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The detection of thermotolerant

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods. The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species. Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively. Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant. A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after oxidative and freeze stress indicated that recovery behaviour was also dependent on the stress applied. A scenario analysis was conducted to evaluate the impact of heterogeneity in outgrowth kinetics of single cells on the reliability of detection outcomes following ISO protocol 10272-1:2017. This revealed that a ‘worst-case’-scenario for successful detection by a combination of the longest lag-duration of 7.6 h and lowest growth rate of 0.47 h−1 still resulted in positive detection outcomes since the detection limit was reached within 32.5 h. This suggests that other factors such as competitive microbiota can act as a causative factor in false-negative outcomes of tested food samples.

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.

Bats are commonly regarded as vectors for viruses, but little is known about bacterial communities in bats and the possible role of bats in the transmission cycle of foodborne diseases. To gain more insight, microbial communities in fecal samples from 37 insectivorous bats of different species from the Netherlands were investigated by polymerase chain reaction and denaturant gradient gel electrophoresis (PCR-DGGE). Subsequently, 10 samples from the following bat species: common pipistrelle (Pipistrellus pipistrellus; n = 3), Daubenton's bat (Myotis daubentonii; n = 3), serotine bat (Eptesicus serotinus; n = 1), whiskered bat (Myotis mystacinus; n = 1), Geoffroy's bat (Myotis emarginatus; n = 1) and Natterer's bat (Myotis nattereri; n = 1) were selected and used in bacterial 16S rDNA cloning and sequencing. The fecal microbiota in bats was found to be diverse with predominant bacterial genera Carnobacterium, Serratia, Pseudomonas, Enterococcus and Yersinia. The presence of opportunistic pathogens Citrobacter freundii, Escherichia coli, Enterococcus faecalis, Serratia fonticola and Rahnella aquatilis was also recorded. Based on cloning results, we found no proof that bats in the Netherlands are a major vector for the transmission of bacterial zoonotic diseases, although previous findings in literature reported isolation of foodborne pathogens from bats.

Consumption of horse milk has become popular in developed countries, especially among people suffering from bowel problems and skin diseases. Since the positive effect is supposedly not observed after pasteurisation, the product is mostly consumed as raw milk. Since the microbiological quality of this milk has not been systematically surveyed, in this study we examined the presence of spoilage- and pathogenic microorganisms in 123 samples of horse milk collected in The Netherlands and Belgium. Hygiene and faecal indicators were found in a wide range of numbers. Although Salmonella, Campylobacter and Listeria were not found, these pathogens showed no reduction in challenge tests with artificially contaminated horse milk stored for 1 week at 7 °C. Since faecal indicators were present and able to grow at 7 °C, combined with the fact that pathogens may easily end up and survive in the milk, it is not advised to consume raw horse milk.

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 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 two days at 41.5°C. Recently the presence of abundantly growing extended-spectrum β-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 and Bolton broth plus clavulanic acid have been suggested to inhibit competitive flora. Therefore these different broths were used as enrichments to measure the growth kinetics of several strains of C. 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. Preston broth and Bolton broth plus clavulanic acid 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 Bolton broth 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.

Background: Hand hygiene is important for interrupting transmission of viruses through hands. Effectiveness of alcohol-based hand disinfectant has been shown for bacteria but their effectiveness in reducing transmission of viruses is ambiguous. Aim: To test efficacy of alcohol hand disinfectant against human enteric and respiratory viruses and to compare efficacy of an alcohol-based hand disinfectant and handwashing with soap and water against norovirus. Methods: Efficacies of a propanol and an ethanol-based hand disinfectant against human enteric and respiratory viruses were tested in carrier tests. Efficacy of an alcohol-based hand disinfectant and handwashing with soap and water against noroviruses GI. 4, GII. 4, and MNV1 were tested using finger pad tests. Findings: The alcohol-based hand disinfectant reduced the infectivity of rotavirus and influenza A virus completely within 30 s whereas poliovirus Sabin 1, adenovirus type 5, parechovirus 1, and MNV1 infectivity were reduced 3.0 +/- 0.4 log(10)) was significantly higher than treating hands with alcohol (2.8 +/- 1.5 log(10)). Washing with soap and water for 30 s removed genomic copies of MNV1 (>5 log(10)), noroviruses GI. 4 (>6 log(10)), and GII. 4 (4 log(10)) completely from all finger pads. Treating hands with propanol-based hand disinfectant showed little or no reduction to complete reduction with mean genomic copy reduction of noroviruses GI. 4, GII. 4, and MNV1 being >2.6, >3.3, and >1.2 log(10) polymerase chain reaction units respectively. Conclusions: Washing hands with soap and water is better than using alcohol-based hand disinfectants in removing noroviruses from hands. (C) 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Prediction of microbial growth kinetics can differ from the actual behavior of the target microorganisms. In the present study, the impact of strain variability on maximum specific growth rate (µmax) (h- 1) was quantified using twenty Listeria monocytogenes strains. The µmax was determined as function of four different variables, namely pH, water activity (aw)/NaCl concentration [NaCl], undissociated lactic acid concentration ([HA]), and temperature (T). The strain variability was compared to biological and experimental variabilities to determine their importance. The experiment was done in duplicate at the same time to quantify experimental variability and reproduced at least twice on different experimental days to quantify biological (reproduction) variability. For all variables, experimental variability was clearly lower than biological variability and strain variability; and remarkably, biological variability was similar to strain variability. Strain variability in cardinal growth parameters, namely pHmin, [NaCl]max, [HA]max, and Tmin was further investigated by fitting secondary growth models to the µmax data, including a modified secondary pH model. The fitting results showed that L. monocytogenes had an average pHmin of 4.5 (5–95% prediction interval (PI) 4.4–4.7), [NaCl]max of 2.0 mM (PI 1.8–2.1), [HA]max of 5.1 mM (PI 4.2–5.9), and Tmin of - 2.2 °C (PI (- 3.3)–(- 1.1)). The strain variability in cardinal growth parameters was benchmarked to available literature data, showing that the effect of strain variability explained around 1/3 or less of the variability found in literature. The cardinal growth parameters and their prediction intervals were used as input to illustrate the effect of strain variability on the growth of L. monocytogenes in food products with various characteristics, resulting in 2–4 log CFU/ml(g) difference in growth prediction between the most and least robust strains, depending on the type of food product. This underlined the importance to obtain quantitative knowledge on variability factors to realistically predict the microbial growth kinetics. Keywords: Quantitative microbiology; Growth parameters; Heterogeneity; Pathogen