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Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 102–108 CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA–metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms. [ABSTRACT FROM AUTHOR]

The metagenomic surveillance of antimicrobial resistance in wastewater has been suggested as a methodological tool to characterize the distribution, status, and trends of antibiotic-resistant bacteria. In this study, a cross-sectional collection of samples of hospital-associated raw and treated wastewater were obtained from February to March 2020. Shotgun metagenomic sequencing and bioinformatic analysis were performed to characterize bacterial abundance and antimicrobial resistance gene analysis. The main bacterial phyla found in all the samples were as follows: Proteobacteria, Bacteroides, Firmicutes, and Actinobacteria. At the species level, ESKAPEE bacteria such as E. coli relative abundance decreased between raw and treated wastewater, but S. aureus, A. baumannii, and P. aeruginosa increased, as did the persistence of K. pneumoniae in both raw and treated wastewater. A total of 172 different ARGs were detected; blaOXA, blaVEB, blaKPC, blaGES, mphE, mef, erm, msrE, AAC(6′), ant(3″), aadS, lnu, PBP-2, dfrA, vanA-G, tet, and sul were found at the highest abundance and persistence. This study demonstrates the ability of ESKAPEE bacteria to survive tertiary treatment processes of hospital wastewater, as well as the persistence of clinically important antimicrobial resistance genes that are spreading in the environment. [ABSTRACT FROM AUTHOR]

Gut microbes thrive by utilising host energy and, in return, provide valuable benefits, akin to a symbiotic relationship. Here, metagenomic sequencing was performed to characterise and compare the community composition, diversity and antibiotic resistance of the gut microbiota of Relict gull (Larus relictus) and Anatidae species. Alpha diversity analysis revealed that the intestinal microbial richness of L. relictus was significantly lower than that of Anatidae, with distinct differences observed in microbial composition. Notably, the intestines of L. relictus harboured more pathogenic bacteria such as clostridium, which may contribute to the decline in their population and endangered status. A total of 117 strains of Escherichia coli were isolated, with 90.60% exhibiting full susceptibility to 21 antibiotics, while 25.3% exhibited significant biofilm formation. Comprehensive Antibiotic Resistance Database data indicated that glycopeptide resistance genes were the most prevalent type carried by migratory birds, alongside quinolone, tetracycline and lincosamide resistance genes. The abundance of resistance genes carried by migratory birds decreased over time. This metagenomic analysis provides valuable insights into the intestinal microbial composition of these wild bird species, offering important guidance for their conservation efforts, particularly for L. relictus, and contributing to our understanding of pathogen spread and antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

The composition of microbiota in the digestive tract gut is essential for insect physiology, homeostasis, and pathogen infection. Little is known about the interactions between microbiota load and oral infection with baculoviruses. CnmeGV is an obligative baculovirus to Cnaphalocrocis medinalis. We investigated the impact of CnmeGV infection on the structure of intestinal microbes of C. medinalis during the initial infection stage. The results revealed that the gut microbiota profiles were dynamically driven by pathogen infection of CnmeGV. The numbers of all the OTU counts were relatively higher at the early and later stages, while the microbial diversity significantly increased early but dropped sharply following the infection. The compositional abundance of domain bacteria Firmicutes developed substantially higher. The significantly enriched and depleted species can be divided into four groups at the species level. Fifteen of these species were ultimately predicted as the biomarkers of CnmeGV infection. CnmeGV infection induces significant enrichment of alterations in functional genes related to metabolism and the immune system, encompassing processes such as carbohydrate, amino acid, cofactor, and vitamin metabolism. Finally, the study may provide an in-depth analysis of the relationship between host microbiota, baculovirus infection, and pest control of C. medinalis. [ABSTRACT FROM AUTHOR]

