Shedding of Mycobacterium caprae by wild red deer (Cervus elaphus) in the Bavarian alpine regions, Germany

The number of natural infections with Mycobacterium caprae in wildlife and in cattle in the Bavarian and Austrian alpine regions has increased over the last decade. Red deer (Cervus elaphus) have been recognized as maintenance reservoir; however, the transmission routes of M. caprae among and from naturally infected red deer are unknown. The unexpected high prevalence in some hot spot regions might suggest an effective indirect transmission of infection. Therefore, this study was undertaken to diagnose the occurrence of M. caprae in faeces and secretions of red deer in their natural habitat. A total of 2,806 red deer hunted in this region during 2014–2016 were included in this study. After pathological examination, organs (lymph nodes, lung, heart), excretions and secretions (faeces, urine, saliva and tonsil swabs) were further investigated by qPCR specific for Mycobacterium tuberculosis complex (MTC), M. bovis and M. caprae. Samples tested positive by qPCR were processed for culturing of mycobacteria. In total, 55 (2.0%) animals were confirmed positive for M. caprae by pathological examination, PCR and culturing of the affected organ material. With the exception of one sample, all of the secretion and excretion samples were negative for mycobacteria of the Mycobacterium tuberculosis complex (MTC). From one red deer, M. caprae could be isolated from the heart sac as well as from the faeces. Whole‐genome sequencing confirmed that both strains were clonally related. This is the first confirmation that M. caprae can be shed with the faeces of a naturally infected red deer. However, further studies focusing on a higher number of infected animals, sample standardization and coordinated multiple sampling are necessary to improve the understanding of transmission routes under natural conditions

Spoilage characteristics of sous-vide beef caused by Clostridium estertheticum

The increasing popularity of sous-vide (SV) cooking necessitates research into the microbiological quality, sensory changes, and shelf life of SV products. Studies show that SV cooking significantly reduces the levels of meat microbiota and pathogens, positively affecting the shelf life and safety of SV products. However, the meat spoilage organism Clostridium estertheticum can survive SV cooking as it can produce heat-tolerant spores. Theses spores can germinate and multiply during storage at refrigerated temperatures, leading to spoilage of SV meat. Therefore, the aim of this study was to characterise the spoilage of SV beef caused by C. estertheticum compared to non-SV beef. In addition to the determination of spoilage characteristics, all beef samples were subjected to culture and qPCR analysis to determine the numbers of total bacteria, lactic acid bacteria, Enterobacteriaceae, yeasts, and C. estertheticum. Species identification of the colonies on the culture media was performed using MALDI-TOF MS. The tests were carried out at three different times (three repetitions). A total of 90 beef samples were analysed, of which 54 samples were artificially contaminated with three strains of C. estertheticum and vacuum-packed. Of these, 27 beef samples underwent SV cooking (55 °C, 70 min). After 28 days of storage at 4 °C, the SV beef samples exhibited significantly higher levels of gas and stronger spoilage odour compared to non-SV samples (p < 0.05). While drip loss and pH levels were also higher in SV beef, these were not considered specific spoilage characteristics caused by C. estertheticum. Microbiological and qPCR analyses revealed that all SV beef samples had very low bacterial and yeast counts but very high numbers of C. estertheticum, which strongly correlated with the sensory changes observed. We concluded that SV beef containing C. estertheticum has a shorter shelf life than contaminated non-SV beef. This is the first study to examine the spoilage of SV beef by C. estertheticum. The results may help raise awareness among meat producers and restaurants about the risk of meat losses due to spoilage caused by these bacteria

Symbiotic Husbandry of Chickens and Pigs Does Not Increase Pathogen Transmission Risk

A symbiotic or mixed animal husbandry (e.g., pigs and chickens) is considered to have a positive effect for animal welfare and sustainable agriculture. On the other hand, a risk of infection and transmission of microorganisms, especially of zoonotic pathogens, between animal species may potentially occur and thus might increase the risk of foodborne illnesses for consumers. To prove these assumptions, two groups of animals and their environmental (soil) samples were investigated in this study. Animals were kept in a free-range system. In the first group, pigs and chickens were reared together (pasture 1), while the other group contained only pigs (pasture 2). During a one-year study, fecal swab samples of 240 pigs and 120 chickens, as well as 120 ground samples, were investigated for the presence of Campylobacter spp., Salmonella spp. and E. coli. Altogether, 438 E. coli and 201 Campylobacter spp. strains were isolated and identified by MALDI-TOF MS. Salmonella spp. was not isolated from any of the sample types. The prevalences of Campylobacter coli and C. jejuni in pigs were 26.7% and 3.3% in pasture 1 and 30.0% and 6.7% in pasture 2, while the prevalences of C. coli and C. jejuni in chickens from pasture 1 were 9.2% and 78.3%, respectively. No correlation between the rearing type (mixed vs. pigs alone) and the prevalence of Campylobacter spp. was observed. All swab samples were positive for E. coli, while the average prevalences in soil samples were 78.3% and 51.7% in pasture 1 and 2, respectively. Results of similarity analysis of the MALDI-TOF MS spectra (for C. coli, C. jejuni and E. coli) and FT-IR spectra (for E. coli) of the same bacterial species showed no recognizable correlations, no matter if strains were isolated from chickens, pig or soil samples or isolated at different sampling periods. The results of the study indicate that the symbiotic husbandry of pigs and chickens neither results in an increased risk of a transmission of Campylobacter spp. or E. coli, nor in a risk of bacterial alteration, as shown by MALDI-TOF MS and FT-IR spectra. In conclusion, the benefits of keeping pigs and chickens together are not diminished by the possible transmission of pathogens

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