16S rRNA Next-generation Sequencing Research Articles

Выбор метода количественной оценки микробиоты кишечника: сравнительный анализ 16S NGS и ПЦР-РВ

Recently, considerable commercialization of services for quantification of gut microbiota aimed to diagnose dysbiosis, the microbial imbalance, is observed. In the context of growing interest to the personalized approaches in medicine and preventive therapy, the diagnosis of dysbiosis is becoming increasingly important. The results of such screening are used to adjust guidelines on correction of the diet, lifestyle modification, or, where necessary, drug therapy prescription. Such assessment requires a reliable and accurate method for evaluation of microbiota, since validity of further recommendations and therapeutic interventions depends on the quality of the data obtained. The paper reports the main aspects of the two approaches used for microbiota quantification: 16S rRNA next-generation sequencing (16S NGS) and real-time PCR (qPCR). The strengths (from our perspective) and weaknesses of the approaches are also provided.

Novel insights on microbiome dynamics during a gill disease outbreak in farmed rainbow trout (Oncorhynchus mykiss)

The generic term “Gill disease” refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors’ knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill’s microbiome with other possible cofactors and establish strong prevention guidelines.

Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer.

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.

Aging-related Alterations in Gastric Microbiota: A Potential Contributor to Gastrointestinal Diseases

Abstract BACKGROUND: Helicobacter pylori is believed to cause several gastrointestinal (GI) diseases. The aging process captures many facets of biological variation of the human body, which leads to functional decline and increased incidence of infection in the gastric of elderly people. However, till date, most of the larger studies have focused on adult populations. The aim of this study is to test the hypothesis that microbial dysbiosis with the progression of age could lead to GI diseases. METHODS: This study involved 48 patients: 13 were 18–35 years old (Group 1), 13 were 36–50 years old (Group 2), 14 were 51–65 years old (Group 3), and 10 were 66–90 years old (Group 4). Gastric microbiome composition was analyzed using 16S rRNA next-generation sequencing, and the resulting operational taxonomic units were compared to study the microbial alterations among the age groups. RESULTS: Firmicutes/Bacteroidetes ratio (F/B ratio) was found to sharply increase from Group 1 to Group 2 and then just as sharply decreased from Group 3 to Group 4. At the phylum level, Patescibacteria was positively correlated and Proteobacteria, Campylobacterota, Actinobacteriota, Firmicutes, Bacteroidetes, and Fusobacteria were negatively correlated with the age. At the genus level, Ralstonia and TM7x were positively correlated and Alloprevotella, Staphylococcus, Veillonella, Rothia, and Prevotella showed negative correlation with the age of the subjects. The correlation of other genera with Ralstonia was studied. CONCLUSION: Our results highlighted that microbial dysbiosis caused by aging can be a factor for causing GI diseases. Further studies are recommended.

Alteration in the gastric microbiota of Erosive Reflux Disease and Non-erosive Reflux Disease patients: An indicator for disease progression

Introduction and Aim: Erosive reflux disease (ERD) and Non-erosive reflux disease (NERD) are common gastrointestinal (GI) diseases in primary healthcare facilities that might result in alterations in gastric microflora. The aim was to characterize the altered composition of the gastric microflora in both groups and their effects on normal metabolic pathways. Materials and Methods: A total of 27 individuals, consisting of 18 patients of ERD, 4 patients of NERD, and 5 controls based on the questionnaire data and clinical diagnosis. The gastric microbiome was sequenced using 16S rRNA next-generation sequencing. Gastric microbial diversity and compositions were analyzed using MicrobiomeAnalyst. Functional analysis was performed using PICRUST. Results: Dysbiosis was observed in both ERD and NERD groups when compared to the control. Alpha diversity was found to be significant at the ACE index, at the order (p = 0.0239) and class (p = 0.019) taxonomic levels. The gastric microbiome composition at the genus level of the study groups represented a significant decrease of Gram-positive bacterial genera such as Streptococcus, Corynebacterium, and Granulicatella along with an increase of Gram-negative genera such as Helicobacter and Veillonella. Significant alterations in the metabolic pathways due to this dysbiosis were also predicted for both groups. Conclusion: The alterations in the gastric microflora are caused by both ERD and NERD, these alterations are further associated with a shift in microbial consortia towards the abundance of gram-negative bacterial genera that might lead to the progression of GI diseases to a chronic state.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution

Introduction: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the “acquired pellicle engineering” concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. Objective: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. Methods: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed. Results: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS. Conclusion: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.

16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.

Background and Objectives: Ascites, often associated with liver cirrhosis, poses diagnostic challenges, particularly in detecting bacterial infections. Traditional methods have limitations, prompting the exploration of advanced techniques such as 16S rDNA next-generation sequencing (NGS) for improved diagnostics in such low-biomass fluids. The aim of this study was to investigate whether the NGS method enhances detection sensitivity compared to a conventional ascites culture. Additionally, we aimed to explore the presence of a microbiome in the abdominal cavity and determine whether it has a sterile condition. Materials and Methods: Ten patients with clinically suspected spontaneous bacterial peritonitis (SBP) were included in this study. A traditional ascites culture was performed, and all ascites samples were subjected to 16S ribosomal RNA gene amplification and sequencing. 16S rRNA gene sequencing results were interpreted by comparing them to positive and negative controls for each sample. Results: Differential centrifugation was applied to all ascites samples, resulting in very small or no bacterial pellets being harvested. The examination of the 16S amplicon sequencing libraries indicated that the target amplicon products were either minimally visible or exhibited lower intensity than their corresponding negative controls. Contaminants present in the reagents were also identified in the ascites samples. Sequence analysis of the 16S rRNA gene of all samples showed microbial compositions that were akin to those found in the negative controls, without any bacteria isolated that were unique to the samples. Conclusions: The peritoneal cavity and ascites exhibit low bacterial biomass even in the presence of SBP, resulting in a very low positivity rate in 16S rRNA gene sequencing. Hence, the 16S RNA sequencing method does little to enhance the rate of positive samples compared to traditional culture methods, including in SBP cases.

Exploring the Relationship between Gut Microbiome Composition and Blood Indole-3-acetic Acid in Hemodialysis Patients.

Indole-3-acetic acid (IAA), a protein-bound uremic toxin resulting from gut microbiota-driven tryptophan metabolism, increases in hemodialysis (HD) patients. IAA may induce endothelial dysfunction, inflammation, and oxidative stress, elevating cardiovascular and cognitive risk in HD patients. However, research on the microbiome-IAA association is limited. This study aimed to explore the gut microbiome's relationship with plasma IAA levels in 72 chronic HD patients aged over 18 (August 2016-January 2017). IAA levels were measured using tandem mass spectrometry, and gut microbiome analysis utilized 16s rRNA next-generation sequencing. Linear discriminative analysis effect size and random forest analysis distinguished microbial species linked to IAA levels. Patients with higher IAA levels had reduced microbial diversity. Six microbial species significantly associated with IAA levels were identified; Bacteroides clarus, Bacteroides coprocola, Bacteroides massiliensi, and Alisteps shahii were enriched in low-IAA individuals, while Bacteroides thetaiotaomicron and Fusobacterium varium were enriched in high-IAA individuals. This study sheds light on specific gut microbiota species influencing IAA levels, enhancing our understanding of the intricate interactions between the gut microbiota and IAA metabolism.

Composition and diversity of meibum microbiota in meibomian gland dysfunction and the correlation with tear cytokine levels.

Meibomian gland dysfunction (MGD) leads to meibum stasis and pathogenic bacteria proliferation. We determined meibum microbiota via next-generation sequencing (NGS) and examined their association with tear cytokine levels in patients with MGD. This cross-sectional study included 44 moderate-severe patients with MGD and 44 healthy controls (HCs). All volunteers underwent assessment with the ocular surface disease index questionnaire, Schirmer without anesthesia, tear break-up time, Oxford grading of ocular surface staining, and lid and meibum features. Sample collection included tears for cytokine detection and meibum for 16S rRNA NGS. No significant differences were observed in the α-diversity of patients with MGD compared with that in HCs. However, Simpson's index showed significantly decreased α-diversity for severe MGD than for moderate MGD (p = 0.045). Principal coordinate analysis showed no significant differences in β-diversity in meibum samples from patients with MGD and HCs. Patients with MGD had significantly higher relative abundances of Bacteroides (8.54% vs. 6.00%, p = 0.015) and Novosphingobium (0.14% vs. 0.004%, p = 0.012) than the HCs. Significantly higher interleukin (IL)-17A was detected in the MGD group than in the HC group, particularly for severe MGD (p = 0.008). Although Bacteroides was more abundant in the MGD group than in the HC group, it was not positively correlated with IL-17A. The relationship between core meibum microbiota and tear cytokine levels remains unclear. However, increased Bacteroides and Novosphingobium abundance may be critical in MGD pathophysiology.

