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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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 ∆log 10 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., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The increasing antimicrobial resistance (AMR) among pathogenic and opportunistic pathogenic microorganisms is one of the main global public health problems. The consumption of food contaminated with such bacteria (ARB), especially of raw products, might result in the direct acquisition of ARB and in a spread of resistant bacteria along the food chain. The aim of the study was to characterize the antimicrobial susceptibility of potentially extended spectrum β-lactamase (ESBL) producing or AmpC resistant Enterobacteriaceae isolated from the surface of 147 muskmelons from wholesale and retail. A phenotypic analysis was carried out by using minimum inhibitory concentration (MIC) test strips for ESBL detection and MIC susceptibility plates against 14 antimicrobials. Furthermore, ESBL genes, sul-genes and plasmid-mediated AmpC resistance were analyzed by real-time PCR. Additionally, a further insight in the AmpC resistance of isolates of the Enterobacter cloacae complex (ECC) was obtained by analyzing the sequence of the ampC regulatory region (n = 15). A total of 73 potentially resistant Enterobacteriaceae were isolated from 56 muskmelons. Of these, 15 isolates of the ECC were suspicious for ESBL/AmpC resistance, and eleven thereof were positive for the AmpC family EBC. Phenotypic analysis showed diminished susceptibility against "critically" and "highly important" antimicrobials, according to the WHO classification. Furthermore, divergence in the ampC regulatory region was detected between the 15 isolates. These findings highlight the important role that raw produce might play in the transmission of antimicrobial resistances along the food chain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Psychrophilic and psychrotolerant clostridia (n = 110) were isolated from vacuum-packed meat (beef and lamb), fresh venison and from skin and fecal samples of wild boars. They were identified to species level using MALDI-TOF MS, sequence and phylogeny analysis of the 16S rRNA and species specific multiplex qPCR. The results of all three methods were concordant. The majority of isolates were identified as C. tagluense-like Group I (n = 34) and Group II (n = 42). Thirty-five isolates could be identified to species level as follows: C. estertheticum (n = 15), C. frigoriphilum (n = 13), C. frigidicarnis (n = 1) and C. bowmanii (n = 5). This is the first report of detection and identification of C. frigoriphilum and C. tagluense-like Group II as causative agents of blown pack spoilage of beef. The species specific multiplex qPCR developed in this study could be applied to identify and to quantify the Clostridium species described above in suspicious meat juice samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Food processing of spoiled meat is prohibited by law, since it is a deception and does not comply with food safety aspects. In general, spoilage of meat is mostly caused by bacteria. However, a high contamination level of fungi could be also found in some meat or meat products with certain preserving conditions. In case that unhygienic meat is used to produce heat processed products, the microorganisms will be deactivated by heat, so that they cannot be detected by a standard cultivation method. Therefore, this study aimed to develop and apply a molecular biological method--polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP)--to reconstruct the original fungal flora of heat processed meat. Twenty primer pairs were tested for their specificity for fungal DNA. Since none of them fully complied with all study criteria (such as high specificity and sensitivity for fungal DNA; suitability of the products for PCR-SSCP) in the matrix "meat", we designed a new reverse primer, ITS5.8R. The primer pair ITS1/ITS5.8R amplified DNA from all tested fungal species, but not DNA from meat-producing animals or from ingredients of plant origin (spices). For the final test, 32 DNA bands in acrylamide gel from 15 meat products and 1 soy sauce were sequenced-all originating from fungal species, which were, in other studies, reported to contaminate meat e.g. Alternaria alternata, Aureobasidium pullulans, Candida rugosa, C. tropicalis, C. zeylanoides, Eurotium amstelodami and Pichia membranifaciens, and/or spices such as Botrytis aclada, Guignardia mangiferae, Itersonilia perplexans, Lasiodiplodia theobromae, Lewia infectoria, Neofusicoccum parvum and Pleospora herbarum. This confirms the suitability of PCR-SSCP to specifically detect fungal DNA in heat processed meat products, and thus provides an overview of fungal species contaminating raw material such as meat and spices., (Copyright © 2013 Elsevier B.V. All rights reserved.)