Level of Detection (LOD50) of Campylobacter Is Strongly Dependent on Strain, Enrichment Broth, and Food Matrix.

The detection of thermotolerant Campylobacter in food may be difficult due to the growth of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during enrichment, resulting in false-negative samples. Therefore, the ISO protocol (ISO 10272-1:2017) suggests that, next to Bolton broth (BB), Preston broth (PB) is used as an enrichment broth to inhibit competitive flora in samples with suspected high levels of background microorganisms, such as ESBL-producing bacteria. However, the application of the strains used for validation of this ISO was not clearly characterized. This study examined the LOD50 (level of detection, the concentration where the probability of detection is 50%) of the validation strains (three C. jejuni and two C. coli strains) in BB and PB using different food matrices, namely, raw milk, chicken skin, frozen minced meat, and frozen spinach. The LOD50 was calculated by inoculating multiple portions with at least two inoculum levels. For each reproduction, eight test portions were used for each inoculum level and the test portion size was 10 g (chicken skin, frozen minced meat, and frozen spinach) or 10 mL (raw milk). Furthermore, the effect of artificially inoculated ESBL-producing E. coli on the LOD50 was examined to mimic the presence of ESBL-producing background microorganisms in the food matrices, namely, raw milk and chicken skin. In BB, the LOD50 of all strains tested in raw milk, chicken skin, and frozen spinach was rather low (0.4-37 CFU/test portion), while the LOD50 in frozen minced meat was higher and much more variable (1-1,500 CFU/test portion), depending on the strain. Generally, enrichment in PB resulted in higher LOD50 than in BB, especially for C. coli. Co-inoculation with ESBL-producing E. coli increased the LOD50 in BB, while PB successfully inhibited the growth of this competitive microorganism. In conclusion, food matrix and enrichment broth may have a large influence on the LOD50 of different Campylobacter strains. Therefore, it is not possible to give an unequivocal advice on when to use which enrichment broth, and this advocates the use of both methods in case of doubt. Furthermore, this study indicates specific strains that would be a good choice to use for Campylobacter method verification as described in ISO 16140-3:2021.

Comparative Analysis of L-Fucose Utilization and Its Impact on Growth and Survival of Campylobacter Isolates.

Campylobacter jejuni and Campylobacter coli were previously considered asaccharolytic, but are now known to possess specific saccharide metabolization pathways, including L-fucose. To investigate the influence of the L-fucose utilization cluster on Campylobacter growth, survival and metabolism, we performed comparative genotyping and phenotyping of the C. jejuni reference isolate NCTC11168 (human isolate), C. jejuni Ca1352 (chicken meat isolate), C. jejuni Ca2426 (sheep manure isolate), and C. coli Ca0121 (pig manure isolate), that all possess the L-fucose utilization cluster. All isolates showed enhanced survival and prolonged spiral cell morphology in aging cultures up to day seven in L-fucose-enriched MEMα medium (MEMαF) compared to MEMα. HPLC analysis indicated L-fucose utilization linked to acetate, lactate, pyruvate and succinate production, confirming the activation of the L-fucose pathway in these isolates and its impact on general metabolism. Highest consumption of L-fucose by C. coli Ca0121 is conceivably linked to its enhanced growth performance up to day 7, reaching 9.3 log CFU/ml compared to approximately 8.3 log CFU/ml for the C. jejuni isolates. Genetic analysis of the respective L-fucose clusters revealed several differences, including a 1 bp deletion in the Cj0489 gene of C. jejuni NCTC11168, causing a frameshift in this isolate resulting in two separate genes, Cj0489 and Cj0490, while no apparent phenotype could be linked to the presumed frameshift in this isolate. Additionally, we found that the L-fucose cluster of C. coli Ca0121 was most distant from C. jejuni NCTC11168, but confirmation of links to L-fucose metabolism associated phenotypic traits in C. coli versus C. jejuni isolates requires further studies.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique.