Seasonal Effects on the Fecal Microbial Composition of Wild Greater Thick-Tailed Galagos (Otolemur crassicaudatus)

AbstractBacterial communities present in the host digestive tract are important for the breakdown and absorption of nutrients required by the host. Changes in diet and the environment are major factors affecting the composition and diversity of the fecal microbiome. In addition to changes in ambient temperature and rainfall, primates living in seasonal temperate environments also need to adapt to seasonal changes in food resource quantity and quality. However, there is a lack of information about the fecal microbiome in African strepsirrhines relative to other primate taxa. We examined the effects of seasonal dietary and environmental changes on fecal microbial alpha diversity and composition in wild greater thick-tailed galagos (Otolemur crassicaudatus) at Lajuma Research Centre, South Africa. We collected fecal samples and assessed food availability and weather in summer and winter across 1 year and used 16S rRNA next-generation sequencing to characterise the fecal microbiome of 49 animals. We found significant increases in rainfall, ambient temperature, and food availability in summer compared with winter. However, we found no significant changes in body mass or in the overall diversity of bacterial species present in fecal samples between the two seasons. We found significant decreases in the abundance of certain bacterial families in winter, suggesting a change in diet. Our findings suggest that greater thick-tailed galagos can find food resources to maintain their body mass throughout the year. Our insights into the seasonal fecal microbiome of greater thick-tailed galagos add to the growing knowledge and understanding of fecal microbiomes in primates and how they help primates cope with changes to their environments.

The effect of ultrasonic and multisonic irrigation on root canal microbial communities: An ex vivo study.

To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an exvivo model that used extracted molars with a history of pulp necrosis. Twenty-three mandibular molars were prepared exvivo for collection of superficial (surface control), pre-treatment and post-treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl (n = 11) and GW group (n = 12). All samples were processed using quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA next-generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t-test (α = .05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal-Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples. Quantitative real-time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively (p = .048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower (p < .001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre-treatment samples (R = .090, p = .070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post-treatment samples (R = -.05, p = .829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation (p = .007 and p = .002 respectively). Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this exvivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study.

Analysis of the bacterial community in female Rhipicephalus microplus ticks from selected provinces in Luzon, Philippines, using next-generation sequencing.

Analysis of the tick microbiome can help understand tick-symbiont interactions and identify undiscovered pathogens, which may aid in implementing control of ticks and tick-borne diseases. The tropical cattle tick Rhipicephalus microplus is a widespread ectoparasite of cattle in the Philippines, negatively affecting animal productivity and health. This study characterized the bacterial community of R. microplus from Luzon, Philippines, through next-generation sequencing of 16s rRNA. DNA was extracted from 45 partially engorged female ticks from nine provinces. The DNA samples were pooled per province and then sequenced and analyzed using an open-source bioinformatics platform. In total, 667 operational taxonomic units (OTUs) were identified. The ticks in all nine provinces were found to have Coxiella, Corynebacterium, Staphylococcus, and Acinetobacter. Basic local alignment search tool (BLAST) analysis revealed the presence of known pathogens of cattle, such as Bartonella, Ehrlichia minasensis, and Dermatophilus congolensis. The tick samples from Laguna, Quezon, and Batangas had the most diverse bacterial species, whereas the tick samples from Ilocos Norte had the lowest diversity. Similarities in the composition of the bacterial community in ticks from provinces near each other were also observed. This is the first study on metagenomic analysis of cattle ticks in the Philippines, providing new insights that may be useful for controlling ticks and tick-borne diseases.

Evaluation of Microbial Dynamics of Kombucha Consortia upon Continuous Backslopping in Coffee and Orange Juice.

