Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test®

Aims: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. Methods and Results: Adhesion assays were conducted in 96‐well, polystyrene microtiter plates using the BioFilm Ring Test^® method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test^® and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food‐processing and clinical cases. Conclusions: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. Significance and Impact of the Study: The adhesion capability of strains could partly explain the cross‐contamination or re‐contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.     

Tracing the source of campylobacteriosis

Campylobacter jejuni is the leading cause of bacterial gastro-enteritis in the developed world. It is thought to infect 2–3 million people a year in the US alone, at a cost to the economy in excess of US $4 billion. C. jejuni is a widespread zoonotic pathogen that is carried by animals farmed for meat and poultry. A connection with contaminated food is recognized, but C. jejuni is also commonly found in wild animals and water sources. Phylogenetic studies have suggested that genotypes pathogenic to humans bear greatest resemblance to non-livestock isolates. Moreover, seasonal variation in campylobacteriosis bears the hallmarks of water-borne disease, and certain outbreaks have been attributed to contamination of drinking water. As a result, the relative importance of these reservoirs to human disease is controversial. We use multilocus sequence typing to genotype 1,231 cases of C. jejuni isolated from patients in Lancashire, England. By modeling the DNA sequence evolution and zoonotic transmission of C. jejuni between host species and the environment, we assign human cases probabilistically to source populations. Our novel population genetics approach reveals that the vast majority (97%) of sporadic disease can be attributed to animals farmed for meat and poultry. Chicken and cattle are the principal sources of C. jejuni pathogenic to humans, whereas wild animal and environmental sources are responsible for just 3% of disease. Our results imply that the primary transmission route is through the food chain, and suggest that incidence could be dramatically reduced by enhanced on-farm biosecurity or preventing food-borne transmission.     

Molecular typing of Campylobacter jejuni and C. coli from chickens and patients with gastritis or Guillain‐Barré syndrome based on multilocus sequence types and pulsed‐field gel electrophoresis patterns

Campylobacter jejuni has recently been noted as the most common cause of bacterial foodborne diseases in Japan. In the present study, we determined ST types of C. jejuni and Campylobacter coli isolated from chickens and patients with enteritis or GBS in Japan and Thailand. C. jejuni from chickens, enteritis, and GBS exhibited divergent ST types and included several novel types in addition to worldwide common types. C. coli from enteritis was also divergent. Novel ST types may represent unidentified native clones in each country. Pulsed‐field gel electrophoresis confirmed the above typing and demonstrated long‐term persistence and transmission.     

Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds

Zoonotic pathogens often infect several animal species, and gene flow among populations infecting different host species may affect the biological traits of the pathogen including host specificity, transmissibility and virulence. The bacterium Campylobacter jejuni is a widespread zoonotic multihost pathogen, which frequently causes gastroenteritis in humans. Poultry products are important transmission vehicles to humans, but the bacterium is common in other domestic and wild animals, particularly birds, which are a potential infection source. Population genetic studies of C. jejuni have mainly investigated isolates from humans and domestic animals, so to assess C. jejuni population structure more broadly and investigate host adaptation, 928 wild bird isolates from Europe and Australia were genotyped by multilocus sequencing and compared to the genotypes recovered from 1366 domestic animal and human isolates. Campylobacter jejuni populations from different wild bird species were distinct from each other and from those from domestic animals and humans, and the host species of wild bird was the major determinant of C. jejuni genotype, while geographic origin was of little importance. By comparison, C. jejuni differentiation was restricted between more phylogenetically diverse farm animals, indicating that domesticated animals may represent a novel niche for C. jejuni and thereby driving the evolution of those bacteria as they exploit this niche. Human disease is dominated by isolates from this novel domesticated animal niche.     

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.     

