Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

Changes in the Microbiota from Fresh to Spoiled Meat, Determined by Culture and 16S rRNA Analysis.

Growth of meat microbiota usually results in spoilage of meat that can be perceived by consumers due to sensory changes. However, a high bacterial load does not necessarily result in sensory deviation of meat; nevertheless, this meat is considered unfit for human consumption. Therefore, the aims of this study were to investigate changes in the microbiota from fresh to spoiled meat and whether the proportions of certain bacteria can probably be used to indicate the hygiene status of meat. For this purpose, 12 fresh pork samples were divided into two groups, and simultaneously aerobically stored at 4°C and 22°C. At each time-temperature point (fresh meat, days 6, 13, and 20 at 4°C, and days 1, 2, 3, and 6 at 22°C), 12 meat subsamples were investigated. Sequences obtained from next-generation sequencing (NGS) were further analyzed down to species level. Plate counting of six bacterial groups and NGS results showed that Pseudomonas spp. and lactic acid bacteria (LAB) were found in a high proportion in all stored meat samples and can therefore be considered as important "spoilage indicator bacteria". On the contrary, sequences belonging to Staphylococcus epidermidis were found in a relatively high proportion in almost all fresh meat samples but were less common in stored meat. In this context, they can be considered as "hygiene indicator bacteria" of meat. Based on these findings, the proportion of the "hygiene indicator bacteria" in relation to the "spoilage indicator bacteria" was calculated to determine a "hygiene index" of meat. This index has a moderate to strong correlation to bacterial loads obtained from culture (p < 0.05), specifically to Pseudomonas spp., LAB and total viable counts (TVCs). Knowledge of the proportions of hygiene and spoilage indicator bacteria obtained by NGS could help to determine the hygiene status even of (heat-) processed composite meat products for the first time, thus enhancing food quality assurance and consumer protection.

A simple method for the isolation of cold-tolerant Clostridium spp. from meat samples.

Pure isolates of Clostridium spp. are required when further investigations are needed, such as the characterisation of the colonies, sporulation and germination, biochemical analysis, growth rate and growth conditions, toxin production, spoilage potential and genomic profile. However, the isolation of cold-tolerant Clostridium spp. from suspicious meat samples is challenging due to their slow growth rates and overgrowth with less fastidious meat microbiota. Therefore, this study aimed to establish a practical method for the isolation of psychrophilic and psychrotolerant Clostridium spp. Primarily, meat drip samples (n = 3), enriched in Peptone Yeast Glucose Starch (PYGS) broth, were heated with different temperatures and durations, before they were subcultured on Columbia blood agar (CBA). The treatment procedure of heating samples at 80 °C for 5 min was evaluated as effective and was further validated using pure cultures of lactic acid bacteria (n = 5), bacteria belonging to the family of Enterobacteriaceae (n = 5), and cold-tolerant Clostridium spp. (n = 34). Almost all other meat microbiota was inactivated by heating at 80 °C for 5 min, while 28 strains of cold-tolerant Clostridium spp. survived. Finally, the treatment procedure was applied with drip of meat samples (n = 41) previously tested positive for 49 cold-tolerant Clostridium spp. using specific multiplex qPCR. All meat drip samples were enriched in PYGS broth by incubating them at 4 °C for 3 weeks, to ensure growth of Clostridium spp. before the enrichments were proceeded to heat treatment and to culturing on CBA. A total of 35 (71 %) from 49 Clostridium spp. strains were isolated using this culturing procedure. The accuracy of the recovery rates obtained from two replicates was 68 %. The method of detection and isolation applied in this study is easy and resulted in a high isolation rate of cold-tolerant Clostridium spp.

Unknown cold-tolerant Clostridium spp.: Characteristics and potential to cause meat spoilage.

Clostridium spp. are ubiquitous bacteria and often found in foods and animals. Some species are pathogenic, others food spoiling or commensals. In this study, 65 cold-tolerant Clostridium spp. strains isolated from variable samples (beef, lamb, venison, feces/skin of wild boars) were investigated. Fifty strains were lecithinase positive; six additionally produced ß-hemolysis. By applying specific qPCR, 16S rRNA gene analysis, RFLP method, and MALDI-TOF MS, they were classified into two major groups: 29 strains were identified as C. tagluense-like, while the other 36 remained unidentified. Subsequently, twenty-two vacuum-packed beef samples were spiked with a single strain from both groups and stored at 4 °C for 8 weeks. The odor of challenged samples was variable (from unchanged, sour/musty, to sulfurous), while color, meat consistency and drip loss were similar to the control group. The ability to produce gas of all tested strains was lower than of C. estertheticum. Even though both groups of cold-tolerant clostridia exhibited similar 16S rRNA genes and biochemical activities, RFLP methods and MALDI-TOF MS are sufficient to differentiate them. In terms of food safety, strains producing lecithinase and hemolysin should be further investigated for their potential to produce substances affecting human and animal health.

High incidence of cold-tolerant Clostridium frigoriphilum and C. algidicarnis in vacuum-packed beef on retail sale in Germany.

Sixty vacuum-packed beef samples retailed in Germany were investigated for the occurrence of cold-tolerant Clostridium spp. After a storage period at 4 °C for eight weeks, meat juice from all samples was processed for culturing, DNA extraction and SYBR green qPCR for Clostridium species. After that, a previously developed multiplex qPCR, sequence analysis of the 16S rRNA gene, and MALDI-TOF MS were applied in order to identify Clostridium spp. found in samples. Subsequently, 23 samples were found positive for C. frigoriphilum (n = 19), C. estertheticum (n = 2), C. tagluense (n = 1) and C. lacusfryxellense/C. frigoris (n = 1). By using a new multiplex qPCR and a new RFLP method developed in this study, a further 15 meat juice samples were revealed to be contaminated with C. algidicarnis. With some samples being co-contaminated with two different species, 53% (n = 32) of all investigated vacuum-packed beef samples were found to be positive for cold-tolerant clostridia. This is the first report of detection and identification of C. algidicarnis in meat samples in Germany and Central Europe.

Development of two specific multiplex qPCRs to determine amounts of Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta and Staphylococcus in meat and heat-treated meat products.

Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae, genus Pseudomonas, genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made "Lyoner" sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log10 cfu/g, the relative accuracy and the t-test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available "Bologna-type" sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas, Enterobacteriaceae and B. thermosphacta could together be used as an indicator for "spoiled meat" used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud.