Intraspecific variability in heat resistance of fungal conidia.

Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries.

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.

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.

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.

Transfer of noroviruses between fingers and fomites and food products.

Human norovirus (NoV) contaminated hands are important routes for transmission. Quantitative data on transfer during contact with surfaces and food are scarce but necessary for a quantitative risk assessment. Therefore, transfer of MNV1 and human NoVs GI.4 and GII.4 was studied by artificially contaminating human finger pads, followed by pressing on stainless steel and Trespa® surfaces and also on whole tomatoes and cucumber slices. In addition, clean finger pads were pressed on artificially contaminated stainless steel and Trespa® surfaces. The transfers were performed at a pressure of 0.8-1.9 kg/cm(2) for approximately 2s up to 7 sequential transfers either to carriers or to food products. MNV1 infectivity transfer from finger pads to stainless steel ranged from 13 ± 16% on the first to 0.003 ± 0.009% on the sixth transfer on immediate transfer. After 10 min of drying, transfer was reduced to 0.1 ± 0.2% on the first transfer to 0.013 ± 0.023% on the fifth transfer. MNV1 infectivity transfer from stainless steel and Trespa® to finger pads after 40 min of drying was 2.0 ± 2.0% and 4.0 ± 5.0% respectively. MNV1 infectivity was transferred 7 ± 8% to cucumber slices and 0.3 ± 0.5% to tomatoes after 10 min of drying, where the higher transfer to cucumber was probably due to the higher moisture content of the cucumber slices. Similar results were found for NoVs GI.4 and GII.4 transfers measured in PCR units. The results indicate that transfer of the virus is possible even after the virus is dried on the surface of hands or carriers. Furthermore, the role of fingers in transmission of NoVs was quantified and these data can be useful in risk assessment models and to establish target levels for efficacy of transmission intervention methods.

Residual viral and bacterial contamination of surfaces after cleaning and disinfection.

Environmental surfaces contaminated with pathogens can be sources of indirect transmission, and cleaning and disinfection are common interventions focused on reducing contamination levels. We determined the efficacy of cleaning and disinfection procedures for reducing contamination by noroviruses, rotavirus, poliovirus, parechovirus, adenovirus, influenza virus, Staphylococcus aureus, and Salmonella enterica from artificially contaminated stainless steel surfaces. After a single wipe with water, liquid soap, or 250-ppm free chlorine solution, the numbers of infective viruses and bacteria were reduced by 1 log(10) for poliovirus and close to 4 log(10) for influenza virus. There was no significant difference in residual contamination levels after wiping with water, liquid soap, or 250-ppm chlorine solution. When a single wipe with liquid soap was followed by a second wipe using 250- or 1,000-ppm chlorine, an extra 1- to 3-log(10) reduction was achieved, and except for rotavirus and norovirus genogroup I, no significant additional effect of 1,000 ppm compared to 250 ppm was found. A reduced correlation between reduction in PCR units (PCRU) and reduction in infectious particles suggests that at least part of the reduction achieved in the second step is due to inactivation instead of removal alone. We used data on infectious doses and transfer efficiencies to estimate a target level to which the residual contamination should be reduced and found that a single wipe with liquid soap followed by a wipe with 250-ppm free chlorine solution was sufficient to reduce the residual contamination to below the target level for most of the pathogens tested.

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.

Survival, elongation, and elevated tolerance of Salmonella enterica serovar Enteritidis at reduced water activity.

Growing microorganisms on dry surfaces, which results in exposure to low water activity (a(w)), may change their normal morphology and physiological activity. In this study, the morphological changes and cell viability of Salmonella enterica serovar Enteritidis challenged to low a(w) were analyzed. The results indicated that exposure to reduced a(w) induced filamentation of the cells. The amount of filamentous cells at a(w) 0.94 was up to 90% of the total number of cells. Surviving filamentous cells maintained their membrane integrity after exposure to low a(w) for 21 days. Furthermore, cells prechallenged to low a(w), obtained with an ionic humectant, demonstrated higher resistance to sodium hypochlorite than control cells. These resistant cells are able to survive disinfection more efficiently and can therefore cause contamination of foods coming in contact with surfaces. This points to the need for increased attention to cleaning of surfaces in household environments and disinfection procedures in processing plants.

Fluorescence microscopy of NaCl-stressed, elongated Salmonella and Listeria cells reveals the presence of septa in filaments.

The cell morphology of Salmonella enteritidis and Listeria monocytogenes after the application of stress was examined. Cells were stressed by plating the bacteria on Tryptone Soya Agar (TSA) plates, with 5-10% NaCl. The plates were subsequently incubated for 6 days at 25 degrees C. Finally, the cells were harvested and subjected to different fluorescent probes in order to visualize the possible presence of septa in elongated cells. Use of the stain 4',6-Diamidino-2-phenylindole (DAPI), which is a blue fluorescent nucleic acid stain that preferentially stains double-stranded DNA, showed clearly the presence of dark spots, probably cellular partitions where no nucleic acids were present, in both Salmonella and Listeria cells. Another stain, FM 4-64, a lipophilic styryl dye for red staining of the inner membrane, showed the presence of highly fluorescent spots in Listeria cells, probably indicating the presence of membranes. For Salmonella, however, FM 4-64 was not successful in revealing septa in filaments. Double staining applied to elongated Listeria cells showed areas with high fluorescence in DAPI-staining (DNA-rich spots) which contained low fluorescence in FM 4-64-staining (membrane spots) and vice versa, which is a confirmation that the elongated cells are indeed composed of several normal size cells.

Listeria monocytogenes: diagnostic problems.

The first isolation methods for the detection of Listeria spp. were generally based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult. In time, new techniques were developed, based on a variety of selective and elective agents in isolation and enrichment media, which gained better and quicker results. Current reference methods allow the recovery of L. monocytogenes from a variety of foods with relative ease. However, more comparative studies are needed to select one horizontal method. It is suggested that the procedure of the International Organization for Standardization is a good base for such comparisons.