Metagenomic next-generation sequencing (mNGS) methodology serves as an excellent supplement in cases where diagnosis is challenging to establish through conventional laboratory tests, and its usage is increasingly prevalent. Examining the causes of infectious diseases in the central nervous system (CNS) is vital for understanding their spread, managing outbreaks, and effective patient care. In a study conducted in the state of São Paulo, Brazil, cerebrospinal fluid (CSF) samples from 500 patients with CNS diseases of indeterminate etiology, collected between 2017 and 2021, were analyzed. Employing a mNGS approach, we obtained the complete coding sequence of Pegivirus hominis (HPgV) genotype 2 in a sample from a patient with encephalitis (named IAL-425/BRA/SP/2019); no other pathogen was detected. Subsequently, to determine the extent of this virus's presence, both polymerase chain reaction (PCR) and/or real-time PCR assays were utilized on the entire collection. The presence of the virus was identified in 4.0% of the samples analyzed. This research constitutes the first report of HPgV detection in CSF samples in South America. Analysis of the IAL-425 genome (9107 nt) revealed a 90% nucleotide identity with HPgV strains from various countries. Evolutionary analyses suggest that HPgV is both endemic and extensively distributed. The direct involvement of HPgV in CNS infections in these patients remains uncertain. [ABSTRACT FROM AUTHOR]

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 102–108 CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA–metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms.

MFCs represent a promising sustainable biotechnology that enables the direct conversion of organic matter from wastewater into electricity using bacterial biofilms as biocatalysts. A crucial aspect of MFCs is how electroactive bacteria (EAB) behave and their associated mechanisms during extracellular electron transfer to the anode. A critical phase in the MFC start-up process is the initial colonization of the anode by EAB. Two MFCs were operated with an external resistance of 1000 ohms, one with an applied electrical voltage of 500 mV during the initial four days of biofilm formation and the other without any additional applied voltage. After stabilization of electricity production, total DNA and protein were extracted and sequenced from both setups. The combined metaproteomic/metagenomic analysis revealed that the application of voltage during the colonization step predominantly increased direct electron transfer via cytochrome c, mediated primarily by Geobacter sp. Conversely, the absence of applied voltage during colonization resulted in a broader diversity of bacteria, including Pseudomonas and Aeromonas, which participated in electricity production via mediated electron transfer involving flavin family members. [ABSTRACT FROM AUTHOR]

The metagenomic surveillance of antimicrobial resistance in wastewater has been suggested as a methodological tool to characterize the distribution, status, and trends of antibiotic-resistant bacteria. In this study, a cross-sectional collection of samples of hospital-associated raw and treated wastewater were obtained from February to March 2020. Shotgun metagenomic sequencing and bioinformatic analysis were performed to characterize bacterial abundance and antimicrobial resistance gene analysis. The main bacterial phyla found in all the samples were as follows: Proteobacteria, Bacteroides, Firmicutes, and Actinobacteria. At the species level, ESKAPEE bacteria such as E. coli relative abundance decreased between raw and treated wastewater, but S. aureus, A. baumannii, and P. aeruginosa increased, as did the persistence of K. pneumoniae in both raw and treated wastewater. A total of 172 different ARGs were detected; blaOXA, blaVEB, blaKPC, blaGES, mphE, mef, erm, msrE, AAC(6′), ant(3″), aadS, lnu, PBP-2, dfrA, vanA-G, tet, and sul were found at the highest abundance and persistence. This study demonstrates the ability of ESKAPEE bacteria to survive tertiary treatment processes of hospital wastewater, as well as the persistence of clinically important antimicrobial resistance genes that are spreading in the environment.

