Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues.

Gabriela Albuquerque ,Emmanuel Dias-Neto,Bruno S Moda,Alexandre Defelicibus,Klas I Udekwu,Elizabeth C Miola,Stephania M Bezerra,Laura C L Claro ,Luiz Paulo Kowalski ,Thais Fernanda Bartelli ,María Galli De Amorim ,Cláudia Zitron ,Fábio Abreu Alves ,Isabella K T M Takenaka ,Gabriela P Branco ,Diana Noronha Nunes ,Felipe J F Coimbra ,Lais Lie Senda De Abrantes ,Valquiria C A Martins ,S.c Zequi ,Adriane Pelosof ,Marianna Sampaio Serpa ,Israel Tojal Da Silva ,Kátia Luz Torres

doi:10.3390/genes13020237

Abstract

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Highlights

The human microbiota consists of trillions of microorganisms, including viruses, archaea, bacteria, fungi, protists, and other eukaryotes that may impact human health.The transition to an unbalanced, dysbiotic state needs to be explored, as it may be either the cause or the result of numerous pathological states
Standard curves were plotted, allowing to correlate the approximate absolute number of genome copies for bacteria and fungi according to the respective Cts achieved in qPCR
In order to simulate real-life analysis of samples that present significant variations in the ratios of target microorganisms and host-derived DNA, we evaluated the Ct fluctuations in face of different human: bacteria and fungi: human DNA ratios and their possible impact on the quantification of these microorganisms

Summary

Introduction

The human microbiota consists of trillions of microorganisms, including viruses, archaea, bacteria, fungi, protists, and other eukaryotes that may impact human health.The transition to an unbalanced, dysbiotic state needs to be explored, as it may be either the cause or the result of numerous pathological states. Advances in the study of human-associated microbiota allowed the investigation of numerous health and disease aspects, ranging from the impact of C-section versus vaginal delivery on the microbiota of newborns [10,11], to neurodegenerative diseases [12,13] and the microbiota impact in various cancers [14,15], including treatment response [16]. The most desirable and informative protocols for microbiota studies involve total DNA extraction, followed by DNA fragmentation, library construction, and sequencing, which is denominated shotgun metagenomics. This approach, depending on the efficacy of the DNA/RNA extraction, would allow the capture of all nucleic acids (total nucleic acids—TNA) available and provide a less skewed view of the microbiota [17]

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