Microbial influencers and cotton leaf curl disease (CLCuD) susceptibility: a network perspective.

Biotic stresses, such as plant viruses, e.g., cotton leaf curl virus (CLCuV), can alter root-associated and leaf-associated microbial diversities in plants. There are complex ecological dynamics at play, with each microbe contributing to a multitude of biotic and abiotic interactions, thus deciding the stability of the plant's ecosystem in response to the disease. Deciphering these networks of interactions is a challenging task. The inferential research in microbiome is also at a nascent stage, often constrained by the underlying analytical assumptions and the limitations with respect to the depth of sequencing. There is also no real consensus on network-wide statistics to identify the influential microbial players in a network. Guided by the latest developments in network science, including recently published metrics such as Integrated View of Influence (IVI) and some other centrality measures, this study provides an exposé of the most influential nodes in the rhizospheric and phyllospheric microbial networks of the cotton leaf curl disease (CLCuD) susceptible, partially tolerant, and resistant cotton varieties. It is evident from our results that the CLCuD-resistant Gossypium arboreum possesses an equal share of keystone species, which helps it to withstand ecological pressures. In the resistant variety, the phyllosphere harbors the most influential nodes, whereas in the susceptible variety, they are present in the rhizosphere. Based on hubness score, spreading score, and IVI, the top 10 occurring keystone species in the FDH-228 (resistant) variety include Actinokineospora, Cohnella, Thermobacillus, Clostridium, Desulfofarcimen, and MDD-D21. Elusimicrobia, Clostridium-sensu-stricto_12, Candidatus woesebacteria, and Dyella were identified as the most influential nodes in the PFV-1 (partially tolerant) variety. In the PFV-2 (susceptible) variety, the keystone species were identified as Georginia, Nesterenkonia, Elusimicrobia MVP-88, Acetivibrio, Tepedisphaerales, Chelatococcus, Nitrosospira, and RCP2-54. This concept deciphers the diseased and healthy plant's response to viral disease, which may be microbially mediated.

CViewer: a Java-based statistical framework for integration of shotgun metagenomics with other omics datasets.

BACKGROUND: Shotgun metagenomics for microbial community survey recovers enormous amount of information for microbial genomes that include their abundances, taxonomic, and phylogenetic information, as well as their genomic makeup, the latter of which then helps retrieve their function based on annotated gene products, mRNA, protein, and metabolites. Within the context of a specific hypothesis, additional modalities are often included, to give host-microbiome interaction. For example, in human-associated microbiome projects, it has become increasingly common to include host immunology through flow cytometry. Whilst there are plenty of software approaches available, some that utilize marker-based and assembly-based approaches, for downstream statistical analyses, there is still a dearth of statistical tools that help consolidate all such information in a single platform. By virtue of stringent computational requirements, the statistical workflow is often passive with limited visual exploration. RESULTS: In this study, we have developed a Java-based statistical framework ( https://github.com/KociOrges/cviewer ) to explore shotgun metagenomics data, which integrates seamlessly with conventional pipelines and offers exploratory as well as hypothesis-driven analyses. The end product is a highly interactive toolkit with a multiple document interface, which makes it easier for a person without specialized knowledge to perform analysis of multiomics datasets and unravel biologically relevant patterns. We have designed algorithms based on frequently used numerical ecology and machine learning principles, with value-driven from integrated omics tools which not only find correlations amongst different datasets but also provide discrimination based on case-control relationships. CONCLUSIONS: CViewer was used to analyse two distinct metagenomic datasets with varying complexities. These include a dietary intervention study to understand Crohn's disease changes during a dietary treatment to include remission, as well as a gut microbiome profile for an obesity dataset comparing subjects who suffer from obesity of different aetiologies and against controls who were lean. Complete analyses of both studies in CViewer then provide very powerful mechanistic insights that corroborate with the published literature and demonstrate its full potential. Video Abstract.

