Development of real-time PCR and hybridization methods for detection and identification of thermophilic Campylobacter spp. in pig faecal samples

AIMS: To develop a real-time (rt) PCR for species differentiation of thermophilic Campylobacter and to develop a method for assessing co-colonization of pigs by Campylobacter spp. METHODS AND RESULTS: The specificity of a developed 5' nuclease rt-PCR for species-specific identification of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and of a hipO gene nucleotide probe for detection of C. jejuni by colony-blot hybridization were determined by testing a total of 75 reference strains of Campylobacter spp. and related organisms. The rt-PCR method allowed species-specific detection of Campylobacter spp. in naturally infected pig faecal samples after an enrichment step, whereas the hybridization approach enhanced the specific isolation of C. jejuni (present in minority to C. coli) from pigs. Conclusions: The rt-PCR was specific for Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis and the colony-blot hybridization approach provided an effective tool for isolation of C. jejuni from pig faecal samples typically dominated by C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Species differentiation between thermophilic Campylobacter is difficult by phenotypic methods and the developed rt-PCR provides an easy and fast method for such differentiation. Detection of C. jejuni by colony hybridization may increase the isolation rate of this species from pig faeces.     

Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.     

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.     

PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

Factors affecting the pressure resistance of some Campylobacter species

AIMS: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. METHODS AND RESULTS: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. CONCLUSIONS: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.     

Identification of Campylobacter jejuni, Campylobacter coli and Campylobacter lari by the nucleic acid amplification system NASBR

M. UYTTENDAELE, R. SCHUKKINK, B. VAN GEMEN AND J. DEBEVERE. 1994. NASBA^R, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBA^R and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni , present in a total count of 4 times 10⁶ cfu of Gram-negative bacteria, resulted in a positive hybridization signal.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay

Campylobacters isolated from mussels and oysters in The Netherlands were analysed by a novel assay, based on DNA amplification with primers, based on semiconserved GTP-binding sites of a putative GTPase gene. Polymerase chain reaction (PCR) was followed by a single step reverse hybridization line probe assay (PCR-LiPA). This permits identification of Campylobacter jejuni, C. coli, C. lari and C. upsaliensis . Among a group of 44 isolates, three C. jejuni , one C. upsaliensis , one double infection with C. jejuni and C. coli , and 38 C. lari strains were identified. These results were in complete agreement with conventional identification methods and whole cell protein analysis. One C. hyointestinalis isolate was not identified by the PCR-LiPA, since the reverse hybridization assay does not comprise specific probes for this particular species. PCR products from 36 C. lari isolates were sequenced and phylogenetic analysis revealed the presence of two major C. lari subgroups: one comprised 11 highly homologous sequences, whereas the 25 sequences in the other subgroup were more heterogeneous. This confirmed earlier findings that C. lari isolates comprise a more heterogeneous group of isolates as compared with C. jejuni , C. coli and C. upsaliensis . Based on the sequence information, a novel PCR-LiPA was developed that permits specific and rapid detection of the different C. lari variants.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Prevalence of Campylobacter jejuni,Campylobacter lari,and Campylobacter coli in different ecological guilds and taxa of migrating birds

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.     

Reliability of nucleotide sequence information of full-length flagellin A gene (flaA) and flaA short variable region (SVR) for molecular discrimination of Campylobacter lari organisms

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n?=?45) including UN C. lari (n?=?17), UPTC (n?=?18), and Campylobacter jejuni (n?=?10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.     

Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

AIM: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. METHODS AND RESULTS: Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. CONCLUSIONS: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. SIGNIFICANCE AND IMPACT OF STUDY: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.     

