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.

Exploring the burden of actinic keratoses through development of a patient decision aid: a mixed methods study.

BACKGROUND: Actinic keratoses (AKs) present on sun-exposed sites and are considered precursors of cutaneous squamous cell carcinoma (cSCC). A better understanding of the experiences of patients with this condition may improve patient-provider relationships and guide the introduction of shared-decision making (SDM) to treatment decisions. OBJECTIVES: To develop a patient decision aid (PDA) for field treatment of multiple actinic keratoses in line with the International Patient Decision Aid Standards (IPDAS), by (i) characterising the burden and lived experiences of patients with multiple AKs, (ii) understanding the decisional needs of patients requiring field treatment and (iii) exploring clinician preferences regarding field treatment for multiple AKs. MATERIALS AND METHODS: This mixed methods study followed the most up-to-date guidelines set out by the IPDAS Collaboration; a voluntary body which aims to enhance the quality of PDAs by developing an evidence-based systematic process for the development of unbiased and effective PDAs. RESULTS: Multiple actinic keratoses have a psychosocial impact on patients. Patients feel supported through the integration of evidence-based information to guide SDM. CONCLUSIONS: We propose that the use of a PDA for multiple AKs provides a key role in supporting informed shared patient-provider decision making and empowers patient involvement in their prospective treatment strategy.

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 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.

Improving breastfeeding support through the implementation of the Baby-Friendly Hospital and Community Initiatives: a scoping review.

BACKGROUND: Improved breastfeeding practices have the potential to save the lives of over 823,000 children under 5 years old globally every year. The Baby-Friendly Hospital Initiative (BFHI) is a global campaign by the World Health Organization and the United Nations Children's Fund, which promotes best practice to support breastfeeding in maternity services. The Baby-Friendly Community Initiative (BFCI) grew out of step 10, with a focus on community-based implementation. The aim of this scoping review is to map and examine the evidence relating to the implementation of BFHI and BFCI globally. METHODS: This scoping review was conducted according to the Joanna Briggs Institute methodology for scoping reviews. Inclusion criteria followed the Population, Concepts, Contexts approach. All articles were screened by two reviewers, using Covidence software. Data were charted according to: country, study design, setting, study population, BFHI steps, study aim and objectives, description of intervention, summary of results, barriers and enablers to implementation, evidence gaps, and recommendations. Qualitative and quantitative descriptive analyses were undertaken. RESULTS: A total of 278 articles were included in the review. Patterns identified were: i) national policy and health systems: effective and visible national leadership is needed, demonstrated with legislation, funding and policy; ii) hospital policy is crucial, especially in becoming breastfeeding friendly and neonatal care settings iii) implementation of specific steps; iv) the BFCI is implemented in only a few countries and government resources are needed to scale it; v) health worker breastfeeding knowledge and training needs strengthening to ensure long term changes in practice; vi) educational programmes for pregnant and postpartum women are essential for sustained exclusive breastfeeding. Evidence gaps include study design issues and need to improve the quality of breastfeeding data and to perform prevalence and longitudinal studies. CONCLUSION: At a national level, political support for BFHI implementation supports expansion of Baby-Friendly Hospitals. Ongoing quality assurance is essential, as is systematic (re)assessment of BFHI designated hospitals. Baby Friendly Hospitals should provide breastfeeding support that favours long-term healthcare relationships across the perinatal period. These results can help to support and further enable the effective implementation of BFHI and BFCI globally.

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.

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.

Beyond Basic Diversity Estimates-Analytical Tools for Mechanistic Interpretations of Amplicon Sequencing Data.

Understanding microbial ecology through amplifying short read regions, typically 16S rRNA for prokaryotic species or 18S rRNA for eukaryotic species, remains a popular, economical choice. These methods provide relative abundances of key microbial taxa, which, depending on the experimental design, can be used to infer mechanistic ecological underpinnings. In this review, we discuss recent advancements in in situ analytical tools that have the power to elucidate ecological phenomena, unveil the metabolic potential of microbial communities, identify complex multidimensional interactions between species, and compare stability and complexity under different conditions. Additionally, we highlight methods that incorporate various modalities and additional information, which in combination with abundance data, can help us understand how microbial communities respond to change in a typical ecosystem. Whilst the field of microbial informatics continues to progress substantially, our emphasis is on popular methods that are applicable to a broad range of study designs. The application of these methods can increase our mechanistic understanding of the ongoing dynamics of complex microbial communities.

Analysis of pit latrine microbiota reveals depth-related variation in composition, and key parameters and taxa associated with latrine fill-up rate.