Gut microbes thrive by utilising host energy and, in return, provide valuable benefits, akin to a symbiotic relationship. Here, metagenomic sequencing was performed to characterise and compare the community composition, diversity and antibiotic resistance of the gut microbiota of Relict gull (Larus relictus) and Anatidae species. Alpha diversity analysis revealed that the intestinal microbial richness of L. relictus was significantly lower than that of Anatidae, with distinct differences observed in microbial composition. Notably, the intestines of L. relictus harboured more pathogenic bacteria such as clostridium, which may contribute to the decline in their population and endangered status. A total of 117 strains of Escherichia coli were isolated, with 90.60% exhibiting full susceptibility to 21 antibiotics, while 25.3% exhibited significant biofilm formation. Comprehensive Antibiotic Resistance Database data indicated that glycopeptide resistance genes were the most prevalent type carried by migratory birds, alongside quinolone, tetracycline and lincosamide resistance genes. The abundance of resistance genes carried by migratory birds decreased over time. This metagenomic analysis provides valuable insights into the intestinal microbial composition of these wild bird species, offering important guidance for their conservation efforts, particularly for L. relictus, and contributing to our understanding of pathogen spread and antibiotic-resistant bacteria.

The composition of microbiota in the digestive tract gut is essential for insect physiology, homeostasis, and pathogen infection. Little is known about the interactions between microbiota load and oral infection with baculoviruses. CnmeGV is an obligative baculovirus to Cnaphalocrocis medinalis. We investigated the impact of CnmeGV infection on the structure of intestinal microbes of C. medinalis during the initial infection stage. The results revealed that the gut microbiota profiles were dynamically driven by pathogen infection of CnmeGV. The numbers of all the OTU counts were relatively higher at the early and later stages, while the microbial diversity significantly increased early but dropped sharply following the infection. The compositional abundance of domain bacteria Firmicutes developed substantially higher. The significantly enriched and depleted species can be divided into four groups at the species level. Fifteen of these species were ultimately predicted as the biomarkers of CnmeGV infection. CnmeGV infection induces significant enrichment of alterations in functional genes related to metabolism and the immune system, encompassing processes such as carbohydrate, amino acid, cofactor, and vitamin metabolism. Finally, the study may provide an in-depth analysis of the relationship between host microbiota, baculovirus infection, and pest control of C. medinalis.

Metagenomic next-generation sequencing (mNGS) methodology serves as an excellent supplement in cases where diagnosis is challenging to establish through conventional laboratory tests, and its usage is increasingly prevalent. Examining the causes of infectious diseases in the central nervous system (CNS) is vital for understanding their spread, managing outbreaks, and effective patient care. In a study conducted in the state of São Paulo, Brazil, cerebrospinal fluid (CSF) samples from 500 patients with CNS diseases of indeterminate etiology, collected between 2017 and 2021, were analyzed. Employing a mNGS approach, we obtained the complete coding sequence of Pegivirus hominis (HPgV) genotype 2 in a sample from a patient with encephalitis (named IAL-425/BRA/SP/2019); no other pathogen was detected. Subsequently, to determine the extent of this virus’s presence, both polymerase chain reaction (PCR) and/or real-time PCR assays were utilized on the entire collection. The presence of the virus was identified in 4.0% of the samples analyzed. This research constitutes the first report of HPgV detection in CSF samples in South America. Analysis of the IAL-425 genome (9107 nt) revealed a 90% nucleotide identity with HPgV strains from various countries. Evolutionary analyses suggest that HPgV is both endemic and extensively distributed. The direct involvement of HPgV in CNS infections in these patients remains uncertain.

MFCs represent a promising sustainable biotechnology that enables the direct conversion of organic matter from wastewater into electricity using bacterial biofilms as biocatalysts. A crucial aspect of MFCs is how electroactive bacteria (EAB) behave and their associated mechanisms during extracellular electron transfer to the anode. A critical phase in the MFC start-up process is the initial colonization of the anode by EAB. Two MFCs were operated with an external resistance of 1000 ohms, one with an applied electrical voltage of 500 mV during the initial four days of biofilm formation and the other without any additional applied voltage. After stabilization of electricity production, total DNA and protein were extracted and sequenced from both setups. The combined metaproteomic/metagenomic analysis revealed that the application of voltage during the colonization step predominantly increased direct electron transfer via cytochrome c, mediated primarily by Geobacter sp. Conversely, the absence of applied voltage during colonization resulted in a broader diversity of bacteria, including Pseudomonas and Aeromonas, which participated in electricity production via mediated electron transfer involving flavin family members.