The validation in an interlaboratory study of the International Standards Organization standard method for the enumeration of Campylobacter in foods (ISO 10272-2) was performed after preparation of the revised Standard based on scientifically sound and validated methods of analysis. The matrices selected for testing in the collaborative trial were frozen spinach, minced meat, raw milk, chicken skin, and broiler caecal material. Each matrix was artificially inoculated with a different Campylobacter strain. Fifteen laboratories participated in the interlaboratory study. As a general indication of repeatability limit (r), the following overall values can be used when testing chicken skin samples: As a general indication of reproducibility limit (R), the following overall values can be used when testing chicken skin samples: The validation data for all matrices were incorporated in the newly published ISO standard EN ISO 10272-2:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 2: colony-count technique.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method.

During the last decade Campylobacter has been the most commonly reported gastrointestinal bacterial infection in humans in the European Union. The use of a sensitive detection method based on enrichment of Campylobacter spp. is often needed when examining foods. However, as background flora developed resistance to third generation ß-lactams used in selective culture media, the ISO method was adapted. It now consists of three different procedures (A, B, and C) depending on the expected concentration and condition of Campylobacter and the background microflora. As the diagnostic sensitivity of the detection test varies between laboratories, this justifies the validation of the method in an interlaboratory study. The matrices selected for testing in the collaborative trials were frozen spinach (procedure A, Bolton enrichment broth), minced meat (procedure A, Bolton enrichment broth), raw milk (procedure B, Preston enrichment broth), chicken skin (procedure B, Preston enrichment broth), and broiler caecal material (procedure C, direct plating on mCCD agar). Each matrix was artificially inoculated with a different Campylobacter strain at a low and high contamination level, and with sterile diluent for 'blanks'. Seventeen laboratories participated in the interlaboratory study. The sensitivity and specificity of the methods for the five selected matrices were determined, as well as the level of detection (LOD50). Calculated LOD50 values ranged from 0.84 cfu/test portion in frozen spinach and 2.2 cfu/test portion in minced meat to 14 cfu/test portion in chicken skin and 57 cfu/test portion in raw milk, all based on test portions of 10 g. The test portion size for broiler caecal material was a 10 µl-loop, yielding a LOD50 of 6.1 cfu/test portion. The validation data were incorporated in the newly published ISO standard EN ISO 10272-1:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 1: Detection method.

Wild, insectivorous bats might be carriers of Campylobacter spp.

BACKGROUND: The transmission cycles of the foodborne pathogens Campylobacter and Salmonella are not fully elucidated. Knowledge of these cycles may help reduce the transmission of these pathogens to humans. METHODOLOGY/PRINCIPAL FINDINGS: The presence of campylobacters and salmonellas was examined in 631 fresh fecal samples of wild insectivorous bats using a specially developed method for the simultaneous isolation of low numbers of these pathogens in small-sized fecal samples (≤ 0.1 g). Salmonella was not detected in the feces samples, but thermotolerant campylobacters were confirmed in 3% (n = 17) of the bats examined and these pathogens were found in six different bat species, at different sites, in different ecosystems during the whole flying season of bats. Molecular typing of the 17 isolated strains indicated C. jejuni (n = 9), C. coli (n = 7) and C. lari (n = 1), including genotypes also found in humans, wildlife, environmental samples and poultry. Six strains showed unique sequence types. CONCLUSION/SIGNIFICANCE: This study shows that insectivorous bats are not only carriers of viral pathogens, but they can also be relevant for the transmission of bacterial pathogens. Bats should be considered as carriers and potential transmitters of Campylobacter and, where possible, contact between bats (bat feces) and food or feed should be avoided.

Prevalence and types of Campylobacter on poultry farms and in their direct environment.