Pit latrines are used by billions of people globally, often in developing countries where they provide a low-tech and low-cost sanitation method. However, health and social problems can arise from a lack of emptying or maintenance of these facilities. A better understanding of the biological and environmental parameters within pit latrines could inform attempts to enhance material decomposition rates, and therefore slow fill-up rate. In this study, we have performed a spatial analysis of 35 Tanzanian pit latrines to identify bacteria and environmental factors that are associated with faster or slower pit latrine fill-up rates. Using ordination of microbial community data, we observed a linear gradient in terms of beta diversity with increasing pit latrine sample depth, corresponding to a shift in microbial community structure from gut-associated families in the top layer to environmental- and wastewater-associated taxa at greater depths. We also investigated the bacteria and environmental parameters associated with fill-up rates, and identified pH, volatile solids, and volatile fatty acids as features strongly positively correlated with pit latrine fill-up rates, whereas phosphate was strongly negatively correlated with fill-up rate. A number of pit latrine microbiota taxa were also correlated with fill-up rates. Using a multivariate regression, we identified the Lactobacillaceae and Incertae_Sedis_XIII taxa as particularly strongly positively and negatively correlated with fill-up rate, respectively. This study therefore increases knowledge of the microbiota within pit latrines, and identifies potentially important bacteria and environmental variables associated with fill-up rates. These new insights may be useful for future studies investigating the decomposition process within pit latrines.

Indigenous Bacillus paramycoides spp. and Alcaligenes faecalis: sustainable solution for bioremediation of hospital wastewater.

Farmers near towns and cities are using a wide range of highly polluted wastewaters for crop irrigation in Pakistan due to severe freshwater shortage. The present study aimed to promote indigenous bacterial strains isolated from domestic, hospital, textile, pharmaceutical and mixed wastewaters to remove contaminants and colour and render these wastewaters safer for irrigation. Thirty seven bacterial strains were isolated from five wastewater samples collected from different sites in Lahore, Pakistan. Under optimal growth conditions, three isolates (D6, D7 and P1) showed >93% decolourisation potential in the treatment of hospital wastewater. 16S rDNA sequencing identified two of these isolates (D6 and D7) as showing 100% and 99.86% homology to Bacillus paramycoides spp. - novel strains from B. cereus group. Isolate P1 showed 97.47% homology to Alcaligenes faecalis. GCMS analysis of the untreated hospital wastewater revealed the presence of pharmaceutic pollutants, i.e. Phenol (876 µg/L), Salicylic acid (48 µg/L), Caffeine (7 µg/L), Naproxen (23 µg/L), Octadecene (185 µg/L) and Diazepam (14 µg/L). The analysis of treated hospital wastewaters showed percentage degradation of pharmaceutic pollutants (100%-43%) and significant reduction in the BOD5 (91%-68%), COD (89%-52%) and heavy metals concentrations. These strains therefore can represent a low-cost and low-tech alternative to bioremediate complex matrices of hospital wastewater prior to crop irrigation to support the achievement of clean re-usable water in developing countries like Pakistan.

First proof of concept for full-scale, direct, low-temperature anaerobic treatment of municipal wastewater.

Municipal wastewater constitutes the largest fraction of wastewater, and yet treatment processes are largely removal-based. High-rate anaerobic digestion (AD) has revolutionised the sustainability of industrial wastewater treatment and could additionally provide an alternative for municipal wastewater. While AD of dilute municipal wastewater is common in tropical regions, the low temperatures of temperate climates has resulted in slow uptake. Here, we demonstrate for the first time, direct, high-rate, low-temperature AD of low-strength municipal wastewater at full-scale. An 88 m3 hybrid reactor was installed at the municipal wastewater treatment plant in Builth Wells, UK and operated for 290 days. Ambient temperatures ranged from 2 to 18 °C, but remained below 15 °C for > 100 days. Influent BOD fluctuated between 2 and 200 mg L-1. However, BOD removal often reached > 85%. 16S rRNA amplicon sequencing of DNA from the biomass revealed a highly adaptable core microbiome. These findings could provide the basis for the next-generation of municipal wastewater treatment.

Hospital wastewater treated with a novel bacterial consortium (Alcaligenes faecalis and Bacillus paramycoides spp.) for phytotoxicity reduction in Berseem clover and tomato crops.