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species (two different hosts- ten nasal mites from two independent samples per host), incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species and its putative endosymbiont. We obtained a metagenome assembled genome (MAG) that was estimated to be 98.1% complete and containing only 0.9% possible contamination. Moreover, the MAG has characteristics typical of endosymbionts (namely, small genome size an AT bias). Overall, our results support the presence of a potential endosymbiont, which is the first described for avian nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites.

Recent acquisitions about the role of the microbiota in the functioning of the human body make it possible to envisage an increasing use of beneficial microbes, and more particularly of probiotics as well as their metabolites, as nutritional supplements. National and EU authorities are engaged in assuring the safety and quality of food supplements and in defining rules to assess and communicate their efficacy on human health. The quality of probiotics, intended as strains’ identification, viability, and stability over time, is a crucial factor of credibility with consumers and health professionals. Analytical technologies for the quality control of probiotics must also be adapted to new preparations, such as those including new multistrains complex combinations. Accredited laboratories face this relevant challenge on a daily basis. Through its close collaboration with the laboratory commissioned to produce the specifications for its ESLP quality label (identification and quantitative analyses) together with its scientific committee, the ESLP has been focusing on this issue for 10 years. Recently, as part of the internationalization of the ESLP quality label, a new and unique initiative in Europe for the evaluation of the quality of probiotic preparations has been carried out. The collaboration between two accredited laboratories in Belgium and in Italy represented a concrete example of supranational collaboration in the assessment of the quality of probiotic preparations. Results show that both laboratories are in line as expected in terms of performance. Common approaches to the qualitative assessment of probiotic preparations, especially for complex and composite recipes, in terms of number of strains and included substances, should be encouraged and promoted all over the EU.

Microbial communities are important players in coastal sediments for the functioning of the ecosystem and the regulation of biogeochemical cycles. They also have great potential as indicators of environmental perturbations. To assess how microbial communities can change their composition and abundance along coastal areas, we analyzed the composition of the microbiome of four locations of the Yucatan Peninsula using 16S rRNA gene amplicon sequencing. To this end, sediment from two conserved (El Palmar and Bocas de Dzilam) and two contaminated locations (Sisal and Progreso) from the coast northwest of the Yucatan Peninsula in three different years, 2017, 2018 and 2019, were sampled and sequenced. Microbial communities were found to be significantly different between the locations. The most noticeable difference was the greater relative abundance of Planctomycetes present at the conserved locations, versus FBP group found with greater abundance in contaminated locations. In addition to the difference in taxonomic groups composition, there is a variation in evenness, which results in the samples of Bocas de Dzilam and Progreso being grouped separately from those obtained in El Palmar and Sisal. We also carry out the functional prediction of the metabolic capacities of the microbial communities analyzed, identifying differences in their functional profiles. Our results indicate that landscape of the coastal microbiome of Yucatan sediment shows changes along the coastline, reflecting the constant dynamics of coastal environments and their impact on microbial diversity.

An increasing body of evidence highlights the role of fecal microbiota in various human diseases. However, more than two-thirds of fecal bacteria cannot be cultivated by routine laboratory techniques. Thus, physicians and scientists use DNA sequencing and statistical tools to identify associations between bacterial subgroup abundances and disease. However, discrepancies between studies weaken these results. In the present study, we focus on biases that might account for these discrepancies. First, three different DNA extraction methods (G’NOME, QIAGEN, and PROMEGA) were compared with regard to their efficiency, i.e., the quality and quantity of DNA recovered from feces of 10 healthy volunteers. Then, the impact of the DNA extraction method on the bacteria identification and quantification was evaluated using our published cohort of sample subjected to both 16S rRNA sequencing and whole metagenome sequencing (WMS). WMS taxonomical assignation employed the universal marker genes profiler mOTU-v2, which is considered the gold standard. The three standard pipelines for 16S RNA analysis (MALT and MEGAN6, QIIME1, and DADA2) were applied for comparison. Taken together, our results indicate that the G’NOME-based method was optimal in terms of quantity and quality of DNA extracts. 16S rRNA sequence-based identification of abundant bacteria genera showed acceptable congruence with WMS sequencing, with the DADA2 pipeline yielding the highest congruent levels. However, for low abundance genera (