Identification of thermophilic Campylobacter spp. by phenotypic and molecular methods

AIMS: The differences between phenotyping and genotyping (polymerase chain reaction- restriction fragment length polymorphism) of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis were assessed. METHODS AND RESULTS: A total of 51, 63 and 88 strains from dogs, pigs and humans, respectively, were examined. The strains were first typed by biochemical methods, then by PCR-RFLP using AluI and Tsp509I. None of the strains were typed as Camp. lari by the PCR-RFLP. The biggest differences were found in the identification of Camp. jejuni and Camp. coli. The main discrepancies were caused with the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Strains which were identified biochemically as Camp. coli and by digestion with AluI as Camp. jejuni (eight strains) were tested for the presence of the hippuricase gene. CONCLUSION: The PCR typing results showed the presence of the hippuricase gene as unique to Camp. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: A reliable identification of Campylobacter spp. should be supplemented with a molecular method.     

Molecular detection of Campylobacter spp. in California gull (Larus californicus) excreta

We examined the prevalence, quantity, and diversity of Campylobacter species in the excreta of 159 California gull (Larus californicus) samples using culture-, PCR-, and quantitative PCR (qPCR)-based detection assays. Campylobacter prevalence and abundance were relatively high in the gull excreta examined; however, C. jejuni and C. lari were detected in fewer than 2% of the isolates and DNA extracts from the fecal samples that tested positive. Moreover, molecular and sequencing data indicated that most L. californicus campylobacters were novel (<97% 16S rRNA gene sequence identity to known Campylobacter species) and not closely related to species commonly associated with human illness. Campylobacter estimates were positively related with those of fecal indicators, including a gull fecal marker based on the Catellicoccus marimammalium 16S rRNA gene.     

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.     

Application of the 5'-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.     

Emerging dynamics of human campylobacteriosis in Southern Ireland

Infections with Campylobacter spp. pose a significant health burden worldwide. The significance of Campylobacter jejuni/Campylobacter coli infection is well appreciated but the contribution of non-C. jejuni/C. coli spp. to human gastroenteritis is largely unknown. In this study, we employed a two-tiered molecular study on 7194 patient faecal samples received by the Microbiology Department in Cork University Hospital during 2009. The first step, using EntericBio(?) (Serosep), a multiplex PCR system, detected Campylobacter to the genus level. The second step, utilizing Campylobacter species-specific PCR identified to the species level. A total of 340 samples were confirmed as Campylobacter genus positive, 329 of which were identified to species level with 33 samples containing mixed Campylobacter infections. Campylobacter jejuni, present in 72.4% of samples, was the most common species detected, however, 27.4% of patient samples contained non-C. jejuni/C. coli spp.; Campylobacter fetus (2.4%), Campylobacter upsaliensis (1.2%), Campylobacter hyointestinalis (1.5%), Campylobacter lari (0.6%) and an emerging species, Campylobacter ureolyticus (24.4%). We report a prominent seasonal distribution for campylobacteriosis (Spring), with C. ureolyticus (March) preceeding slightly C. jejuni/C. coli (April/May).     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Evaluation of the effect of temperature and nutrients on the survival of Campylobacter spp. in water microcosms

Batch microcosms containing various water types (de-ionized and river water with or without sediment), incubated at a range of temperatures (5-37 degrees C), were used to facilitate a comparative evaluation of the significance of such variables and their interactions upon the collective and individual survival of four species of thermophilic Campylobacter. All variables significantly influenced (P < = 0.031) population decay rates. Minimal decay for the group was identified at low temperatures (5 degrees C) in river water, i.e. nutrient-containing microcosms. Collective decay rates within river water microcosms were significantly decreased (P = 0.03) from those observed in de-ionized water, particularly at environmental temperatures (5 and 15 degrees C). However, the increased nutrient levels observed in sediment-containing microcosms did not significantly (P = 0.41) reduce population decay rates. Overall, Camp. jejuni populations demonstrated the most resilience to the environmental stressors evaluated, with the exception of 15 degrees C where Camp. lari was the most persistent. Campylobacter coli and Camp. upsaliensis demonstrated comparable survival characteristics but were less resilient than Camp. jejuni and Camp. lari. These observations identify the suitability of water systems as a reservoir and medium for Campylobacter infection, and potentially identifies Camp. jejuni and Camp. lari as the main protagonists of water-mediated campylobacteriosis.