Network analysis of gut microbial communities reveal key genera for a multiple sclerosis cohort with Mycobacterium avium subspecies paratuberculosis infection.

BACKGROUND: In gut ecosystems, there is a complex interplay of biotic and abiotic interactions that decide the overall fitness of an individual. Divulging the microbe-microbe and microbe-host interactions may lead to better strategies in disease management, as microbes rarely act in isolation. Network inference for microbial communities is often a challenging task limited by both analytical assumptions as well as experimental approaches. Even after the network topologies are obtained, identification of important nodes within the context of underlying disease aetiology remains a convoluted task. We therefore present a network perspective on complex interactions in gut microbial profiles of individuals who have multiple sclerosis with and without Mycobacterium avium subspecies paratuberculosis (MAP) infection. Our exposé is guided by recent advancements in network-wide statistical measures that identify the keystone nodes. We have utilised several centrality measures, including a recently published metric, Integrated View of Influence (IVI), that is robust against biases. RESULTS: The ecological networks were generated on microbial abundance data (n = 69 samples) utilising 16 S rRNA amplification. Using SPIEC-EASI, a sparse inverse covariance estimation approach, we have obtained networks separately for MAP positive (+), MAP negative (-) and healthy controls (as a baseline). Using IVI metric, we identified top 20 keystone nodes and regressed them against covariates of interest using a generalised linear latent variable model. Our analyses suggest Eisenbergiella to be of pivotal importance in MS irrespective of MAP infection. For MAP + cohort, Pyarmidobacter, and Peptoclostridium were predominately the most influential genera, also hinting at an infection model similar to those observed in Inflammatory Bowel Diseases (IBDs). In MAP- cohort, on the other hand, Coprostanoligenes group was the most influential genera that reduces cholesterol and supports the intestinal barrier. CONCLUSIONS: The identification of keystone nodes, their co-occurrences, and associations with the exposome (meta data) advances our understanding of biological interactions through which MAP infection shapes the microbiome in MS individuals, suggesting the link to the inflammatory process of IBDs. The associations presented in this study may lead to development of improved diagnostics and effective vaccines for the management of the disease.

Faecal volatile organic compounds differ according to inflammatory bowel disease sub-type, severity, and response to treatment in paediatric patients.

BACKGROUND: Faecal volatile organic compounds (VOCs) differ with disease sub-type and activity in adults with established inflammatory bowel disease (IBD) taking therapy. OBJECTIVE: To describe patterns of faecal VOCs in children newly presented with IBD according to disease sub-type, severity, and response to treatment. METHODS: Children presenting with suspected IBD were recruited from three UK hospitals. Children in whom IBD was diagnosed were matched with a non-IBD child for age, sex, and recruitment site. Faecal VOCs were characterised by gas chromatography-mass spectrometry at presentation and 3 months later in children with IBD. RESULTS: In 132 case/control pairs, median (inter-quartile range) age in IBD was 13.3 years (10.2-14.7) and 38.6% were female. Compared with controls, the mean abundance of 27/62 (43.6%) faecal VOCs was statistically significantly decreased in Crohn's disease (CD), ulcerative colitis (UC) or both especially amongst ketones/diketones, fatty acids, and alcohols (p < 0.05). Short-chain, medium chain, and branched chain fatty acids were markedly reduced in severe colitis (p < 0.05). Despite clinical improvement in many children with IBD, the number and abundance of almost all VOCs did not increase following treatment, suggesting persistent dysbiosis. Oct-1-en-3-ol was increased in CD (p = 0.001) and UC (p = 0.012) compared with controls and decreased following treatment in UC (p = 0.01). In CD, propan-1-ol was significantly greater than controls (p < 0.001) and extensive colitis (p = 0.001) and fell with treatment (p = 0.05). Phenol was significantly greater in CD (p < 0.001) and fell with treatment in both CD (p = 0.02) and UC (p = 0.01). CONCLUSION: Characterisation of faecal VOCs in an inception cohort of children with IBD reveals patterns associated with diagnosis, disease activity, and extent. Further work should investigate the relationship between VOCs and the microbiome in IBD and their role in diagnosis and disease monitoring.