Metagenomic next-generation sequencing (mNGS) methodology serves as an excellent supplement in cases where diagnosis is challenging to establish through conventional laboratory tests, and its usage is increasingly prevalent. Examining the causes of infectious diseases in the central nervous system (CNS) is vital for understanding their spread, managing outbreaks, and effective patient care. In a study conducted in the state of São Paulo, Brazil, cerebrospinal fluid (CSF) samples from 500 patients with CNS diseases of indeterminate etiology, collected between 2017 and 2021, were analyzed. Employing a mNGS approach, we obtained the complete coding sequence of Pegivirus hominis (HPgV) genotype 2 in a sample from a patient with encephalitis (named IAL-425/BRA/SP/2019); no other pathogen was detected. Subsequently, to determine the extent of this virus's presence, both polymerase chain reaction (PCR) and/or real-time PCR assays were utilized on the entire collection. The presence of the virus was identified in 4.0% of the samples analyzed. This research constitutes the first report of HPgV detection in CSF samples in South America. Analysis of the IAL-425 genome (9107 nt) revealed a 90% nucleotide identity with HPgV strains from various countries. Evolutionary analyses suggest that HPgV is both endemic and extensively distributed. The direct involvement of HPgV in CNS infections in these patients remains uncertain.

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species (two different hosts- ten nasal mites from two independent samples per host), incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species and its putative endosymbiont. We obtained a metagenome assembled genome (MAG) that was estimated to be 98.1% complete and containing only 0.9% possible contamination. Moreover, the MAG has characteristics typical of endosymbionts (namely, small genome size an AT bias). Overall, our results support the presence of a potential endosymbiont, which is the first described for avian nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites. [ABSTRACT FROM AUTHOR]

Recent acquisitions about the role of the microbiota in the functioning of the human body make it possible to envisage an increasing use of beneficial microbes, and more particularly of probiotics as well as their metabolites, as nutritional supplements. National and EU authorities are engaged in assuring the safety and quality of food supplements and in defining rules to assess and communicate their efficacy on human health. The quality of probiotics, intended as strains' identification, viability, and stability over time, is a crucial factor of credibility with consumers and health professionals. Analytical technologies for the quality control of probiotics must also be adapted to new preparations, such as those including new multistrains complex combinations. Accredited laboratories face this relevant challenge on a daily basis. Through its close collaboration with the laboratory commissioned to produce the specifications for its ESLP quality label (identification and quantitative analyses) together with its scientific committee, the ESLP has been focusing on this issue for 10 years. Recently, as part of the internationalization of the ESLP quality label, a new and unique initiative in Europe for the evaluation of the quality of probiotic preparations has been carried out. The collaboration between two accredited laboratories in Belgium and in Italy represented a concrete example of supranational collaboration in the assessment of the quality of probiotic preparations. Results show that both laboratories are in line as expected in terms of performance. Common approaches to the qualitative assessment of probiotic preparations, especially for complex and composite recipes, in terms of number of strains and included substances, should be encouraged and promoted all over the EU. [ABSTRACT FROM AUTHOR]

