Evaluation of milk sample fractions for characterization of milk microbiota from healthy and clinical mastitis cows.

Svetlana Ferreira Lima,Marcela Lucas De Souza Bicalho,Rodrigo Carvalho Bicalho,Juan J Loor

doi:10.1371/journal.pone.0193671

Abstract

Amplicon sequencing technique has been increasingly applied to the clinical setting as a sensitive diagnostic tool. Therefore, it is of great importance to develop a DNA extraction method that accurate isolates DNA from complex host-associated microbiota. Given the multifactorial etiology of clinical mastitis and the diversified lifestyle of bacterial species harboring in milk, here four distinct milk sample fractions: raw whole milk, milk fat, casein-pellet, and casein-pellet + fat from healthy cows and cows with clinical mastitis, were subjected to bead-beating DNA extraction, followed by high-throughput sequencing. We aimed to identify the best approach for characterization of the milk microbiota and detection of mastitis pathogens (Klebsiella spp., Streptococcus spp. and Escherichia coli). DNA from each milk fraction tested was extracted by two commercial kits, which include physical, mechanical and chemical lysis; in total 280 DNA samples from 35 cows were analyzed. Milk-health-status were categorized into four groups (healthy group; E. coli-mastitis group; Klebsiella spp.-mastitis group; and Streptococcus spp.–mastitis group). Bacterial phyla and families were described for each milk-health-status group across milk sample fractions and DNA extraction kits. For the mastitis groups the relative abundance of f__Enterobacteriaceae and f__Streptococcaceae were compared to determine the efficacy of procedures in detecting the mastitis pathogens. The four milk fractions used allowed efficiently and uniformly detection of the causative agent of mastitis. Only 27% of the families detected in healthy milk were shared among the samples extracted from all fractions of milk samples; followed by 3, 4, and 12% for the samples from E. coli-mastitis, Klebsiella spp.-mastitis and Streptococcus spp-mastitis, respectively. However, the shared families comprised a mean relative abundance greater than 85%, regardless of milk-health-status, milk fraction and DNA isolation method. Taxonomic data at the family level showed that sequences from mastitis milk samples cultured positive for E. coli and Klebsiella spp. were predominantly affiliated with f__Enterobacteriaceae, while for Streptococcus spp. were dominated by f__Streptococcacea, followed by f__Pseudomonadaceae and f__Enterococcaceae. Microbial community analysis revealed that most of the microbial community composition corresponded to milk bacterial species irrespective of the DNA isolation method and milk fraction evaluated.

Highlights

Inflammation of the mammary gland, known as mastitis, is arguably the most important disease affecting dairy herds worldwide [1]
Equal volumes of each of the 35 milk samples were aliquoted in eight microcentrifuge tubes, which were processed as whole milk, fat, fat + pellet, and pellet by two different DNA extraction kits (PowerFood or PowerSoil), giving a total of 280 study samples
Four distinct milk sample fractions: raw whole milk, milk fat, casein-pellet, and casein-pellet + fat, obtained from milk samples of healthy cows and cows diagnosed with clinical mastitis, were subjected to DNA isolation by beat-beading method to evaluate the best approach for milk microbiota characterization and direct detection of Klebsiella spp., Streptococcus spp. and E. coli bacteria

Summary

Introduction

Inflammation of the mammary gland, known as mastitis, is arguably the most important disease affecting dairy herds worldwide [1]. Because cultures of mastitic milk samples may not always result in bacterial growth, an increasing number of studies has shown the potential of molecular techniques to improve the diagnosis of mastitis, with high sensitivity and specificity [10,11,12,13]. For any PCR-based approach, generating high-quality DNA is both critical and a challenge for accurate taxonomic profiling. Clinical samples from an infected mammary gland contain additional PCR inhibitory factors such as bacterial and mammalian cellular debris [14], whereas non-clinical milk samples typically have low bacterial loads [15]. Different bacterial species may possess distinct cell-structural characteristics that may affect DNA recovery, the treatment applied to the sample could bias the results of the downstream analysis and the taxonomic profiling

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