Dataset of 569 metagenome-assembled genomes from the caeca of multiple chicken breeds from commercial and backyard farming setups of Pakistan.

This article focuses the recovery of prokaryotic organisms including bacteria and archaea from 9 different groups of chicken raised in different farm setups in Pakistan. The groups comprise of three different breeds (Broilers, White Layers, and Black Australorp) of chicken raised in different farming setups that include antibiotic-free control, commercial (open and controlled shed), and backyard farms. We have recovered 569 Metagenomics-Assembled Genomes (MAGs) with a completeness of ≥50 % and contamination of ≤10 %. For each MAG, functional annotations were obtained that include KEGG modules, carbohydrate active enzymes (CAZymes), peptidases, geochemical cycles, antibiotic resistance genes, stress genes, and virulence genes. Furthermore, two different sets of Single Copy Genes (SCGs) were used to construct the phylogenetic trees. Based on the reconstructed phylogeny, phylogenetic gain of each MAG is calculated to give an account of novelty.

Low-abundant but highly transcriptionally active uncharacterised Nitrosomonas drive ammonia-oxidation in the Brouage mudflat, France.

Exploring differences in nitrification within adjacent sedimentary structures of ridges and runnels on the Brouage mudflat, France, we quantified Potential Nitrification Rates (PNR) alongside amoA genes and transcripts. PNR was lower in ridges (≈1.7 fold-lower) than runnels, despite higher (≈1.8 fold-higher) ammonia-oxidizing bacteria (AOB) abundance. However, AOB were more transcriptionally active in runnels (≈1.9 fold-higher). Sequencing of amoA genes and transcripts revealed starkly contrasting profiles with transcripts from ridges and runnels dominated (≈91 % in ridges and ≈98 % in runnels) by low abundant (≈4.6 % of the DNA community in runnels and ≈0.8 % in ridges) but highly active phylotypes. The higher PNR in runnels was explained by higher abundance of this group, an uncharacterised Nitrosomonas sp. cluster. This cluster is phylogenetically similar to other active ammonia-oxidizers with worldwide distribution in coastal environments indicating its potential, but previously overlooked, contribution to ammonia oxidation globally. In contrast DNA profiles were dominated by highly abundant but low-activity clusters phylogenetically distinct from known Nitrosomonas (Nm) and Nitrosospira (Ns). This cluster is also globally distributed in coastal sediments, primarily detected as DNA, and often classified as Nitrosospira or Nitrosomonas. We therefore propose to classify this cluster as Ns/Nm. Our work indicates that low abundant but highly active AOB could be responsible for the nitrification globally, while the abundant AOB Ns/Nm may not be transcriptionally active, and as such account for the lack of correlation between rate processes and gene abundances often reported in the literature. It also raises the question as to what this seemingly inactive group is doing?

Dataset of 130 metagenome-assembled genomes of healthy and diseased broiler chicken caeca from Pakistan.

This article presents metagenomic-assembled genomes (MAGs) of prokaryotic organisms originating from chicken caeca. The samples originate from broiler chickens, one group was infected with Newcastle Disease Virus (NDV) and one uninfected control group. There were four birds per group. Both groups were raised on commercially available antibiotic free feed under a semi-controlled setup. The binning step of the samples identified 130 MAGs with ≥50 % completion, and ≤10 % contamination. The data presented includes sequences in FASTA format, tables of functional annotation of genes, and data from two different approaches for phylogenetic tree construction using these MAGs. Major geochemical cycles at community level including carbon, sulfur, and nitrogen cycles are also presented.

Gut metabolome and microbiota signatures predict response to treatment with exclusive enteral nutrition in a prospective study in children with active Crohn's disease.