To study whether broiler and layer farms contribute to the environmental Campylobacter load, environmental matrices at or close to farms, and caecal material from chickens, were examined. Similarity between Campylobacter from poultry and environment was tested based on species identification and Multilocus Sequence Typing. Campylobacter prevalence in caecal samples was 97% at layer farms (n = 5), and 93% at broiler farms with Campylobacter-positive flocks (n = 2/3). Campylobacter prevalence in environmental samples was 24% at layer farms, and 29% at broiler farms with Campylobacter-positive flocks. Campylobacter was detected in soil and surface water, not in dust and flies. Campylobacter prevalence in adjacent and remote surface waters was not significantly (P > 0.1) different. Detected species were C. coli (52%), C. jejuni (40%) and C. lari (7%) in layers, and C. jejuni (100%) in broilers. Identical sequence types (STs) were detected in caecal material and soil. A deviating species distribution in surface water adjacent to farms indicated a high background level of environmental Campylobacter. STs from layer farms were completely deviant from surface water STs. The occasional detection of identical STs in broilers, wastewater at broiler farms and surface water in the farm environment suggested a possible contribution of broiler farms to the aquatic environmental Campylobacter load.

Quantification of Growth of Campylobacter and Extended Spectrum ß-Lactamase Producing Bacteria Sheds Light on Black Box of Enrichment Procedures.

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide, and is routinely found in meat originating from poultry, sheep, pigs, and cattle. Effective monitoring of Campylobacter contamination is dependent on the availability of reliable detection methods. The method of the International Organization for Standardization for the detection of Campylobacter spp. in food (ISO 10272-1:2006) recommends the use of Bolton broth (BB) as selective enrichment medium, including a pre-enrichment step of 4-6 h at 37°C to revive sublethally damaged cells prior to incubation for 2 days at 41.5°C. Recently the presence of abundantly growing extended spectrum ß-lactamase producing Enterobacteriaceae (ESBL bacteria) has become one of the most important factors that interfere with the isolation of Campylobacter, resulting in false-negative detection. However, detailed growth dynamics of Campylobacter and its competitors remain unclear, where these would provide a solid base for further improvement of the enrichment procedure for Campylobacter. Other enrichment broths, such as Preston broth (PB) and BB plus clavulanic acid (BBc) have been suggested to inhibit competitive flora. Therefore, these different broths were used as enrichments to measure the growth kinetics of several strains of Campylobacter jejuni and ESBL bacteria separately, in co-culture and of strains in chicken samples. The maximum cell numbers and often the growth rates of Campylobacter in mixed culture with ESBL bacteria were significantly lower than in single cultures, indicating severe suppression of Campylobacter by ESBL bacteria, also in naturally contaminated samples. PB and BBc successfully diminished ESBL bacteria and might therefore be a better choice as enrichment medium in possibly ESBL-bacteria contaminated samples. The efficacy of a pre-enrichment step in the BB ISO-procedure was not supported for cold-stressed and non-stressed cells. Therefore, omission of this step (4-6 h at 37°C) might be advised to obtain a less troublesome protocol.

Comparison of Campylobacter levels in crops and ceca of broilers at slaughter.

A considerable fraction of the poultry carcasses becomes contaminated with Campylobacter by cross-contamination from the digestive tract of colonized broilers at slaughter. Campylobacter in the crop may serve as a possible source of cross-contamination, because the crop may contain high numbers of Campylobacter and is more likely to rupture during the slaughtering process than intestines. In this study, the correlation between Campylobacter colonization levels in crop and cecum was assessed in 48 broilers of 31 days of age. In addition, the effect of drinking water supplemented with 0.2% volatile fatty acid (VFA) on these Campylobacter colonization levels was studied. No correlation between crop and cecal colonization levels was found (p = 0.09; P = 0.71), indicating that future studies on cross-contamination should include an examination of not only cecal colonization levels but also crop colonization levels. Supplementation of drinking water with VFA did not result in a significant reduction of colonization levels in either the crop (P = 0.50) or the ceca (P = 0.92), indicating that this is not an effective measure to reduce cross-contamination at slaughter.

Medium chain fatty acid feed supplementation reduces the probability of Campylobacter jejuni colonization in broilers.