Genotyping and PCR detection of potential virulence genes in <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Campylobacter coli</Emphasis> isolates from different sources in Poland

The prevalence of potential virulence markers was determined among the population of Polish Campylobacter jejuni and Campylobacter coli isolates from children, chickens, pigs and dogs. The presence of the flaA, flaB, cdtA, cdtB, cdtC, cdtABC, virB11, and cj0588 genes among 74 C. jejuni and 15 C. coli isolates was detected by PCR. High prevalence of five different putative virulence and toxin genes (flaA, cdtA, cdtB, cdtC, and cj0588) was found among isolates obtained from children, chickens and dogs. The occurrence of these genes among isolates obtained from pigs was significantly different than for strains isolated from other sources. Two methods for genotyping Campylobacter spp. strains were applied — flaA-typing, and ADSRRS-fingerprinting method, which was used for the first time for Campylobacter spp. strains. Similarity of the genetic profiles was demonstrated in strains isolated from chickens and dogs, and in isolates from chickens and children. Strains isolated from pigs, both C. jejuni as well as C. coli, did not group with isolates from other sources.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Gene pool transmission of multidrug resistance among Campylobacter from livestock,sewage and human disease

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.     

Frequency and Spatial Distribution of Environmental Campylobacter spp.

Humans are exposed to Campylobacter spp. in a range of sources via both food and environmental pathways. For this study, we explored the frequency and distribution of thermophilic Campylobacter spp. in a 10- by 10-km square rural area of Cheshire, United Kingdom. The area contains approximately 70, mainly dairy, farms and is used extensively for outdoor recreational activities. Campylobacter spp. were isolated from a range of environmental samples by use of a systematic sampling grid. Livestock (mainly cattle) and wildlife feces and environmental water and soil samples were cultured, and isolates were presumptively identified by standard techniques. These isolates were further characterized by PCR. Campylobacter jejuni was the most prevalent species in all animal samples, ranging from 11% in samples from nonavian wildlife to 36% in cattle feces, and was isolated from 15% of water samples. Campylobacter coli was commonly found in water (17%) and sheep (21%) samples, but rarely in other samples. Campylobacter lari was recovered from all sample types, with the exception of sheep feces, and was found in moderate numbers in birds (7%) and water (5%). Campylobacter hyointestinalis was only recovered from cattle (7%) and birds (1%). The spatial distribution and determinants of C. jejuni in cattle feces were examined by the use of model-based spatial statistics. The distribution was consistent with very localized within-farm or within-field transmission and showed little evidence of any larger-scale spatial dependence. We concluded that there is a potentially high risk of human exposure to Campylobacter spp., particularly C. jejuni, in the environment of our study area. The prevalence and likely risk posed by C. jejuni-positive cattle feces in the environment diminished as the fecal material aged. After we took into account the age of the fecal material, the absence or presence of rain, and the presence of bird feces, there was evidence of significant variation in the prevalence of C. jejuni-positive cattle feces between grazing fields but no evidence of spatial clustering beyond this resolution. The spatial pattern of C. jejuni is therefore consistent with that for an organism that is ubiquitous in areas contaminated with cattle feces, with a short-scale variation in infection intensity that cannot be explained solely by variations in the age of the fecal material. The observed pattern is not consistent with large-scale transmission attributable to watercourses, wildlife territories, or other geographical features that transcend field and farm boundaries.     

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C)

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

Prevalence and Genetic Diversity of Campylobacter spp. in Environmental Water Samples from a 100-Square-Kilometer Predominantly Dairy Farming Area

Water samples were taken systematically from a 100-km² area of mainly dairy farmland in northwestern England and examined for Campylobacter spp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) and flaA strain typing of Campylobacter jejuni and Campylobacter coli isolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables. Campylobacter spp. were isolated from 40.5% (n = 119) of the water samples tested. C. jejuni was isolated from 14.3%, C. coli was isolated from 18.5%, and Campylobacter lari was isolated from 4.2% of the samples. Campylobacter hyointestinalis was not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P = 0.001). C. jejuni was the species most commonly isolated from trough-water and running-water sources, while C. coli was the most frequently isolated from standing water (P < 0.001). No association was found between the presence of Escherichia coli and that of Campylobacter spp. The final multivariable logistic regression model for Campylobacter spp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population of C. jejuni and the presence of a common C. coli flaA type that was widely distributed throughout the area. Most of the isolates within the common flaA type were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology of Campylobacter spp. in a farming environment.     

