The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

Ojo-Okunola, Anna; Claassen-Weitz, Shantelle; Mwaikono, Kilaza Samson; Gardner-Lubbe, Sugnet; Zar, Heather J.; Nicol, Mark P.; Toit, Elloise du

doi:10.3390/mps3020039

Cited by 23 publications

(44 citation statements)

References 53 publications

(78 reference statements)

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“…Although kit NM recovered a significantly higher abundance of R. glycinis, this species was also found in the negative extraction control for this kit. The application of different DNA extraction methods resulted in variation in DNA yield and bacterial profiles, which is consistent with previous studies of HM (Douglas et al 2020;Ojo-Okunola et al 2020). Similarly, studies with other sample types have also reported variation in bacterial DNA profiles following DNA extraction with different kits (Yuan et al 2012;Wesolowska-Andersen et al 2014;Fouhy et al 2016;Glassing et al 2016;Hallmaier-Wacker et al 2018;Stinson et al 2018).…”

Section: Discussionsupporting

confidence: 88%

“…This has important implications for the interpretation of data from previous studies. This results reported here, together with those reported previously in HM studies (Douglas et al 2020;Ojo-Okunola et al 2020), suggest that it is problematic to compare data from different HM microbiome studies that have utilized different extraction methods. Extraction efficiency is an important consideration for low-biomass samples.…”

Section: Discussionsupporting

confidence: 41%

“…However, the HM microbiome varies significantly between studies with respect to composition, richness and diversity. These variations likely reflect differences in methodologies used (de Boer et al 2010;Yuan et al 2012;Kennedy et al 2014;Fouhy et al 2016;Hallmaier-Wacker et al 2018;Douglas et al 2020;Ojo-Okunola et al 2020) as well as geographical and demographic factors associated with the study populations (Lackey et al 2019). Importantly, unlike other commonly analysed samples such as stool, at present there are no standardized protocols for microbiome studies of HM (Wesolowska-Andersen et al 2014;Wu et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…They observed significant differences in DNA yield and purity, 16S rRNA gene sequence read depth and microbiota profiles. Similarly, Ojo-Okunola et al compared four commercial DNA extraction kits (QIAamp DNA Microbiome Kit; ZR Fungal/Bacterial DNA MiniPrep, QIAsymphony DSP DNA Kit and Zymo-BIOMICS DNA Miniprep Kit) using mock microbial communities and 10 HM samples (Ojo-Okunola et al 2020). These authors also reported that extraction method substantially influenced DNA yield and bacterial DNA profiles generated from HM samples.…”

Section: Introductionmentioning

confidence: 99%

“…These authors also reported that extraction method substantially influenced DNA yield and bacterial DNA profiles generated from HM samples. To date, no single DNA extraction methodology has been found to be ideal for use with HM samples (Douglas et al 2020;Ojo-Okunola et al 2020). Further work is needed to define an optimal DNA extraction method for HM.…”

Section: Introductionmentioning

confidence: 99%

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DNA extraction method influences human milk bacterial profiles

Cheema

Stinson

Lai

et al. 2020

J. Appl. Microbiol.

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Aims: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). Methods and Results: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0Á68 ng ll À1) and QM (mean = 0Á55 ng ll À1). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. Conclusions: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. Significance and Impact of the Study: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 41%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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DNA extraction method influences human milk bacterial profiles

Cheema

Stinson

Lai

et al. 2020

J. Appl. Microbiol.

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Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits

Janssen¹,

Low

Wang

et al. 2021

Appl Plant Sci

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Premise Within a broader study on leaf fossilization in freshwater environments, a long‐term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis. Methods We extracted surface‐associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits. Results Kit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition. Discussion The success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram‐positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.

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Effect of storage, temperature, and extraction kit on the phylogenetic composition detected in the human milk microbiota

et al. 2020

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Human milk is considered the optimum feeding regime for newborns and is a source of bacteria for the developing infant gastrointestinal tract. However, as with all low biomass samples, standardization across variabilities such as sample collection, storage, and extraction methods is needed to eliminate discrepancies in microbial composition across studies. The aim of this study was to investigate how different storage methods, temperatures, preservatives, and extraction kits influence the human milk microbiome, compared to fresh samples. Breast milk samples were processed via six different methods: fresh (Method 1), frozen at −80°C (Method 2), treated with RNAlater and stored at 4°C or −80°C (Methods 3 and 4), and treated with Milk Preservation Solution at room temperature (Methods 5 and 6). Methods 1‐5 were extracted using PowerFood TM Microbial DNA Isolation kit (Mobio), and Method 6 was extracted using Milk DNA Preservation and Isolation kit (Norgen BioTek). At genus level, the most abundant genera were shared across Methods 1‐5. Samples frozen at −80°C had fewest significant changes while samples treated and extracted using Milk Preservation and Isolation kit had the most significant changes when compared to fresh samples. Diversity analysis indicated that variation in microbiota composition was related to the method and extraction kit used. This study highlighted that, when extraction from fresh milk samples is not an option, freezing at −80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrate that choice of extraction kit had a profound impact on the microbiota populations detected in milk.

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The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

Cited by 23 publications

References 53 publications

DNA extraction method influences human milk bacterial profiles

DNA extraction method influences human milk bacterial profiles

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits

Effect of storage, temperature, and extraction kit on the phylogenetic composition detected in the human milk microbiota

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