工卫生不良,会对消费者的健康构成潜在威胁.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Campylobacter jejuni inactivation in New Zealand soils

AIM: The study was undertaken to determine the inactivation rate of Campylobacter jejuni in New Zealand soils. METHODS AND RESULTS: Farm dairy effluent (FDE) inoculated at c. 10(5) ml(-1) with C. jejuni was applied to intact soil cores at a rate of 2 l m(-2). Four soils were used: Hamilton (granular); Taupo (pumice); Horotiu and Waihou (allophanic). After FDE application cores were incubated at 10 degrees C for up to 32 days. For all four soils all the FDE remained within the cores and at least 99% of C. jejuni were retained in the top 5 cm. Campylobacter jejuni had declined to the limit of detection (two C. jejuni 100 g(-1)) by 25 days in Hamilton and Taupo soils and by 32 days in Waihou soil. In contrast, in Horotiu soil the decline was only three orders of magnitude after 32 days. Simulated heavy rainfall was applied 4 and 11 days after FDE application and only about 1% of the applied C. jejuni were recovered in leachates. CONCLUSIONS: This study demonstrated that at least 99% of applied C. jejuni were retained in the top 5 cm of four soils where they survived for at least 25 days at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil retention of C. jejuni is efficient at FDE application rates that prevent drainage losses. The low infectious dose of C. jejuni and its ability to survive up to 25 days have implications for stock management on dairy farms.     

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

Aims: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. Methods and Results: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which – in combination with MLST – increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37·5, 33·1 and 8·1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone‐sensitive human isolates showed overlapping MLST‐flaB types with those of chicken origin, resistant strains showed only 39% of matching types. Conclusion: Mainly quinolone‐sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. Significance and Impact of the Study: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.