Systematic Evaluation of the Viable Microbiome in the Human Oral and Gut Samples with Spike-in Gram+/– Bacteria

Liu, Feng; Lü, Hui; Dong, Biao; Huang, Xiaowan; Cheng, Hongyu; Qu, Ru; Hu, Yichen; Zhong, Luyun; Guo, Zhen‐Ni; You, Yuehua; Xu, Zhenjiang Zech

doi:10.1128/msystems.00738-22

Cited by 5 publications

(2 citation statements)

References 51 publications

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“…PMA is a compound extensively employed in the surveillance of viable microorganisms [ 42 ]. At present, PMA combined with amplicon sequencing technology has been successfully applied to the analysis of the structure of living microbial communities in soil, sewage and human gut [ 20 , 43 , 44 ]. This study established the optimal pre-treatment protocol for PMA in microbes from fermented grain samples.…”

Section: Discussionmentioning

confidence: 99%

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing

Bo,

Zhang,

Zong

et al. 2024

Foods

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The fermentation process of Chinese Baijiu’s fermented grains involves the intricate succession and metabolism of microbial communities, collectively shaping the Baijiu’s quality. Understanding the composition and succession of these living microbial communities within fermented grains is crucial for comprehending fermentation and flavor formation mechanisms. However, conducting high-throughput analysis of living microbial communities within the complex microbial system of fermented grains poses significant challenges. Thus, this study addressed this challenge by devising a high-throughput analysis framework using light-flavor Baijiu as a model. This framework combined propidium monoazide (PMA) pretreatment technology with amplicon sequencing techniques. Optimal PMA treatment parameters, including a concentration of 50 μM and incubation in darkness for 5 min followed by an exposure incubation period of 5 min, were identified. Utilizing this protocol, viable microorganism biomass ranging from 8.71 × 106 to 1.47 × 108 copies/μL was successfully detected in fermented grain samples. Subsequent amplicon sequencing analysis revealed distinct microbial community structures between untreated and PMA-treated groups, with notable differences in relative abundance compositions, particularly in dominant species such as Lactobacillus, Bacillus, Pediococcus, Saccharomycopsis, Issatchenkia and Pichia, as identified by LEfSe analysis. The results of this study confirmed the efficacy of PMA-amplicon sequencing technology for analyzing living microbial communities in fermented grains and furnished a methodological framework for investigating living microbial communities in diverse traditional fermented foods. This technical framework holds considerable significance for advancing our understanding of the fermentation mechanisms intrinsic to traditional fermented foods.

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Section: Discussionmentioning

confidence: 99%

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing

Bo,

Zhang,

Zong

et al. 2024

Foods

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“…As an example, PMA dye treatment was tested on suspensions of pure bacterial broth cultures [ 11 , 14 , 15 ], wastewater treatment plant influents and effluents [ 16 ], artificially contaminated food [ 17 , 18 ], urine [ 19 ], and piggery effluents [ 20 ]. A few studies have tested the applicability of PMA dye treatment on complex, heterogenous matrices such as stools and biosolids, i.e., on human [ 21 , 22 , 23 ] and equine [ 24 ] donor fecal slurries prepared for transplantation to assess the viability of the donor microbiome.…”

Section: Introductionmentioning

confidence: 99%

A Pilot Study to Detect Viable Salmonella spp. in Diarrheal Stool Using Viability Real-Time PCR as a Culture-Independent Diagnostic Tool in a Clinical Setting

Thilakarathna

Chui

2023

IJMS

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Frontline laboratories are adopting culture-independent diagnostic testing (CIDT) such as nucleic acid amplification tests (NAATs) due to numerous advantages over culture-based testing methods. Paradoxically, the viability of pathogens, a crucial factor determining active infections, cannot be confirmed with current NAATs alone. A recent development of viability PCR (vPCR) was introduced to mitigate this limitation associated with real-time PCR (qPCR) by using a DNA-intercalating dye to remove residual and dead cell DNA. This study assessed the applicability of the vPCR assay on diarrheal stools. Eighty-five diarrheal stools confirmed for Salmonellosis were tested via qPCR and vPCR using in-house primers and probe targeting the invA gene. vPCR-negative stools (Ct cut off > 31) were enriched in mannitol selenite broth (MSB) to verify low bacterial loads. vPCR assay showed ~89% sensitivity (qPCR- and vPCR-positive stools: 76/85). vPCR-negative stools (9/85; qPCR-positive: 5; qPCR-negative: 4) were qPCR- and culture-positive post-MSB-enrichment and confirmed the presence of low viable bacterial loads. Random sampling error, low bacterial loads, and receiving stools in batches could contribute to false negatives. This is a pilot study and further investigations are warranted to explore vPCR to assess pathogen viability in a clinical setting, especially when culture-based testing is unavailable.

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Next Steps: Studying Diabetic Foot Infections with Next-Generation Molecular Assays

Sande,

Boston,

Kalan

et al. 2023

Curr Infect Dis Rep

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Purpose of Review In 2019, the International Working Group on the Diabetic Foot voiced six concerns regarding the use of molecular microbiology techniques for routine diagnosis of infection complicating diabetic foot ulcers. The purpose of this review is to evaluate contemporary evidence addressing each of these concerns and describe promising avenues for continued development of molecular microbiology assays. Recent Findings Since 2019, the feasibility of conducting metagenomic and metatranscriptomic studies on diabetic foot ulcer samples has been shown. However, these preliminary studies used small samples with concerns for selection bias. We await larger-scale, longitudinal studies, potentially using the recently formed Diabetic Foot Consortium, to identify microbiome profiles associated with infection and patient outcomes. How these results would translate into a clinical diagnostic requires further clarification. Summary High-throughput molecular microbiology techniques are not yet ready for clinical adoption as first-line diagnostics. However, moving from amplicon sequencing to metagenomic and metatranscriptomic studies has the potential to significantly accelerate development of assays that might meaningfully impact patient care.

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Systematic Evaluation of the Viable Microbiome in the Human Oral and Gut Samples with Spike-in Gram+/– Bacteria

Cited by 5 publications

References 51 publications

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing

A Pilot Study to Detect Viable Salmonella spp. in Diarrheal Stool Using Viability Real-Time PCR as a Culture-Independent Diagnostic Tool in a Clinical Setting

Next Steps: Studying Diabetic Foot Infections with Next-Generation Molecular Assays

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