BACKGROUND: Predicting response to exclusive enteral nutrition (EEN) in active Crohn's disease (CD) could lead to therapy personalization and pretreatment optimization. OBJECTIVES: This study aimed to explore the ability of pretreatment parameters to predict fecal calprotectin (FCal) levels at EEN completion in a prospective study in children with CD. METHODS: In children with active CD, clinical parameters, dietary intake, cytokines, inflammation-related blood proteomics, and diet-related metabolites, metabolomics and microbiota in feces, were measured before initiation of 8 wk of EEN. Prediction of FCal levels at EEN completion was performed using machine learning. Data are presented with medians (IQR). RESULTS: Of 37 patients recruited, 15 responded (FCal < 250 µg/g) to EEN (responders) and 22 did not (nonresponders). Clinical and immunological parameters were not associated with response to EEN. Responders had lesser (µmol/g) butyrate [responders: 13.2 (8.63-18.4) compared with nonresponders: 22.3 (12.0-32.0); P = 0.03], acetate [responders: 49.9 (46.4-68.4) compared with nonresponders: 70.4 (57.0-95.5); P = 0.027], phenylacetate [responders: 0.175 (0.013-0.611) compared with nonresponders: 0.943 (0.438-1.35); P = 0.021], and a higher microbiota richness [315 (269-347) compared with nonresponders: 243 (205-297); P = 0.015] in feces than nonresponders. Responders consumed (portions/1000 kcal/d) more confectionery products [responders: 0.55 (0.38-0.72) compared with nonresponders: 0.19 (0.01-0.38); P = 0.045]. A multicomponent model using fecal parameters, dietary data, and clinical and immunological parameters predicted response to EEN with 78% accuracy (sensitivity: 80%; specificity: 77%; positive predictive value: 71%; negative predictive value: 85%). Higher taxon abundance from Ruminococcaceae, Lachnospiraceae, and Bacteroides and phenylacetate, butyrate, and acetate were the most influential variables in predicting lack of response to EEN. CONCLUSIONS: We identify microbial signals and diet-related metabolites in feces, which could comprise targets for pretreatment optimization and personalized nutritional therapy in pediatric CD.

Gut microbial ecology and exposome of a healthy Pakistani cohort.

BACKGROUND: Pakistan is a multi-ethnic society where there is a disparity between dietary habits, genetic composition, and environmental exposures. The microbial ecology of healthy Pakistani gut in the context of anthropometric, sociodemographic, and dietary patterns holds interest by virtue of it being one of the most populous countries, and also being a Lower Middle Income Country (LMIC). METHODS: 16S rRNA profiling of healthy gut microbiome of normo-weight healthy Pakistani individuals from different regions of residence is performed with additional meta-data collected through filled questionnaires. The current health status is then linked to dietary patterns through [Formula: see text] test of independence and Generalized Linear Latent Variable Model (GLLVM) where distribution of individual microbes is regressed against all recorded sources of variability. To identify the core microbiome signature, a dynamic approach is used that considers into account species occupancy as well as consistency across assumed grouping of samples including organization by gender and province of residence. Fitting neutral modeling then revealed core microbiome that is selected by the environment. RESULTS: A strong determinant of disparity is by province of residence. It is also established that the male microbiome is better adapted to the local niche than the female microbiome, and that there is microbial taxonomic and functional diversity in different ethnicities, dietary patterns and lifestyle habits. Some microbial genera, such as, Megamonas, Porphyromonas, Haemophilus, Klebsiella and Finegoldia showed significant associations with consumption of pickle, fresh fruits, rice, and cheese. Our analyses suggest current health status being associated with the diet, sleeping patterns, employment status, and the medical history. CONCLUSIONS: This study provides a snapshot of the healthy core Pakistani gut microbiome by focusing on the most populous provinces and ethnic groups residing in predominantly urban areas. The study serves a reference dataset for exploring variations in disease status and designing personalized dietary and lifestyle interventions to promote gut health, particularly in LMICs settings.

Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated with Gossypium arboreum.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum. Using salicylic acid-producing Serratia spp. and Fictibacillus spp. isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

intI1 gene abundance from septic tanks in Thailand using validated intI1 primers.

IMPORTANCE: Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies.

Temporal stability and community assembly mechanisms in healthy broiler cecum.

In recent years, there has been an unprecedented advancement in in situ analytical approaches that contribute to the mechanistic understanding of microbial communities by explicitly incorporating ecology and studying their assembly. In this study, we have analyzed the temporal profiles of the healthy broiler cecal microbiome from day 3 to day 35 to recover the stable and varying components of microbial communities. During this period, the broilers were fed three different diets chronologically, and therefore, we have recovered signature microbial species that dominate during each dietary regime. Since broilers were raised in multiple pens, we have also parameterized these as an environmental condition to explore microbial niches and their overlap. All of these analyses were performed in view of different parameters such as body weight (BW-mean), feed intake (FI), feed conversion ratio (FCR), and age (days) to link them to a subset of microbes that these parameters have a bearing upon. We found that gut microbial communities exhibited strong and statistically significant specificity for several environmental variables. Through regression models, genera that positively/negatively correlate with the bird's age were identified. Some short-chain fatty acids (SCFAs)-producing bacteria, including Izemoplasmatales, Gastranaerophilales, and Roseburia, have a positive correlation with age. Certain pathogens, such as Escherichia-Shigella, Sporomusa, Campylobacter, and Enterococcus, negatively correlated with the bird's age, which indicated a high disease risk in the initial days. Moreover, the majority of pathways involved in amino acid biosynthesis were also positively correlated with the bird's age. Some probiotic genera associated with improved performance included Oscillospirales; UCG-010, Shuttleworthia, Bifidobacterium, and Butyricicoccaceae; UCG-009. In general, predicted antimicrobial resistance genes (piARGs) contributed at a stable level, but there was a slight increase in abundance when the diet was changed. To the best of the authors' knowledge, this is one of the first studies looking at the stability, complexity, and ecology of natural broiler microbiota development in a temporal setting.

A normative microbiome is not restored following kidney transplantation.

Dialysis and kidney transplantation (Ktx) mitigate some of the physiological deficits in chronic kidney disease (CKD), but it remains to be determined if these mitigate microbial dysbiosis and the production of inflammatory microbial metabolites, which contribute significantly to the uraemic phenotype. We have investigated bacterial DNA signatures present in the circulation of CKD patients and those receiving a KTx. Our data are consistent with increasing dysbiosis as CKD progresses, with an accompanying increase in trimethylamine (TMA) producing pathobionts Pseudomonas and Bacillus. Notably, KTx patients displayed a significantly different microbiota compared with CKD5 patients, which surprisingly included further increase in TMA producing Bacillus and loss of salutogenic Lactobacilli. Only two genera (Viellonella and Saccharimonidales) showed significant differences in abundance following KTx that may reflect a reciprocal relationship between TMA producers and utilisers, which supersedes restoration of a normative microbiome. Our metadata analysis confirmed that TMA N-oxide (TMAO) along with one carbon metabolism had significant impact upon both inflammatory burden and the composition of the microbiome. This indicates that these metabolites are key to shaping the uraemic microbiome and might be exploited in the development of dietary intervention strategies to both mitigate the physiological deficits in CKD and enable the restoration of a more salutogenic microbiome.

Process stability in expanded granular sludge bed bioreactors enhances resistance to organic load shocks.

