Clostridium tyrobutyricum, a Gram-positive, anaerobic, spore-forming bacterium, is considered as one of the main causative agents for spoilage of hard and semihard cheeses. Growth of C. tyrobutyricum in cheese is critically influenced by ripening temperature and time, pH, salt and lactic acid concentration, moisture and fat content, and the presence of other microorganisms. Previous studies revealed high intraspecies diversity of C. tyrobutyricum strains and variable tolerance toward pH, temperatures, and salt concentrations. These findings indicate that strain-dependent characteristics may be relevant to assess the risk for cheese spoilage if clostridial contamination occurs. In this study, we aimed to compare the phenotypes of 12 C. tyrobutyricum strains which were selected from 157 strains on the basis of genotypic and proteotypic variability. The phenotypic analysis comprised the assessment of gas production and organic acid concentrations in an experimental cheese broth incubated at different temperatures (37, 20, and 14 °C). For all tested strains, delayed gas production at lower incubation temperatures and a strong correlation between gas production and the change in organic acid concentrations were observed. However, considering the time until gas production was visible at different incubation temperatures, a high degree of heterogeneity was found among the tested strains. In addition, variation among replicates of the same strain and differences due to different inoculum levels became evident. This study shows, that, among other factors, strain-specific germination and growth characteristics should be considered to evaluate the risk of cheese spoilage by C. tyrobutyricum.
Clostridium tyrobutyricum has been recognized as the main cause of late blowing defects (LBD) in cheese leading to considerable economic losses for the dairy industry. Although differences in spoilage ability among strains of this species have been acknowledged, potential links to the genetic diversity and functional traits remain unknown. In the present study, we aimed to investigate and characterize genomic variation, pan-genomic diversity and key traits of C. tyrobutyricum by comparing the genomes of 28 strains. A comparative genomics analysis revealed an “open” pangenome comprising 9,748 genes and a core genome of 1,179 genes shared by all test strains. Among those core genes, the majority of genes encode proteins related to translation, ribosomal structure and biogenesis, energy production and conversion, and amino acid metabolism. A large part of the accessory genome is composed of sets of unique, strain-specific genes ranging from about 5 to more than 980 genes. Furthermore, functional analysis revealed several strain-specific genes related to replication, recombination and repair, cell wall, membrane and envelope biogenesis, and defense mechanisms that might facilitate survival under stressful environmental conditions. Phylogenomic analysis divided strains into two clades: clade I contained human, mud, and silage isolates, whereas clade II comprised cheese and milk isolates. Notably, these two groups of isolates showed differences in certain hypothetical proteins, transcriptional regulators and ABC transporters involved in resistance to oxidative stress. To the best of our knowledge, this is the first study to provide comparative genomics of C. tyrobutyricum strains related to LBD. Importantly, the findings presented in this study highlight the broad genetic diversity of C. tyrobutyricum, which might help us understand the diversity in spoilage potential of C. tyrobutyricum in cheese and provide some clues for further exploring the gene modules responsible for the spoilage ability of this species.
Bee pollen can be considered as perfect food with a great nutritional value, highprotein and essential amino acid content, vitamins and minerals. It can be a sourceof healthy nutrients, but as an animal product also of harmful microbialcontaminants. The aim of our study was to determine potential health risks andbenefits of Slovenian bee pollen. We determined its i) microbiological burden:aerobic mesophilic microorganisms, yeast, molds, and coliform bacteria in CFU/g;ii) polyphenolic content: the Folin-Ciocalteu method (mgGA/g); iii) antioxidativepotential (AOP): DPPH• scavenging assay (EC50 in mgGA/L); and iv)antimicrobial activity (MIC): microdilution method on Escherichia coli, Listeriamonocytogenes and Campylobacter jejuni. We analyzed 14 samples of bee pollengathered from 7 Slovenian geographical regions, from April until May 2017. Themicrobiological burden was high, with all indicator tests reaching up to 6.78log10CFU/g of bee pollen, but the number of coliform bacteria in all samples from2.00 to 4.48 log10CFU/g. The polyphenolic content and AOP of the samples wasgood, with up to 13.1 mg GA/g and as low as 2.4 mgGA/L (EC50), respectively.Interestingly, antimicrobial activity was not always in correlation withpolyphenolic content, but always strongly against E. coli, substantial against C.jejuni, and negligible against L. monocytogenes. Our results show a great healthpotential of bee pollen for human health, but also the need of bee pollen processingimprovement for its standardized quality and safety.
Clostridium tyrobutyricum is recognized as the main causative agent of late blowing defect—severe spoilage of hard and semihard cheeses. In this work, we present the draft genome sequences of 12 C. tyrobutyricum strains isolated from raw milk and cheese.
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