Vertical transmission of microbiomes into embryo culture media and its association with assisted reproductive outcomes.

RESEARCH QUESTION: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment? DESIGN: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility. RESULTS: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate. CONCLUSION: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

ARGs-OAP v2.0 with an expanded SARG database and Hidden Markov Models for enhancement characterization and quantification of antibiotic resistance genes in environmental metagenomes.

Motivation: Much global attention has been paid to antibiotic resistance in monitoring its emergence, accumulation and dissemination. For rapid characterization and quantification of antibiotic resistance genes (ARGs) in metagenomic datasets, an online analysis pipeline, ARGs-OAP has been developed consisting of a database termed Structured Antibiotic Resistance Genes (the SARG) with a hierarchical structure (ARGs type-subtype-reference sequence). Results: The new release of the database, termed SARG version 2.0, contains sequences not only from CARD and ARDB databases, but also carefully selected and curated sequences from the latest protein collection of the NCBI-NR database, to keep up to date with the increasing number of ARG deposited sequences. SARG v2.0 has tripled the sequences of the first version and demonstrated improved coverage of ARGs detection in metagenomes from various environmental samples. In addition to annotation of high-throughput raw reads using a similarity search strategy, ARGs-OAP v2.0 now provides model-based identification of assembled sequences using SARGfam, a high-quality profile Hidden Markov Model (HMM), containing profiles of ARG subtypes. Additionally, ARGs-OAP v2.0 improves cell number quantification by using the average coverage of essential single copy marker genes, as an option in addition to the previous method based on the 16S rRNA gene. Availability and implementation: ARGs-OAP can be accessed through http://smile.hku.hk/SARGs. The database could be downloaded from the same site. Source codes for this study can be downloaded from https://github.com/xiaole99/ARGs-OAP-v2.0. Supplementary information: Supplementary data are available at Bioinformatics online.

Toward an Intensive Longitudinal Understanding of Activated Sludge Bacterial Assembly and Dynamics.

Temporal microbial community studies have broadened our knowledge of the dynamics and correlations among microbes in both natural and artificial engineering systems. Using activated sludge as a model system, we utilized the intensive longitudinal sampling method to identify overlooked diversity and the hidden dynamics of microbes, detect cross-associations among microbes after detrending, and reveal the central microbial dynamics during sludge bulking and foaming. We discovered that the accumulative alpha diversity in activated sludge sampled daily over 392 days could be as high as 14 000 OTUs, and that the bacterial community dynamics followed a gradual succession, drifting away from the initial observed day and displaying a significant time-dependent trend. Cross-associations among bacteria were modulated after removing potential spurious correlations based on autocorrelation in microbial time series. Moreover, clusters of bacteria displaying rapid turnover were discovered during the beginning, ongoing, and fading of sludge bulking and foaming, and their physicochemical parameters are resolved. These identified groups of bacteria and their related environmental factors could potentially supply clues to form hypotheses for treating operational problems, such as sludge bulking and foaming.

Temporal dynamics of activated sludge bacterial communities in two diversity variant full-scale sewage treatment plants.

Bacterial community in activated sludge (AS) is diverse and highly dynamic. Little is known about the mechanism shaping bacterial community composition and dynamics of AS and no study had quantitatively compared the contribution of abiotic environmental factors and biotic associations to the temporal dynamics of AS microbial communities with significantly different diversity. In this study, two full-scale sewage treatment plants (STPs) with distinct operational parameters and influent composition were sampled biweekly over 1 year to reveal the correlating factors to whole and sub-groups of AS bacterial community diversity and dynamics. The results show that the bacterial communities of the two STPs were entirely different and correlated with the influent composition and operating configurations. Bacterial associations represented by cohesion metrics and the environmental factor temperature were the primary correlated factors to the temporal bacterial community dynamics within each STP. The STP with high diversity and evenness could treat influent with higher suspended solid and a shorter sludge retention time, and was less correlated with environmental factors, implying the importance of diversity for AS system.

Cultivation-dependent and high-throughput sequencing approaches studying the co-occurrence of antibiotic resistance genes in municipal sewage system.

During the past years, antibiotic-resistant bacteria (ARB) leading for the spreading of antibiotic resistance genes (ARGs) became a global problem, especially multidrug-resistant (MDR) bacteria are considered the prime culprit of antibiotic resistance. However, the correlation between the antibiotic-resistant phenotype and the ARG profiles remains poorly understood. In the present study, metagenomic functional screening and metagenomic analysis of coliforms were combined to explore the phenotype and genotype of the ARBs from municipal sewage. Our results showed that the ARG co-occurrence was widespread in the municipal sewage. The present study also highlighted the high abundance of ARGs from antibiotic resistance coliforms especially the MDR coliforms with ARG level of 33.8 ± 4.2 copies per cell. The ARG profiles and the antibiotic resistance phenotypes of the isolated antibiotic resistant coliforms were also correlated and indicated that the resistance to the related antibiotic (ampicillin, kanamycin, erythromycin, chloramphenicol, and tetracycline) was mostly contributed by the ARGs belonging to the subtypes of ß-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc.

Comparison of microbial diversity determined with the same variable tag sequence extracted from two different PCR amplicons.