Environmental perturbations such as changes in organic loading rate (OLR) can have deleterious effects on the anaerobic digestion process, leading to VFA accumulation and process failure. However, the operational history of a reactor, such as prior exposure to VFA build up, can impact a reactor's resistance to shock loads. In the present study, the effects of long term (>100 days) bioreactor (un)stability on OLR shock resistance were assessed. Three 4 L EGSB bioreactors were subjected to varying levels of process stability. Operational conditions such as OLR, temperature and pH were maintained stable in R1; R2 was subjected to a series of minor OLR perturbations and R3 was subjected to a series of non-OLR perturbations, including ammonium, temperature, pH and sulfide. The effect of these different operational histories on each reactor's resistance to a sudden 8-fold increase in OLR were assessed by monitoring COD removal efficiency and biogas production. The microbial communities of each reactor were monitored using 16S rRNA gene sequencing to understand the relationship between microbial diversity and reactor stability. It was determined that the stable (un-perturbed) reactor performed best in terms of its resistance to a large OLR shock, despite its lower microbial community diversity.

A prospective study on linking diarrheagenic E. coli with stunted childhood growth in relation to gut microbiome.

Stunted growth is an emerging global challenge affecting children under the age of 5 years in low- and middle-income countries. Despite such a high global prevalence of stunting, the mechanism of pathogenesis and the role of associated gut microbiota is poorly understood. The present study was designed to investigate the association of pathogenic strains of E. coli with the residential gut microbiota of stunted growth children. A total of 64 stool sample were collected from children aged ≤ 5 years, and were processed for isolation and molecular characterization of diarrheagenic E. coli. Selected stool samples (n = 39 including three normal controls) were then analysed for microbial community profiling using 16S ribosomal RNA (rRNA) gene sequencing. Furthermore, associations between changes in the microbiota in the presence of different E. coli strains was explored. Pathotyping of the isolated E. coli (n = 64) has shown that 39.68% belonged to one of the five pathotypes of E. coli whilst the remaining ones were non-typeable. Amongst the different pathotypes, EPEC was found to be the most prevalent (52%; n = 13), followed by EAEC (20%; n = 5), EIEC (12%; n = 3), EHEC (8%; n = 2) and ETEC 2 (8%; n = 2). Phylogrouping analysis has shown that majority of the strains belonged to B2 (28.12%). Microbial diversity is shown to be significant and varied when the samples are organized under the recovered phylogroups. Moreover, based on predictive metabolism, the colonization of these strains were found to be significantly associated with energy utilization pathways such as Denovoprine-2 and glyoxylate-by. Differential analysis has shown that Escherichia-Shigella and Enterococcus were altered for the children with stunted growth.

Microbial electrosynthesis of acetate from CO2 in three-chamber cells with gas diffusion biocathode under moderate saline conditions.

The industrial adoption of microbial electrosynthesis (MES) is hindered by high overpotentials deriving from low electrolyte conductivity and inefficient cell designs. In this study, a mixed microbial consortium originating from an anaerobic digester operated under saline conditions (∼13 g L-1 NaCl) was adapted for acetate production from bicarbonate in galvanostatic (0.25 mA cm-2) H-type cells at 5, 10, 15, or 20 g L-1 NaCl concentration. The acetogenic communities were successfully enriched only at 5 and 10 g L-1 NaCl, revealing an inhibitory threshold of about 6 g L-1 Na+. The enriched planktonic communities were then used as inoculum for 3D printed, three-chamber cells equipped with a gas diffusion biocathode. The cells were fed with CO2 gas and operated galvanostatically (0.25 or 1.00 mA cm-2). The highest production rate of 55.4 g m-2 d-1 (0.89 g L-1 d-1), with 82.4% Coulombic efficiency, was obtained at 5 g L-1 NaCl concentration and 1 mA cm-2 applied current, achieving an average acetate production of 44.7 kg MWh-1. Scanning electron microscopy and 16S rRNA sequencing analysis confirmed the formation of a cathodic biofilm dominated by Acetobacterium sp. Finally, three 3D printed cells were hydraulically connected in series to simulate an MES stack, achieving three-fold production rates than with the single cell at 0.25 mA cm-2. This confirms that three-chamber MES cells are an efficient and scalable technology for CO2 bio-electro recycling to acetate and that moderate saline conditions (5 g L-1 NaCl) can help reduce their power demand while preserving the activity of acetogens.