Campylobacteriosis in humans is associated with handling and consumption of contaminated broiler meat. Reduction of the number of Campylobacter-colonized broiler flocks could potentially be realized by decreasing their susceptibility for colonization. The aim of this study was to determine the effect of feed supplementation with a mixture of medium chain fatty acids (C(8)-C(12)) on susceptibility of broilers for Campylobacter colonization, feed conversion and body weight gain. Two experiments were carried out with individually housed commercial broilers. The birds were fed with medium chain fatty acids supplemented feed (n=227), or received feed without supplement (n=87). The birds were inoculated with a dose of Campylobacter jejuni varying between log(10)1.19-5.47 CFU. During 14 days after inoculation, cecal or fecal samples were collected, in which the presence of C. jejuni was determined by bacterial culture. Beta-binomial dose-response modeling of the colonization status at 14 days post-inoculation was performed to estimate the C. jejuni dose necessary to colonize 50% of inoculated broilers, which was estimated to be 200 times higher in broilers fed with supplemented feed (log(10)4.8 CFU) than in control broilers (log(10)2.5 CFU). Feed conversion was not affected by feed supplementation, while body weight gain was 49 g higher in broilers fed with supplemented feed. These findings indicate that susceptibility of broilers for Campylobacter colonization is decreased by supplementation with medium chain fatty acids, and that feed supplemented with this mixture may be a promising tool for the reduction of Campylobacter colonization in commercial broiler flocks.

Quantifying transmission of Campylobacter jejuni in commercial broiler flocks.

Since meat from poultry colonized with Campylobacter spp. is a major cause of bacterial gastroenteritis, human exposure should be reduced by, among other things, prevention of colonization of broiler flocks. To obtain more insight into possible sources of introduction of Campylobacter into broiler flocks, it is essential to estimate the moment that the first bird in a flock is colonized. If the rate of transmission within a flock were known, such an estimate could be determined from the change in the prevalence of colonized birds in a flock over time. The aim of this study was to determine the rate of transmission of Campylobacter using field data gathered for 5 years for Australian broiler flocks. We used unique sampling data for 42 Campylobacter jejuni-colonized flocks and estimated the transmission rate, which is defined as the number of secondary infections caused by one colonized bird per day. The estimate was 2.37 +/- 0.295 infections per infectious bird per day, which implies that in our study population colonized flocks consisting of 20,000 broilers would have an increase in within-flock prevalence to 95% within 4.4 to 7.2 days after colonization of the first broiler. Using Bayesian analysis, the moment of colonization of the first bird in a flock was estimated to be from 21 days of age onward in all flocks in the study. This study provides an important quantitative estimate of the rate of transmission of Campylobacter in broiler flocks, which could be helpful in future studies on the epidemiology of Campylobacter in the field.

Darkling beetles (Alphitobius diaperinus) and their larvae as potential vectors for the transfer of Campylobacter jejuni and Salmonella enterica serovar paratyphi B variant Java between successive broiler flocks.

Broiler flocks often become infected with Campylobacter and Salmonella, and the exact contamination routes are still not fully understood. Insects like darkling beetles and their larvae may play a role in transfer of the pathogens between consecutive cycles. In this study, several groups of beetles and their larvae were artificially contaminated with a mixture of Salmonella enterica serovar Paratyphi B Variant Java and three C. jejuni strains and kept for different time intervals before they were fed to individually housed chicks. Most inoculated insects were positive for Salmonella and Campylobacter just before they were fed to the chicks. However, Campylobacter could not be isolated from insects that were kept for 1 week before they were used to mimic an empty week between rearing cycles. All broilers fed insects that were inoculated with pathogens on the day of feeding showed colonization with Campylobacter and Salmonella at levels of 50 to 100%. Transfer of both pathogens by groups of insects that were kept for 1 week before feeding to the chicks was also observed, but at lower levels. Naturally contaminated insects that were collected at a commercial broiler farm colonized broilers at low levels as well. In conclusion, the fact that Salmonella and Campylobacter can be transmitted via beetles and their larvae to flocks in successive rearing cycles indicates that there should be intensive control programs for exclusion of these insects from broiler houses.

Effectiveness and efficiency of controlling Campylobacter on broiler chicken meat.