The kombucha market is diverse, and competitors constantly test new components and flavours to satisfy customers' expectations. Replacing the original brewing base, adding flavours, or using "backslopping" influence the composition of the symbiotic starter culture of bacteria and yeast (SCOBY). Yet, deep characterisation of microbial and chemical changes in kombucha consortia in coffee and orange juice during backslopping has not been implemented. This study aimed to develop new kombucha beverages in less-conventional matrices and characterise their microbiota. We studied the chemical properties and microbial growth dynamics of lactic-acid-bacteria-tailored (LAB-tailored) kombucha culture by 16S rRNA next-generation sequencing in coffee and orange juice during a backslopping process that spanned five cycles, each lasting two to four days. The backslopping changed the culture composition and accelerated the fermentation. This study gives an overview of the pros and cons of backslopping technology for the production of kombucha-based beverages. Based on research conducted using two different media, this work provides valuable information regarding the aspects to consider when using the backslopping method to produce novel kombucha drinks, as well as identifying the main drawbacks that need to be addressed.

The Impact of HPP-Assisted Biocontrol Approach on the Bacterial Communities' Dynamics and Quality Parameters of a Fermented Meat Sausage Model.

Traditional foods are increasingly valued by consumers, whose attention and purchase willingness are highly influenced by other claims such as 'natural', 'sustainable', and 'clean label'. The purpose of the present study was to evaluate the impact of a novel non-thermal food processing method (i.e., HPP-assisted biocontrol combining mild high hydrostatic pressure, listeriophage Listex, and pediocin PA-1 producing Pediococcus acidilactici) on the succession of bacterial communities and quality of a fermented sausage model. A comparative analysis of instrumental color, texture, and lipid peroxidation revealed no significant differences (p > 0.05) in these quality parameters between non- and minimally processed fermented sausages throughout 60-day refrigerated storage (4 °C). The microbiota dynamics of biotreated and untreated fermented sausages were assessed by 16S rRNA next-generation sequencing, and the alpha and beta diversity analyses revealed no dissimilarity in the structure and composition of the bacterial communities over the analyzed period. The innovative multi-hurdle technology proposed herein holds valuable potential for the manufacture of traditional fermented sausages while preserving their unique intrinsic characteristics.

Listeria monocytogenes infection in pregnant macaques alters the maternal gut microbiome†.

The bacterium Listeria monocytogenes (Lm) is associated with adverse pregnancy outcomes. Infection occurs through consumption of contaminated food that is disseminated to the maternal-fetal interface. The influence on the gastrointestinal microbiome during Lm infection remains unexplored in pregnancy. The objective of this study was to determine the impact of listeriosis on the gut microbiota of pregnant macaques. A non-human primate model of listeriosis in pregnancy has been previously described. Both pregnant and non-pregnant cynomolgus macaques were inoculated with Lm and bacteremia and fecal shedding were monitored for 14days. Non-pregnant animal tissues were collected at necropsy to determine bacterial burden, and fecal samples from both pregnant and non-pregnant animals were evaluated by 16S rRNA next-generation sequencing. Unlike pregnant macaques, non-pregnant macaques did not exhibit bacteremia, fecal shedding, or tissue colonization by Lm. Dispersion of Lm during pregnancy was associated with a significant decrease in alpha diversity of the host gut microbiome, compared to non-pregnant counterparts. The combined effects of pregnancy and listeriosis were associated with a significant loss in microbial richness, although there were increases in some genera and decreases in others. Although pregnancy alone is not associated with gut microbiome disruption, we observed dysbiosis with listeriosis during pregnancy. The macaque model may provide an understanding of the roles that pregnancy and the gut microbiota play in the ability of Lm to establish intestinal infection and disseminate throughout the host, thereby contributing to adverse pregnancy outcomes and risk to the developing fetus.

Pre- and post-LEEP: analysis of the female urogenital tract microenvironment and its association with sexual dysfunction.