A Cross-Sectional Study of Potential Antimicrobial Resistance and Ecology in Gastrointestinal and Oral Microbial Communities of Young Normoweight Pakistani Individuals.

Antimicrobial resistance (AMR) is a major global public health concern mainly affecting low- and middle-income countries (LMICs) due to lack of awareness, inadequate healthcare and sanitation infrastructure, and other environmental factors. In this study, we aimed to link microbial assembly and covariates (body mass index, smoking, and use of antibiotics) to gut microbiome structure and correlate the predictive antimicrobial gene prevalence (piARG) using PICRUSt2. We examined the gastrointestinal and oral microbial profiles of healthy adults in Pakistan through 16S rRNA gene sequencing with a focus on different ethnicities, antibiotic usage, drinking water type, smoking, and other demographic measures. We then utilised a suite of innovative statistical tools, driven by numerical ecology and machine learning, to address the above aims. We observed that drinking tap water was the main contributor to increased potential AMR signatures in the Pakistani cohort compared to other factors considered. Microbial niche breadth analysis highlighted an aberrant gut microbial signature of smokers with increased age. Moreover, covariates such as smoking and age impact the human microbial community structure in this Pakistani cohort.

The Effects of Smoking on Human Pharynx Microbiota Composition and Stability.

The oral microbiota is essential to the health of the host, yet little is known about how it responds to disturbances. We examined the oropharyngeal microbiota of 30 individuals over 40 weeks. As the oropharynx is an important gateway to pathogens, and as smoking is associated with increased incidence and severity of respiratory infections, we compared the microbiota of smokers and nonsmokers to shed light on its potential for facilitating infections. We hypothesized that decreased species diversity, decreased community stability, or increased differences in community structure could facilitate invading pathogens. We found that smoking is associated with reduced alpha diversity, greater differences in community structure, and increased environmental filtering. The effects of short-term perturbations (antibiotic use and participants exhibiting cold symptoms) were also investigated. Antibiotic use had a negative effect on alpha diversity, irrespective of smoking status, and both antibiotic use and cold symptoms were associated with highly unique bacterial communities. A stability analysis of models built from the data indicated that there were no differences in local or global stability in the microbial communities of smokers, compared to nonsmokers, and that their microbiota are equally resistant to species invasions. Results from these models suggest that smoker microbiota are perturbed but characterized by alternative stable states that are as stable and invasion-resistant as are the microbiota of nonsmokers. Smoking is unlikely to increase the risk of infectious disease through the altered composition and ecological function of the microbiota; this is more likely due to the effects of smoking on the local and systemic immune system. IMPORTANCE Smoking is associated with an increased risk of respiratory infections. Hypothetically, the altered community diversity of smokers' pharyngeal microbiota, together with changes in their ecological stability properties, could facilitate their invasion by pathogens. To address this question, we analyzed longitudinal microbiota data of baseline healthy individuals who were either smokers or nonsmokers. While the results indicate reduced biodiversity and increased species turnover in the smokers' pharyngeal microbiota, their ecological stability properties were not different from those of the microbiota of nonsmokers, implying, in ecological terms, that the smokers' microbial communities are not less resistant to invasions. Therefore, the study suggests that the increased propensity of respiratory infections that is seen in smokers is more likely associated with changes in the local and systemic immune system than with ecological changes in the microbial communities.

Molecular insights informing factors affecting low temperature anaerobic applications: Diversity, collated core microbiomes and complexity stability relationships in LCFA-fed systems.