Epidemiological studies link Sarcoptes scabiei infection and impetigo. Scabies mites can promote Streptococcus pyogenes (Group A Streptococcus) and Staphylococcus aureus infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. However, little is known about the composition and the function of the scabies-associated microbiota. Here, high-throughput whole-metagenome sequencing was used to explore the scabies-associated microbiome. Scabies mites including their immediate microenvironments were isolated from two patients with severe scabies in Northern Australia. Two ~45–50 million paired-end reads Illumina libraries were generated of which ~2 (5.1%) and 0.7 million (1.3%) microbial reads were filtered out by mapping to human (hg19) and mite draft genomes. Taxonomic profiling revealed a microbial community dominated by the phylum Firmicutes (A: 79% and B: 59%) and genera that comprise Streptococcus, Staphylococcus, Acinetobacter, and Corynebacterium. Assembly of the metagenome reads resulted in genome bins representing reference genomes of Acinetobacter baumannii, Streptococcus dysgalactiae (Group C/G), Proteus mirablis and Staphylococcus aureus. The contigs contained genes relevant to pathogenicity and antibiotics resistance. Confocal microscopy of a patient skin sample confirmed A. baumannii, Streptococci and S. aureus in scabies mite gut and faeces and the surrounding skin. The study provides fundamental evidence for the association of opportunistic pathogens with scabies infection. [ABSTRACT FROM AUTHOR]

Microbial communities are important players in coastal sediments for the functioning of the ecosystem and the regulation of biogeochemical cycles. They also have great potential as indicators of environmental perturbations. To assess how microbial communities can change their composition and abundance along coastal areas, we analyzed the composition of the microbiome of four locations of the Yucatan Peninsula using 16S rRNA gene amplicon sequencing. To this end, sediment from two conserved (El Palmar and Bocas de Dzilam) and two contaminated locations (Sisal and Progreso) from the coast northwest of the Yucatan Peninsula in three different years, 2017, 2018 and 2019, were sampled and sequenced. Microbial communities were found to be significantly different between the locations. The most noticeable difference was the greater relative abundance of Planctomycetes present at the conserved locations, versus FBP group found with greater abundance in contaminated locations. In addition to the difference in taxonomic groups composition, there is a variation in evenness, which results in the samples of Bocas de Dzilam and Progreso being grouped separately from those obtained in El Palmar and Sisal. We also carry out the functional prediction of the metabolic capacities of the microbial communities analyzed, identifying differences in their functional profiles. Our results indicate that landscape of the coastal microbiome of Yucatan sediment shows changes along the coastline, reflecting the constant dynamics of coastal environments and their impact on microbial diversity. [ABSTRACT FROM AUTHOR]

An increasing body of evidence highlights the role of fecal microbiota in various human diseases. However, more than two-thirds of fecal bacteria cannot be cultivated by routine laboratory techniques. Thus, physicians and scientists use DNA sequencing and statistical tools to identify associations between bacterial subgroup abundances and disease. However, discrepancies between studies weaken these results. In the present study, we focus on biases that might account for these discrepancies. First, three different DNA extraction methods (G'NOME, QIAGEN, and PROMEGA) were compared with regard to their efficiency, i.e., the quality and quantity of DNA recovered from feces of 10 healthy volunteers. Then, the impact of the DNA extraction method on the bacteria identification and quantification was evaluated using our published cohort of sample subjected to both 16S rRNA sequencing and whole metagenome sequencing (WMS). WMS taxonomical assignation employed the universal marker genes profiler mOTU-v2, which is considered the gold standard. The three standard pipelines for 16S RNA analysis (MALT and MEGAN6, QIIME1, and DADA2) were applied for comparison. Taken together, our results indicate that the G'NOME-based method was optimal in terms of quantity and quality of DNA extracts. 16S rRNA sequence-based identification of abundant bacteria genera showed acceptable congruence with WMS sequencing, with the DADA2 pipeline yielding the highest congruent levels. However, for low abundance genera (<0.5% of the total abundance) two pipelines and/or validation by quantitative polymerase chain reaction (qPCR) or WMS are required. Hence, 16S rRNA sequencing for bacteria identification and quantification in clinical and translational studies should be limited to diagnostic purposes in well-characterized and abundant genera. Additional techniques are warranted for low abundant genera, such as WMS, qPCR, or the use of two bio-informatics pipelines. [ABSTRACT FROM AUTHOR]