The loop electrosurgical excision procedure (LEEP) to treat cervical dysplasia (CD) is known to alter the cervical microbiota, the community of bacteria that play a central role in female genital health. Perturbations to the microbiota of the female urogenital tract (FUT), including the urethra, vagina, and cervix, have been linked with symptoms of sexual dysfunction (SD), though correlations among LEEP, the microenvironment, and SD have not yet been described. To characterize the FUT microbiota before and after LEEP and investigate possible associations with SD. Females undergoing LEEP for CD were recruited to participate in the study. Urinary samples and vaginal and cervical swabs were collected immediately before and 3 months after treatment. Bacterial communities were characterized by 16S rRNA next-generation sequencing. Self-report surveys assessing demographics, medical history, and sexual function were completed at the same intervals. Microbiota taxonomy and Female Sexual Function Index (FSFI) scores. Alpha diversity revealed a significant decrease in species richness in the FUT microbiota post-LEEP. Beta diversity demonstrated significant differences among the cervical, urinary, and vaginal microenvironments pre- and post-LEEP. Lactobacillus spp were the dominant microbial genus in the cervical microenvironment pre- and post-LEEP. Although the vaginal and urinary microenvironments were characterized by Prevotella pre-LEEP, they were colonized by Lactobacillus post-LEEP. Following LEEP, some participants experienced a significant increase in proinflammatory bacteria, including the genera Gardnerella, Megasphaera, Sneathia, Parvimonas, and Peptostreptococcus. Others experienced significant decreases in inflammatory and protective bacteria post-LEEP, including Butyricicoccus, Terriporobacter, Intestinimonas, and Negativibacillus. Overall there were no significant changes in pre- and post-LEEP FSFI scores. However, post-LEEP FSFI scores were seemingly associated with changes in inflammatory bacteria in some participants. There is an overall reduction in FUT microbiota dysbiosis post-LEEP. However, we show variability as some participants experienced persistent dysbiosis of FUT microbiota and elevated FSFI scores, suggesting that therapies to treat dysbiosis of FUT microbiota may reduce FSFI scores, thereby improving SD symptoms. We demonstrate novel associations among urogenital sites, microbiota changes, LEEP, and SD. The small sample size and inability of species classification are limitations. Diverse inflammatory microbiota characterizes CD in the FUT, and LEEP mostly returns microenvironments to a healthy state. However, some participants have persistent inflammatory bacteria post-LEEP, suggesting a non-uniform healing response. This study provides an impetus for future longitudinal studies to monitor and restore FUT microenvironments post-LEEP, aimed at mitigating postoperative SD symptoms.

AquaSens: exploring the use of 16S rRNA next-generation sequencing to determine bacterial composition of various water matrices

Access to clean water, one of the United Nation’s Sustainable Development Goals, is challenged by an increase in the presence of emerging microbial and other contaminants due to urbanization, among other factors. Traditionally, the presence of indicator microorganisms is determined using culturing methods. However, these classical methods cannot be used to determine the identities of ‘unknown’ bacteria and is limited to isolating the culturable state of microorganisms. Thus with culturing, the identities of many bacteria, particularly novel or non-culturable, may remain unknown. The use of a DNA-based method, 16S rRNA next-generation sequencing (NGS), can assist with determining the identities of bacterial populations in a water sample. The objective of this 16S rRNA NGS study was to investigate the bacterial community composition and diversity in a range of water sources. Water samples comprising of potable, surface, ground, marine, aquaculture, rain, wetland and swimming bath water matrices were subjected to 16S rRNA NGS using the Illumina 16S rRNA Metagenomics analysis pipeline. Operational taxonomic units were analysed and the identities of bacterial genera determined. In this study, genera of Acinetobacter, Mycobacterium, Pseudomonas, Legionella, Burkholderia, Yersinia, Staphylococcus and Vibrio were spread across the water matrices. Alpha (within sample) and beta (between samples) diversities for each bacterial community within the tested samples were also determined.