Fats, oil and grease, and their hydrolyzed counterparts-long chain fatty acids (LCFA) make up a large fraction of numerous wastewaters and are challenging to degrade anaerobically, more so, in low temperature anaerobic digestion (LtAD) systems. Herein, we perform a comparative analysis of publicly available Illumina 16S rRNA datasets generated from LCFA-degrading anaerobic microbiomes at low temperatures (10 and 20 °C) to comprehend the factors affecting microbial community dynamics. The various factors considered were the inoculum, substrate and operational characteristics, the reactor operation mode and reactor configuration, and the type of nucleic acid sequenced. We found that LCFA-degrading anaerobic microbiomes were differentiated primarily by inoculum characteristics (inoculum source and morphology) in comparison to the other factors tested. Inoculum characteristics prominently shaped the species richness, species evenness and beta-diversity patterns in the microbiomes even after long term operation of continuous reactors up to 150 days, implying the choice of inoculum needs careful consideration. The generalised additive models represented through beta diversity contour plots revealed that psychrophilic bacteria RBG-13-54-9 from family Anaerolineae, and taxa WCHB1-41 and Williamwhitmania were highly abundant in LCFA-fed microbial niches, suggesting their role in anaerobic treatment of LCFAs at low temperatures of 10-20 °C. Overall, we showed that the following bacterial genera: uncultured Propionibacteriaceae, Longilinea, Christensenellaceae R7 group, Lactivibrio, candidatus Caldatribacterium, Aminicenantales, Syntrophus, Syntrophomonas, Smithella, RBG-13-54-9, WCHB1-41, Trichococcus, Proteiniclasticum, SBR1031, Lutibacter and Lentimicrobium have prominent roles in LtAD of LCFA-rich wastewaters at 10-20 °C. This study provides molecular insights of anaerobic LCFA degradation under low temperatures from collated datasets and will aid in improving LtAD systems for treating LCFA-rich wastewaters.

Smartphone Color Vision Testing as an Alternative to the Conventional Ishihara Booklet.

Introduction Color vision testing was first seen as a parameter to be tested in the 1700s. Nowadays, it is a well-known phenomenon with significant quality-of-life implications. Structures involved in color vision include the lens, pupil, retinal cone photopigments, and several photoreceptor processes that translate the incoming spectrum of different light wavelengths into a processed colored image. An initial color vision assessment was made simply by comparing the color perception of the individual to that of the examiner. The most commonly used tools to screen for color vision defects today are color plates, such as the Ishihara color plates. In the modern age, smartphones have evolved to become an essential part of our everyday lives with applications such as Eye Handbook, which allow easier access to color vision testing using color plates displayed on smartphone screens. In this study, we compared color vision testing on Android and iOS devices to the standard Ishihara booklet. Materials and methods A cross-sectional validation study was performed on patients presenting to the Armed Forces Institute of Ophthalmology, Rawalpindi, Pakistan for six months. The sample size collected was 162 with a 95% confidence interval. The age range of the sample population was kept at 12-70 years. A patient was selected for participation in the study, and a color vision assessment was performed using the Ishihara color plates and Android and iOS smartphones. The collected data was then entered into IBM SPSS (Statistical Package for the Social Sciences) Statistics 25 for analysis, with the p-value being kept at 0.05. Results The sample size was 162, with the gender distribution being predominantly male (69.14%). The average age of the participants was 35.94 (SD = 12.04). The result of the two-tailed paired sample z-testwas not significant based on a p-value of 0.565, indicating the null hypothesis cannot be rejected. This finding suggests the difference between the mean of Ishihara and the mean of the iPhone was not significantly different from zero. Similar results were found for comparisons between Android smartphones and the Ishihara booklet. Conclusions Previous studies conducted showed nearly 60% of subjects with normal color vision correctly identified all colors on standard Ishihara color plates. The two-tailed paired sample t-test conducted in our study showed no significant difference between either of the smartphone groups (iPhone or Android) and the Ishihara booklet group, indicating that smartphones present a viable alternative to standard Ishihara booklet testing. However, there are certain limitations to our study. Different types of smartphone screens present a challenge in standardization while testing color vision, something that is not a problem when using the Ishihara booklet. However, smartphones are more widely available, more versatile, and present far greater ease of access. Both these factors should be considered when comparing the two in future studies.