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Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

ABSTRACT: Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic mesophilic bacterial isolates in each sample. A high biodiversity of 70 and 110 species per sample was determined, of which 25-28% corresponded to yet unknown taxa. The isolates were dominated by Gram-positive members of the genera Staphylococcus, Corynebacterium, Streptococcus, or Janibacter, whilst Chryseobacterium and Acinetobacter were most abundant among the Gram-negative taxa. At the second time point, samples of the same farms were analyzed via both cultivation (1,500 individual colonies each) and high-throughput 16S rRNA gene amplicon sequencing. The latter revealed a threefold higher biodiversity at the genus level, as anaerobic or fastidious species were also detected. However, cultivation identified genera not captured by sequencing, indicating that both approaches are complementary. Using amplicon sequencing, the relative abundance of a few genera was distorted, which seems to be an artifact of sample preparation. Therefore, attention needs to be paid to the library preparation procedure with special emphasis on cell lysis and PCR.

SUBMITTER: Breitenwieser F

PROVIDER: S-EPMC7365021 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

Breitenwieser Franziska F Doll Etienne V EV Clavel Thomas T Scherer Siegfried S Wenning Mareike M

Frontiers in microbiology 20200709

Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic m ...[more]

PMID: 32742267

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Project description:In pasture-based systems, changes in dairy herd habitat due to seasonality results in the exposure of animals to different environmental niches. These niches contain distinct microbial communities that may be transferred to raw milk, with potentially important food quality and safety implications for milk producers. It is postulated that the extent to which these microorganisms are transferred could be limited by the inclusion of a teat preparation step prior to milking. High-throughput sequencing on a variety of microbial niches on farms was used to study the patterns of microbial movement through the dairy production chain and, in the process, to investigate the impact of seasonal housing and the inclusion/exclusion of a teat preparation regime on the raw milk microbiota from the same herd over two sampling periods, i.e., indoor and outdoor. Beta diversity and network analyses showed that environmental and milk microbiotas separated depending on whether they were sourced from an indoor or outdoor environment. Within these respective habitats, similarities between the milk microbiota and that of teat swab samples and, to a lesser extent, fecal samples were apparent. Indeed, SourceTracker identified the teat surface as the most significant source of contamination, with herd feces being the next most prevalent source of contamination. In milk from cows grazing outdoors, teat prep significantly increased the numbers of total bacteria present. In summary, sequence-based microbiota analysis identified possible sources of raw milk contamination and highlighted the influence of environment and farm management practices on the raw milk microbiota. IMPORTANCE:The composition of the raw milk microbiota is an important consideration from both a spoilage perspective and a food safety perspective and has implications for milk targeted for direct consumption and for downstream processing. Factors that influence contamination have been examined previously, primarily through the use of culture-based techniques. We describe here the extensive application of high-throughput DNA sequencing technologies to study the relationship between the milk production environment and the raw milk microbiota. The results show that the environment in which the herd was kept was the primary driver of the composition of the milk microbiota composition.

Project description:BackgroundNext-generation sequencing (NGS) methods and especially 16S rRNA gene amplicon sequencing have become indispensable tools in microbial ecology. While they have opened up new possibilities for studying microbial communities, they also have one drawback, namely providing only relative abundances and thus compositional data. Quantitative PCR (qPCR) has been used for years for the quantification of bacteria. However, this method requires the development of specific primers and has a low throughput. The constraint of low throughput has recently been overcome by the development of high-throughput qPCR (HT-qPCR), which allows for the simultaneous detection of the most prevalent bacteria in moderately complex systems, such as cheese and other fermented dairy foods. In the present study, the performance of the two approaches, NGS and HT-qPCR, was compared by analyzing the same DNA samples from 21 Raclette du Valais protected designation of origin (PDO) cheeses. Based on the results obtained, the differences, accuracy, and usefulness of the two approaches were studied in detail.ResultsThe results obtained using NGS (non-targeted) and HT-qPCR (targeted) show considerable agreement in determining the microbial composition of the cheese DNA samples studied, albeit the fundamentally different nature of these two approaches. A few inconsistencies in species detection were observed, particularly for less abundant ones. The detailed comparison of the results for 15 bacterial species/groups measured by both methods revealed a considerable bias for certain bacterial species in the measurements of the amplicon sequencing approach. We identified as probable origin to this PCR bias due to primer mismatches, variations in the number of copies for the 16S rRNA gene, and bias introduced in the bioinformatics analysis.ConclusionAs the normalized microbial composition results of NGS and HT-qPCR agreed for most of the 21 cheese samples analyzed, both methods can be considered as complementary and reliable for studying the microbial composition of cheese. Their combined application proved to be very helpful in identifying potential biases and overcoming methodological limitations in the quantitative analysis of the cheese microbiota.

Project description:Simple Summary Idiazabal is a traditional cheese produced from raw ewe milk in the Basque Country (Southwestern Europe). The sensory properties of raw milk cheeses have been attributed, among other factors, to microbial shifts that occur during the production and ripening processes. In this study, we used high-throughput sequencing technologies to investigate the microbiota of Latxa ewe raw milk and the dynamics during cheese production and ripening processes. The microbiota of raw milk was composed of lactic acid bacteria (LAB), environmental bacteria and non-desirable bacteria. Throughout the cheese making and ripening processes, the growth of LAB was promoted, whereas that of non-desirable and environmental bacteria was inhibited. Moreover, some genera not reported previously in raw ewe milk were detected and clear differences were observed in the bacterial composition of raw milk and cheese among producers, in relation to LAB and environmental or non-desirable bacteria, some of which could be attributed to the production of flavour related compounds. Abstract In this study, we used high-throughput sequencing technologies (sequencing of V3–V4 hypervariable regions of 16S rRNA gene) to investigate for the first time the microbiota of Latxa ewe raw milk and the bacterial shifts that occur during the production and ripening of Idiazabal cheese. Results revealed several bacterial genera not reported previously in raw ewe milk and cheese, such as Buttiauxella and Obesumbacterium. Both the cheese making and ripening processes had a significant impact on bacterial communities. Overall, the growth of lactic acid bacteria (LAB) (Lactococcus, Lactobacillus, Leuconostoc, Enterococcus, Streptococcus and Carnobacterium) was promoted, whereas that of non-desirable and environmental bacteria was inhibited (such as Pseudomonas and Clostridium). However, considerable differences were observed among producers. It is noteworthy that the starter LAB (Lactococcus) predominated up to 30 or 60 days of ripening and then, the growth of non-starter LAB (Lactobacillus, Leuconostoc, Enterococcus and Streptococcus) was promoted. Moreover, in some cases, bacteria related to the production of volatile compounds (such as Hafnia, Brevibacterium and Psychrobacter) also showed notable abundance during the first few weeks of ripening. Overall, the results of this study enhance our understanding of microbial shifts that occur during the production and ripening of a raw ewe milk-derived cheese (Idiazabal), and could indicate that the practices adopted by producers have a great impact on the microbiota and final quality of this cheese.

Dataset Information

Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

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Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

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