Comparison of five assays for DNA extraction from bacterial cells in human faecal samples.

AIM: To determine the most effective DNA extraction method for bacteria in faecal samples. MATERIALS AND RESULTS: This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. CONCLUSION: From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose.

Assessment of microbial communities on freshly killed wild boar meat by MALDI-TOF MS and 16S rRNA amplicon sequencing.

Wild boars (Sus scrofa) are the most widely distributed large mammals and recent increase in consumption of wild boar meat urges the need of microbiological quality criteria. The aim of the study was to characterize the initial bacterial contamination on freshly-killed wild boar meat using a culture-dependent approach with ISO-methods combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification and 16S rRNA amplicon sequencing. Moreover, the presence of foodborne pathogens was examined using Real-Time-PCR and confirmed by classical isolation. Analysing 22 unrelated wild boar meat samples showed a higher bacterial contamination level compared to pork, with Salmonella present in almost one third of the samples. A great variability of the microbial contamination between the samples was recorded, as well as complementary results between culturing and 16S rRNA amplicon sequencing as frequently isolated genera were not always detected, and vice versa. Furthermore, the foodborne pathogen Salmonella was never detected with 16S rRNA amplicon sequencing, demonstrating the necessity for a cautious approach in the implementation of new analysis techniques in food safety. The present work determines that attention should be paid to the trade of non-inspected meat directly to retail or consumers.

Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis.

Routine evaluation of bacterial contamination in minced pork is still mainly performed by the enumeration of indicator bacteria, including total aerobic colony count and E. coli, using standardized isolation methods. However, the bacterial community structure as well as the effect of the storage time and temperature on the aerobic plate count are largely unknown for this matrix. The aim of the study was to characterize the microbial community in minced pork by 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA gene sequencing. Analysis of 14 unrelated samples showed that total aerobic counts determined at 30 °C and 7 °C showed no significant difference, but the richness was higher on PCA at 30 °C for 7 samples, equal in 5, and higher at 7 °C for 2 samples. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among both the mesophilic and psychrotrophic population. Comparing to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Comparison of different sample preparation techniques and DNA extraction methods demonstrated also in this matrix that different results on the microbial composition and complexity are obtained. Present data illustrate that the combined isolation and identification of isolates using MALDI TOF MS and 16S gene sequencing and overall community profiling using 16S rRNA amplicon sequencing provides complementary results and yields important insights into the complex relationship between microorganisms in a food.