Methodological flaws introduce strong bias into molecular analysis of microbial populations

Krakat, Niclas; Anjum, Reshma; Demirel, Burak; Schröder, Patrick

doi:10.1111/jam.13365

Cited by 16 publications

(9 citation statements)

References 46 publications

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“…2A ). Slower growth was reported with plant slurry preparations, and may be attributed to their concentrated nutritional contents creating osmotic stress environments ( 8 , 24 , 39 ).…”

Section: Resultsmentioning

confidence: 99%

“…Advances in molecular biology techniques with the development of culture-independent techniques have highlighted the joint genomic, transcriptomic, and proteomic interactions that influence the culturability of microorganisms ( 47 ). Despite the high-throughput nature of these culture-independent approaches for community profiling ( 42 ), previous studies demonstrated their inherent bias that results in a superficial view of microbial community compositions ( 24 ). Unculturable populations account for more than 90% of a given ecosystem, and represent diverse groups of microbes with overlooked functional niches.…”

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confidence: 99%

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Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for <i>in vitro</i> Culturing of the Plant Microbiota

Mourad

Sarhan

Daanaa

et al. 2018

Microbes and environments

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In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S′, H′, and D′) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

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“…2A ). Slower growth was reported with plant slurry preparations, and may be attributed to their concentrated nutritional contents creating osmotic stress environments ( 8 , 24 , 39 ).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for <i>in vitro</i> Culturing of the Plant Microbiota

Mourad

Sarhan

Daanaa

et al. 2018

Microbes and environments

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“…How is it that a single extraction procedure can portend to achieve a uniform and balanced extraction? In the process of establishing this ddPCR procedure it was found that Listeria DNA was poorly extracted, quite probably due to the stronger cell wall, compared to gram–bacteria (Krakat et al, 2017 ). Optimization was eventually achieved by sonication.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, QPCR data representative of each pathogen level in a sample requires efficient extraction and amplification of DNA from each of the pathogens. However, the DNA of L. monocytogenes , like most gram positive organisms, is difficult to extract with the same efficiency as gram negative organisms (Krakat et al, 2017 ). This problem impacts not only QPCR of pathogens but also several other quantification methods, such as metagenomics, where samples are known to include a host of unknown organisms and probably both gram + and gram –bacteria.…”

Section: Introductionmentioning

confidence: 99%

Optimized Co-extraction and Quantification of DNA From Enteric Pathogens in Surface Water Samples Near Produce Fields in California

2018

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Pathogen contamination of surface water is a health hazard in agricultural environments primarily due to the potential for contamination of crops. Furthermore, pathogen levels in surface water are often unreported or under reported due to difficulty with culture of the bacteria. The pathogens are often present, but require resuscitation, making quantification difficult. Frequently, this leads to the use of quantitative PCR targeted to genes unique to the pathogens. However, multiple pathogen types are commonly in the same water sample, both gram + and gram –, leading to problems with DNA extraction. With Shiga toxin-producing Escherichia coli (STEC), Salmonella enterica and Listeria monocytogenes as target, a method was optimized to co-extract all three and quantify the level of each using droplet digital PCR (ddPCR). Multiplexed target genes in STEC were virulence genes, shiga toxin 2 (stx2) and hemolysin (ehx). Likewise, multiplexed targets in Listeria and Salmonella were the virulence genes listeriolysin (hly) and invasion protein A (invA). Water samples were processed using microbiological techniques for each of the pathogens and duplicate water samples were quantified by ddPCR. A significant correlation was found between culture and ddPCR results indicating detection primarily of culturable cells by ddPCR. Average virulence gene levels were 923, 23 k, 69 and 152 copies per sample for stx2, ehx, hly and invA, respectively. Additionally, stx2, ehx and inv levels were significantly correlated (P < 0.05, R = 0.34) with generic E. coli MPN levels in the duplicate samples. Indirect quantification with ddPCR will improve understanding of prevalence of the pathogens and may reduce risks associated with contaminated surface water.

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“…While this information complements nicely the DNA‐based information detailed above, abundance differences for the same phylogenetic group using different methods (e.g., single cell based versus sequencing based) might appear. This can be assigned to the sample processing, as for instance the DNA‐based sequencing methods are partly biased due to different DNA extraction and amplification, and FISH can be biased due to fixation and hybridization efficiency differences (Amann and Fuchs, ; Krakat et al ., ).…”

Section: Application Of Microbial Ecology To Metmentioning

confidence: 97%

Microbial ecology‐based engineering of Microbial Electrochemical Technologies

Koch

Korth

2017

Microbial Biotechnology

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SummaryMicrobial ecology is devoted to the understanding of dynamics, activity and interaction of microorganisms in natural and technical ecosystems. Bioelectrochemical systems represent important technical ecosystems, where microbial ecology is of highest importance for their function. However, whereas aspects of, for example, materials and reactor engineering are commonly perceived as highly relevant, the study and engineering of microbial ecology are significantly underrepresented in bioelectrochemical systems. This shortfall may be assigned to a deficit on knowledge and power of these methods as well as the prerequisites for their thorough application. This article discusses not only the importance of microbial ecology for microbial electrochemical technologies but also shows which information can be derived for a knowledge‐driven engineering. Instead of providing a comprehensive list of techniques from which it is hard to judge the applicability and value of information for a respective one, this review illustrates the suitability of selected techniques on a case study. Thereby, best practice for different research questions is provided and a set of key questions for experimental design, data acquisition and analysis is suggested.

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Methodological flaws introduce strong bias into molecular analysis of microbial populations

Cited by 16 publications

References 46 publications

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for <i>in vitro</i> Culturing of the Plant Microbiota

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for <i>in vitro</i> Culturing of the Plant Microbiota

Optimized Co-extraction and Quantification of DNA From Enteric Pathogens in Surface Water Samples Near Produce Fields in California

Microbial ecology‐based engineering of Microbial Electrochemical Technologies

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