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Culture independent assessment of human milk microbial community in lactational mastitis.

ABSTRACT: Breastfeeding undoubtedly provides important benefits to the mother-infant dyad and should be encouraged. Mastitis, one of the common but major cause of premature weaning among lactating women, is an inflammation of connective tissue within the mammary gland. This study reports the influence of mastitis on human milk microbiota by utilizing 16?S rRNA gene sequencing approach. We sampled and sequenced microbiome from 50 human milk samples, including 16 subacute mastitis (SAM), 16 acute mastitis (AM) and 18 healthy-controls. Compared to controls, SAM and AM microbiota were quite distinct and drastically reduced. Genera including, Aeromonas, Staphylococcus, Ralstonia, Klebsiella, Serratia, Enterococcus and Pseudomonas were significantly enriched in SAM and AM samples, while Acinetobacter, Ruminococcus, Clostridium, Faecalibacterium and Eubacterium were consistently depleted. Further analysis of our samples revealed positive aerotolerant odds ratio, indicating dramatic depletion of obligate anaerobes and enrichment of aerotolerant bacteria during the course of mastitis. In addition, predicted functional metagenomics identified several gene pathways related to bacterial proliferation and colonization (e.g. two-component system, bacterial secretion system and motility proteins) in SAM and AM samples. In conclusion, our study confirmed previous hypothesis that mastitis women have lower microbial diversity, increased abundance of opportunistic pathogens and depletion of commensal obligate anaerobes.

SUBMITTER: Patel SH

PROVIDER: S-EPMC5552812 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Culture independent assessment of human milk microbial community in lactational mastitis.

Patel Shriram H SH Vaidya Yati H YH Patel Reena J RJ Pandit Ramesh J RJ Joshi Chaitanya G CG Kunjadiya Anju P AP

Scientific reports 20170810 1

Breastfeeding undoubtedly provides important benefits to the mother-infant dyad and should be encouraged. Mastitis, one of the common but major cause of premature weaning among lactating women, is an inflammation of connective tissue within the mammary gland. This study reports the influence of mastitis on human milk microbiota by utilizing 16 S rRNA gene sequencing approach. We sampled and sequenced microbiome from 50 human milk samples, including 16 subacute mastitis (SAM), 16 acute mastitis ( ...[more]

PMID: 28798374

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Project description:IntroductionRapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging.MethodsHere, we tested 24 bovine milk samples (18 mastitis and six non-mastitis) using four different commercial kits (Qiagens' DNeasy® PowerFood® Microbial, Norgens' Milk Bacterial DNA Isolation, and Molzyms' MolYsis™ Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection.ResultsThe results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsis™ Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and Staphylococcus aureus reads, respectively. This combination also effectively enriched other mastitis pathogens, including Escherichia coli and Streptococcus dysgalactiae. Furthermore, using this approach, we identified important AMR genes such as Tet (A), Tet (38), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene.ConclusionWe implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplification-independent study using nanopore-based metagenomic sequencing for real-time detection of the pathogen (within 5 hours) and the AMR profile (within 5-9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.

Dataset Information

Culture independent assessment of human milk microbial community in lactational mastitis.

Publications

Culture independent assessment of human milk microbial community in lactational mastitis.

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