Hospital wastewaters are produced in large volumes in Pakistan (∼362-745 L/bed.day) and are discharged without proper treatment. They are widely used by farmers for crop irrigation and induce a phytotoxic effect on plant growth. The study was conducted to evaluate the effect of untreated and treated hospital wastewater on seed germination of a fodder crop Trifolium alexandrinum (Berseem clover) and a food crop Solanum lycopersicum (tomato). A bacterial consortium was formed with three bacterial strains, i.e., Alcaligenes faecalis and Bacillus paramycoides spp., which were individually proven efficient in previous studies. The concentrations of untreated and treated hospital wastewater (25, 50, 75 and 100%) were used to irrigate these crop seeds. To assess the efficiency of treatment, the germination percentage, delay index, germination index, stress tolerance indices, seedling vigour index and phytotoxicity index were calculated and were statistically proven significant. The seeds grown in treated wastewater concentrations showed negative values of phytotoxicity indices (tomato: -0.36, -0.47, -0.78 and -1.11; Berseem clover: -0.23) which indicate a stimulatory or non-toxic effect on seedling growth. Our work proposes that this bacterial consortium is efficient for hospital wastewater treatment before crop irrigation.

Loss of SigB in Listeria monocytogenes Strains EGD-e and 10403S Confers Hyperresistance to Hydrogen Peroxide in Stationary Phase under Aerobic Conditions.

UNLABELLED: SigB is the main stress gene regulator in Listeria monocytogenes affecting the expression of more than 150 genes and thus contributing to multiple-stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain, as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that unlike for all other stresses, loss of sigB results in hyperresistance to H2O2 (more than 8 log CFU ml(-1) compared to the wild type) in aerobically grown stationary-phase cultures of L. monocytogenes strains 10403S and EGD-e. Furthermore, growth at 30°C resulted in higher resistance to oxidative stress than that at 37°C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, the loss of SigB in 10403S did not affect survival against H2O2, while in EGD-e, it resulted in a sensitive phenotype. During exponential phase, minor differences occurred, and this result was expected due to the absence of sigB transcription. Catalase tests were performed under all conditions, and stronger catalase results corresponded well with a higher survival rate, underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates, which corresponded with the catalase tests and survival. In addition, reverse transcription-PCR (RT-PCR) showed no differences in transcription between the wild type and the ΔsigB mutant in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are under way. IMPORTANCE: SigB is the most important stress gene regulator in L. monocytogenes and other Gram-positive bacteria. Its increased expression during stationary phase results in resistance to multiple stresses. However, despite its important role in general stress resistance, its expression is detrimental for the cell in the presence of oxidative stress, as it promotes hypersensitivity against hydrogen peroxide. This peculiar phenotype is an important element of the physiology of L. monocytogenes, and it might help us explain the behavior of this organism in environments where oxidative stress is present.

Biological Phosphorus Removal During High-Rate, Low-Temperature, Anaerobic Digestion of Wastewater.

We report, for the first time, extensive biologically mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (∼2%) within the sludge bed and fixed-film biofilms. 4', 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4 and 1.5 kg COD m(-3) d(-1) and hydraulic retention times of 8-24 h, while phosphate removal efficiency ranged from 28 to 78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12°C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterized polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter, and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

Viscosupplementation in lumbar facet joint arthropathy: a pilot study.

BACKGROUND: Degeneration of the spine is a prevalent problem that generally advances with age, although its occurrence is not restricted to the elderly. Chronic low back pain is a common problem with several treatments, but rigorous evaluation of many interventions is still lacking. One of the most common methods of treating lumbar facet arthropathy is intra-articular injection of local anesthetic and steroid. However, in large joints, such as the knee, viscosupplementation has been shown to compare favorably to steroid in terms of symptom relief and duration of benefit. It is suggested that viscosupplementation may supersede steroid in treatment of symptomatic knee osteoarthritis. Hyaluronic acid (HA) therapy is still in its infancy in joints outside the knee, although some preliminary results are promising. OBJECTIVE: This was a pilot study to test the potential effectiveness of HA injection therapy in the treatment of lumbar facet joint arthritis. METHODS: Thirteen patients with symptomatic lumbar facet joint arthritis who met the inclusion criteria were prospectively recruited. Pretreatment evaluation of patients was by questionnaire, including the Visual Analog Score and Oswestry Disability Questionnaire. A single injection of HA into affected facet joints was then performed, with correct placement confirmed on fluoroscopy. The patients were similarly evaluated 6 weeks after treatment. RESULTS: Eighteen facets in 13 patients were injected with HA. At 6-week follow up, there was no significant improvement in pain when measured on the Visual Analog Score. There was also no significant improvement in the Oswestry Disability Questionnaire. CONCLUSIONS: Preliminary results from this pilot study do not demonstrate any benefit of viscosupplementation in the management of symptomatic lumbar facet arthropathy.