Activation of trehalase by heat shock in yeast ascospores: Correlation with total cellular cyclic-AMP content

An animal model resembling the human disease caused byCampylobacter jejuni has been developed. Characteristic illness followed intragastric challenge of neonatal mice with strains ofC. jejuni enhanced for virulence by serial intraperitoneal passage in weanling mice of organisms suspended in either mucin or iron dextran. Such passage lowered the LD₅₀ in weanlings from 2×10¹¹ colony-forming units (CFU) to 2×10⁵ CFU per mouse. Neonatal mice chellenged by intragastric intubation with 2×10⁹ CFU of the virulence-enhanced organisms suspended in mucin or iron dextran showed signs of infection by day 5, including severe diarrhea, increased musus discharge occasionally with blood, and reduced weight gain. Diarrhea contionued for eight days, after which most animals recovered. This mouse infection model provides a means for assessing the determinants of virulence among strains ofC. jejuni.     

Survey of Campylobacter jejuni in stray cats in southern Italy

Aim: To evaluate the presence of thermotolerant Campylobacter spp. in stray cats in southern Italy. Methods and Results: One hundred and thirteen animals were trapped in two different environments (urban area, harbour area) of the city of Naples. From each cat, rectal swabs were collected. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Campylobacter jejuni was isolated from 19/113 (16·8%) stray cats, whereas Campylobacter coli was not isolated. The cat data (age, environment and sex) were analysed by two statistical analyses using the C. jejuni status (positive/negative) as a dependent variable. As regards statistical regression model results, age and environment were risk factors for C. jejuni positivity. In particular, cats older than 1 year had a significantly higher risk of being positive for C. jejuni than cats aged up to 1 year (OR = 10·440; P = 0·000). Moreover, cats living in the harbour area had a significantly higher risk to be a carrier of C. jejuni than cats living in urban area (OR = 17·911; P = 0·008). Conclusion: The findings of the present survey confirm stray cats as potential carriers of C. jejuni. Significance and Impact of the Study: This is the first study on the prevalence of C. jejuni in stray cats in Europe.     

Identification of genomic differences between Campylobacter jejuni subsp. jejuni and C. jejuni subsp. doylei at the nap locus leads to the development of a C. jejuni subspeciation multiplex PCR method

Background  

The human bacterial pathogen Campylobacter jejuni contains two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Although Cjd strains are isolated infrequently in many parts of the world, they are obtained primarily from human clinical samples and result in an unusual clinical symptomatology in that, in addition to gastroenteritis, they are associated often with bacteremia. In this study, we describe a novel multiplex PCR method, based on the nitrate reductase (nap) locus, that can be used to unambiguously subspeciate C. jejuni isolates.     

Potential Intervention of Campylobacter jejuni in the Modulation of Murine Immune Response

Campylobacter jejuni has been reported to produce different toxins that may modulate the immune response in both animals and humans. The effect of C. jejuni enterotoxin on the immune response was investigated in two groups of Balb/c mice. One of them was inoculated intraperitoneally with 10¹⁰ colony forming units (CFU) of an enterotoxigenic strain (CCUG 7580), and the second one with a non-enterotoxigenic strain (CCUG 7440). The number of polymorphonuclear (PMN) cells from spleen increased in both enterotoxigenic and non-enterotoxigenic strains as a consequence of C. jejuni infection. Notwithstanding, lymphocyte proliferation stimulated by lipopolysaccharide (LPS) was increased by both enterotoxigenic and non-enterotoxigenic strains. Interleukin-2 (IL-2) production from splenic cells was increased significantly by infection with the enterotoxigenic strain. Both enterotoxigenic and non-enterotoxigenic strains reduced the splenic response to sheep erythrocytes; the response was significantly suppressed for immunoglobulin M (Ig M) and for immunoglobulin G (Ig G) synthesis. These results suggest that C. jejuni is able to modify some components of the immune response in mice, and also that the enterotoxigenic strain has more immunomodulating activity than the non-enterotoxigenic strain. Received: 4 December 2000/Accepted: 6 February 2001     

