Metagenomic approach based on 16S rRNA gene for evaluation of bacterial

population of Brazilian milk and dairy products

Diego A. Frazílio1, Fabian C. N. Arias1, Vanessa M. de Souza1, Luiza T. Chaul2, Virgínia Farias Alves2, Elaine C.P. De Martinis1*

INTRODUCTION MATERIAL AND METHODS RESULTS

• It is important to determine the microbial species present in
industrial environments, especially in industrial or small food • To evaluate the main bacterial groups present in Brazilian dairy
factories, since microorganisms in biofilms may be more tolerant to products, metagenomic analysis targeting the 16S rRNA gene was
cleaning and sanitizing procedures (Rasamiravakaet al., 2015). done, using the platform Illumina® Miseq. Dairy plants from
Brazilian midwest region were sampled for raw material, food
• The microbiology control in dairy products is of great importance to contact surfaces, food non-contact surfaces and ready to eat
avoid contamination by pathogen microorganisms, such as products, in a total of 12 representative samples. Total DNA was
Staphylococcus aureus and Listeria monocytogenes (El-Ashker et extracted directly from the samples using the commercial kit
al., 2015; Mackiwet al., 2016). PowerLyzer® PowerSoil® DNA isolation (MoBio®), according to
• Metagenomics uses next-generation DNA sequencing to analyze instructions of the manufacturer and, DNA libraries were prepared
complex microbial communities, allowing the genomic mapping of according to the protocol of Illumina®. DNA sequencing was done
species, determining which is the dominant community present in in the equipment Illumina® MiSeq, at the FCFRP-USP and
the sample of interest. It is important to note that classical analyzes were done with the dedicated software available in the
laboratory culture techniques are ineffective for almost 99% of sequencer.
bacteria because of its complex metabolism (Mardis, 2008;
Ramamurthy et al., 2014).
• Environmental and food samples previously collected in dairy
PROCEDURE
products were carried out, through the sequencing of new
generation DNA (MiSeq sequencer Illumina) and the results
Etapa 1 Etapa 2 Etapa 3 Etapa 4
obtained with culture methods and metagenomic analysis were
compared in order to elucidate possible correlations between the
accompanying microbiota and presence of pathogens, evaluating if
there is any combination of microorganisms that would be related to Figura 1. Dendograma demonstrando a relação dos tipos de gênero entre as
the persistence or absence of the pathogen. amostras, onde é possível observar que mesmo algumas das amostras terem
sido feito em duplicata, apenas com diferença da repurificação,que existiram
Samples Non cultivable The DNA was The results diferenças entre aquelas as amostras repurificadas, e aquelas que não foram
OBJECTIVE collected in samples had amplified using were analyzed repurificadas.
dairy factories the DNA the the region using the app
• The objective of this work is the qualitative characterization of the extracted. 16S, and sent BaseSpace
microbiota present in food and milk processing environments, in to sequencing Mteagenomics CONCLUSION
order to contribute to the improvement of strategies to guarantee Illumina® by Illumina®
• The most prevalent species in the dairy samples were
food safety. The work consisted of molecular analysis of bacterial Enterococcus casseliflavus (28%), Enterococcus faecium (6%),
isolates previously obtained from food and dairy environments, for Lactococcus lactis (6%), Staphylococcus agnetis (4%),
genetic confirmation of the identity of the pathogens Listeria Staphylococcus hiyicus (4%). Staphylococcus aureus was detected
monocytogenes and Staphylococcus aureus, by 16S rDNA only in raw material and other pathogens were not found in the
sequencing samples evaluated.

PROJECT OVERVIEW REFERENCES

INTRODUCTION: Bacterial populations in milk and dairy products
are of great interest from a food safety point of view and also due
to the presence of cultures with potential technological
applications. To evaluate the main bacterial groups present in
Brazilian dairy products, metagenomic analysis targeting the 16S
rRNA gene was done, using the platform Illumina® Miseq.
• Rasamiravaka, T., Labtani, Q., Duez, P., Jaziri, M.E. 2015. The formation of biofilms by Pseudomonas aeruginosa: a review of the natural synthetic
compounds interfering with control mechanisms. Biomed. Resear. Internat, 2015: 1-17.
• El-ashker, M., Gwida, M., Tomaso, H., Monecke, S., Enricht, R., El-gohary, F., Hotzel, H. 2015. Staphylococci in cattle and buffaloes with mastites in
Dakahlia Governorate, Egypt. J. Dairy Sci. 98: 7450-7459.
• Mackiw, E., Modzelewska, M., Maka, L., Sciezynska, H.P., Postupolski, J., Korsak, D. 2016. Antimicrobial resistance profiles of Listeria monocytogenes
isolated from ready-to-eat products in Poland in 2007-2011. Food Control, 59:7-11.
• Mardis, E. R. 2008. Next-Generation DNA sequencing Methods. Annu. Ver. Genomics Hum genetic. 9: 387-402.