Impact of Pipe Material and Temperature on Drinking Water Microbiome and Prevalence of Legionella, Mycobacterium, and Pseudomonas Species

In drinking water distribution systems (DWDSs), pipe material and water temperature are some of the critical factors affecting the microbial flora of water. Six model DWDSs consisting of three pipe materials (galvanized steel, copper, and PEX) were constructed. The temperature in three systems was maintained at 22 °C and the other 3 at 32 °C to study microbial and elemental contaminants in a 6-week survey using 16S rRNA next-generation sequencing (NGS) and inductively coupled plasma-optical emission spectrometry (ICP-OES). Pipe material and temperature were preferentially linked with the composition of trace elements and the microbiome of the DWDSs, respectively. Proteobacteria was the most dominant phylum across all water samples ranging from 60.9% to 91.1%. Species richness (alpha diversity) ranking was PEX < steel ≤ copper system and elevated temperature resulted in decreased alpha diversity. Legionellaceae were omni-prevalent, while Mycobacteriaceae were more prevalent at 32 °C (100% vs. 58.6%) and Pseudomonadaceae at 22 °C (53.3% vs. 62.9%). Heterogeneity between communities was disproportionately driven by the pipe material and water temperature. The elevated temperature resulted in well-defined microbial clusters (high pseudo-F index) in all systems, with the highest impact in PEX (10.928) followed by copper (9.696) and steel (5.448). Legionellaceae and Mycobacteriaceae are preferentially prevalent in warmer waters. The results suggest that the water temperature has a higher magnitude of impact on the microbiome than the pipe material.

Influence of Lactobacillus reuteri, Bifidobacterium animalis subsp. lactis, and prebiotic inulin on dysbiotic dental biofilm composition ex vivo.

Probiotic bacterial supplementation has shown promising results in the treatment of periodontitis and the maintenance of periodontal health. The purpose of this investigation was to evaluate the influence of Lactobacillus reuteri or Bifidobacterium animalis subsp. lactis supplementation with and without prebiotic inulin on biofilm composition using an ex vivo biofilm model. Subgingival plaque specimens from three periodontitis-affected human donors were used to grow biofilms on hydroxyapatite disks in media supplemented with varying combinations of prebiotic inulin, Lactobacillus reuteri, and Bifidobacterium animalis subsp. lactis. Relative abundances of bacterial genera present in mature biofilms were evaluated using 16S rRNA next-generation sequencing. Diversity metrics of microbial communities were evaluated using a next-generation microbiome bioinformatics platform. Inulin supplementation produced statistically significant dose-dependent increases in relative abundances of Lactobacillus and Bifidobacterium species (p<0.001) with concomitant decreases in relative abundances of Streptococcus, Veillonella, Fusobacterium, Parvimonas, and Prevotella species (p<0.001). Inoculation with L. reuteri or B. animalis subsp. lactis increased the relative abundance of only the supplemented probiotic genera (p<0.05). Supplemental inulin led to a statistically significant decrease in biofilm alpha diversity (p<0.001). The described ex vivo model appears suitable for investigating the effects of probiotic bacteria, prebiotic oligosaccharides, and combinations thereof on biofilm composition and complexity. Within the limitations imposed by this model, results from the present study underscore the potential for prebiotic inulin to modify biofilm composition favorably. Additional research further elucidating biologic rationale and controlled clinical research defining therapeutic benefits is warranted.

Composition of the gut microbiota in patients with inflammatory bowel disease in Saudi Arabia: A pilot study.

The results of this study provide an overview of the variations in microbiota diversity present in Saudi IBD patients compared to healthy controls. The key finding was three negative bacterial biomarkers, Paraprevotellaceae, the Muribaculaceae families of Bacteroidetes phylum, and the Leuconostocaceae family of Firmicutes phylum, which had a higher relative abundance in healthy individuals compared to IBD patients. It was also found that primary microbiota signatures at certain genera and species levels, including Prevotella copri, Bifidobacterium adolescentis, Ruminococcus callidus, Coprococcus sp., Ruminococcus gnavus, Dorea formicigenerans, Leuconostoc, Dialister, Catenibacterium, Eubacterium biforme, and Lactobacillus mucosae, were absent in almost all IBD patients, while Veillonella dispar was absent in all healthy individuals. After obtaining an informed consent, fecal samples were collected from 11 participants with IBD (patients) and 10 healthy individuals (controls). The bacterial components of the microbial population were identified by next-generation sequencing of partial 16S rRNA. Statistically significant dissimilarities were observed between samples for all metrics. Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition attributed to a complex interaction between imbalances in the gut microbiome, environmental conditions, and a deregulated immune response. The aim of the study was to investigate the composition of the gut microbiome of Saudi patients with IBD.