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1‐year period in Lithuania

Aims: The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. Methods and Results: During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter‐positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73·8%) were positive for Camp. jejuni and 17 flocks (40·48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1‐year period. Conclusions: Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm‐specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. Significance and Impact of the Study: For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.     

Cloning of an Outer Membrane Protein Gene from Campylobacter jejuni

An antigen in the outer membrane protein (OMP) fraction of Campylobacter jejuni was identified and characterized. Western blot analysis demonstrated antigenic differences in this protein between two congenic C. jejuni strains. Strain A74/C, which colonizes chickens, expressed the antigen at 34 kDa, while strain A74/O, which poorly colonizes chickens, expressed the antigen at 32 and 34 kDa. A genomic library was constructed in λgt11 with DNA from A74/O and screened with antibody raised against C. jejuni OMPs. A clone that possessed a 1.3-kb insert and expressed an immunoreactive protein fused to β-galactosidase was isolated and purified. DNA sequence analysis revealed the insert contained one open reading frame 864 bases long. The deduced amino acid sequence demonstrated 56.3% similarity with Bacillus steorothermophilus glnH, a glutamine-binding protein, and 54.0% similarity with C. jejuni PEB1, a putative colonization adhesin. Southern hybridization, Northern hybridization, and DNA sequence analyses of the congenic colonizing and noncolonizing strains of C. jejuni failed to distinguish the two strains and revealed only one copy of the gene. Post-translational modification may be an alternate explanation for the antigenic differences seen between the two strains. Received: 15 October 1996 / Accepted: 3 December 1996     

Differentiation of Campylobacter Isolates on the Basis of Sensitivity to Boiling in Water as Measured by PCR-Detectable DNA

Differential sensitivity for the release of PCR-detectable genomic DNA upon boiling in water is reported for 45 Campylobacter jejuni and Campylobacter coli strains isolated in Egypt. All of the strains released PCR-detectable DNA when treated with proteinase K and sodium dodecyl sulfate. When DNA was extracted from these strains by boiling in water, nine (20%) of the strains were PCR negative or resistant to boiling, suggesting the presence of boiling-sensitive and boiling-resistant phenotypes.     

Comparison of Campylobacter jejuni genotypes from dairy cattle and human sources from the Matamata-Piako District of New Zealand

Aims: To identify the prevalence and types of Campylobacter jejuni carried by dairy cattle and the extent of overlap of these types with those causing disease in humans. Methods and Results: Faecal samples from 410 dairy cattle were collected from 36 farms in the Matamata-Piako district in New Zealand. Campylobacter jejuni was isolated on all 36 farms, with a prevalence of 51% (95% CI 45–57) in dairy cattle and 65% (95% CI 58–72) in calves. Eighty-nine of these isolates were typed using Penner serotyping and pulsed field gel electrophoresis and were compared with 58 human C. jejuni isolates from people resident within this study area. Conclusions: Campylobacter jejuni were found in the faeces of over half of the dairy cows and calves examined. Twenty-one per cent of the bovine isolates and 43% of the human isolates formed indistinguishable clusters of at least one bovine and one human isolate. Significance and Impact of the Study: While a direct link between bovine isolates and human cases was not demonstrated, the finding of indistinguishable genotypes among C. jejuni isolates from bovine and human sources confirms that dairy cows and calves are a potential source of human campylobacteriosis. Barriers to separate bovine faecal material from the general public are therefore important public health measures.