Campylobacter bacteria are an important cause of foodborne infections. We estimated the potential costs and benefits of a large number of possible interventions to decrease human exposure to Campylobacter by consumption of chicken meat, which accounts for 20-40% of all cases of human campylobacteriosis in the Netherlands. For this purpose, a farm-to-fork risk assessment model was combined with economic analysis and epidemiological data. Reduction of contamination at broiler farms could be efficient in theory. However, it is unclear which hygienic measures need to be taken and the costs can be very high. The experimental treatment of colonized broiler flocks with bacteriophages has proven to be effective and could also be cost efficient, if confirmed in practice. Since a major decrease of infections at the broiler farm is not expected in the short term, additional measures in the processing plant were also considered. At this moment, guaranteed Campylobacter-free chicken meat at the retail level is not realistic. The most promising interventions in the processing plant are limiting fecal leakage during processing and separation of contaminated and noncontaminated flocks (scheduling), followed by decontamination of the contaminated flock. New (faster and more sensitive) test methods to detect Campylobacter colonization in broilers flocks are a prerequisite for successful scheduling scenarios. Other methods to decrease the contamination of meat of colonized flocks such as freezing and heat treatment are more expensive and/or less effective than chemical decontamination.

A risk assessment model for Campylobacter in broiler meat.

A quantitative microbiological risk assessment model describes the transmission of Campylobacter through the broiler meat production chain and at home, from entering the processing plant until consumption of a chicken breast fillet meal. The exposure model is linked to a dose-response model to allow estimation of the incidence of human campylobacteriosis. The ultimate objective of the model is to serve as a tool to assess the effects of interventions to reduce campylobacteriosis in the Netherlands. The model describes some basic mechanistics of processing, including the nonlinear effects of cross-contamination between carcasses and their leaking feces. Model input is based on the output of an accompanying farm model and Dutch count data of Campylobacters on the birds' exterior and in the feces. When processing data are lacking, expert judgment is used for model parameter estimation. The model shows that to accurately assess of the effects of interventions, numbers of Campylobacter have to be explicitly incorporated in the model in addition to the prevalence of contamination. Also, as count data usually vary by several orders of magnitude, variability in numbers within and especially between flocks has to be accounted for. Flocks with high concentrations of Campylobacter in the feces that leak from the carcasses during industrial processing seem to have a dominant impact on the human incidence. The uncertainty in the final risk estimate is large, due to a large uncertainty at several stages of the chain. Among others, more quantitative count data at several stages of the production chain are needed to decrease this uncertainty. However, this uncertainty is smaller when relative risks of interventions are calculated with the model. Hence, the model can be effectively used by risk management in deciding on strategies to reduce human campylobacteriosis.

Assessing interventions to reduce the risk of Campylobacter prevalence in broilers.

As part of a comprehensive risk assessment on the Campylobacter prevalence in the chicken production chain (from young born chicken till chicken fillet) in the Netherlands, we formulated a quantitative model on the transmission dynamics of Campylobacter at Dutch broiler farms. This model is used to quantify the risk of Campylobacter prevalence in broilers at the time that flocks leave the farm for processing. To this end, we assumed that the Campylobacter prevalence is primarily determined by two parameters, that is, the within- and between-flock transmission. The within-flock transmission was assessed fitting experimental data to a logistic growth model and the between-flock transmission was assessed fitting field data to a generalized linear model (GLM), which included three possible infection routes: (1) via an infected flock in the previous cycle, (2) via other infected flocks present on the farm, and (3) from other sources. This model was applied to assess the efficacy of three control scenarios; (1) a ban on other livestock on broiler farms, (2) a ban on thinning, and (3) a reduction of the between-flock transmission. In contrast to the other scenarios, the third one was shown to be most effective. Theoretically, this is accomplished by improved biosecurity. However, the impact of improved biosecurity cannot be specified into specific control measures, and therefore it is not clear what investments are needed. Finally, we also assessed the efficacy of scheduled treatment, that is, fresh meat production solely from test-negative flocks. We found that the reliability of negative test results, which is crucial, strongly depends on the length of time between testing and slaughter. The sensitivity and specificity of the test appeared to be of minor importance.