BACKGROUND: Deep sequencing of the variable region of 16S rRNA genes has become the predominant tool for studying microbial ecology. As sequencing datasets have accumulated, meta-analysis of sequences obtained with different variable 16S rRNA gene targets and by different sequencing methods has become an intriguing prospect that remains to be evaluated experimentally. RESULTS: We amplified a group of fecal samples using both V4F-V6R and V6F-V6R primer sets, excised the same V6 fragment from the two sets of Illumina sequencing data, and compared the resulting data in terms of the α-diversity, ß-diversity, and community structure. Principal component analysis (PCA) comparing the microbial community structures of different datasets, including those with simulated sequencing errors, was very reliable. Procrustes analysis showed a high degree of concordance between the different datasets for both abundance-weighted and binary Jaccard distances (P < 0.05), and a meta-analysis of individual datasets resulted in similar conclusions. The Shannon's diversity index was consistent as well, with comparable values obtained for the different datasets and for the meta-analysis of different datasets. In contrast, richness estimators (OTU and Chao) varied significantly, and the meta-analysis of richness estimators was also biased. The community structures of the two datasets were obviously different and led to significant changes in the biomarkers identified by the LEfSe statistical tool. CONCLUSIONS: Our results suggest that beta-diversity analysis and Shannon's diversity are relatively reliable for meta-analysis, while community structures and biomarkers are less consistent. These results should be useful for future meta-analyses of microbiomes from different data sources.

Illumina sequencing of 16S rRNA tag revealed spatial variations of bacterial communities in a mangrove wetland.

The microbial community plays an essential role in the high productivity in mangrove wetlands. A proper understanding of the spatial variations of microbial communities will provide clues about the underline mechanisms that structure microbial groups and the isolation of bacterial strains of interest. In the present study, the diversity and composition of the bacterial community in sediments collected from four locations, namely mudflat, edge, bulk, and rhizosphere, within the Mai Po Ramsar Wetland in Hong Kong, SAR, China were compared using the barcoded Illumina paired-end sequencing technique. Rarefaction results showed that the bulk sediment inside the mature mangrove forest had the highest bacterial α-diversity, while the mudflat sediment without vegetation had the lowest. The comparison of ß-diversity using principal component analysis and principal coordinate analysis with UniFrac metrics both showed that the spatial effects on bacterial communities were significant. All sediment samples could be clustered into two major groups, inner (bulk and rhizosphere sediments collected inside the mangrove forest) and outer mangrove sediments (the sediments collected at the mudflat and the edge of the mangrove forest). With the linear discriminate analysis scores larger than 3, four phyla, namely Actinobacteria, Acidobacteria, Nitrospirae, and Verrucomicrobia, were enriched in the nutrient-rich inner mangrove sediments, while abundances of Proteobacteria and Deferribacterias were higher in outer mangrove sediments. The rhizosphere effect of mangrove plants was also significant, which had a lower α-diversity, a higher amount of Nitrospirae, and a lower abundance of Proteobacteria than the bulk sediment nearby.

Second-line levofloxacin-based quadruple therapy versus bismuth-based quadruple therapy for Helicobacter pylori eradication and long-term changes to the gut microbiota and antibiotic resistome: a multicentre, open-label, randomised controlled trial.

BACKGROUND: Levofloxacin-based therapy or bismuth-based quadruple therapy are the recommended second-line regimens for Helicobacter pylori eradication after failure of clarithromycin-based therapy. However, resistance to levofloxacin has increased in the past decade. Furthermore, little is known about the long-term effects of H pylori eradication on the antibiotic resistome. In this study, we compared these second-line eradication therapies for efficacy, tolerability, and short-term and long-term effects on the gut microbiota, antibiotic resistome, and metabolic parameters. METHODS: We did a multicentre, open-label, parallel group, randomised controlled trial at eight hospitals in Taiwan. Adult patients (age ≥20 years) with persistent H pylori infection after first-line clarithromycin-based therapy were randomly assigned (1:1, permuted block sizes of four) to receive levofloxacin-based sequential quadruple therapy for 14 days (EAML14; esomeprazole 40 mg and amoxicillin 1 g for 7 days, followed by esomeprazole 40 mg, metronidazole 500 mg, and levofloxacin 250 mg for 7 days, all twice-daily) or bismuth-based quadruple therapy for 10 days (BQ10; esomeprazole 40 mg twice daily, bismuth tripotassium dicitrate 300 mg four times a day, tetracycline 500 mg four times a day, and metronidazole 500 mg three times a day). All investigators were masked to the randomisation sequence. The primary endpoint was H pylori eradication rate measured by 13C urea breath test 6 weeks after second-line treatment according to both intention-to-treat (ITT) and per-protocol analysis. The microbiota composition and antibiotic resistome of faecal samples collected at baseline (before treatment) and at 2 weeks, 8 weeks, and 1 year after eradication therapy was profiled by shotgun metagenomic sequencing and 16S rRNA gene sequencing. The frequency of adverse effects and changes in the gut microbiota and antibiotic resistome were assessed in all participants with available data. The trial is complete and registered with ClinicalTrails.gov, NCT03148366. FINDINGS: Between Feb 25, 2015, and Dec 11, 2020, 560 patients were randomly assigned to receive EAML14 or BQ10 (n=280 per group; 261 [47%] men and 299 [53%] women). Mean age was 55·9 years (SD 12·7) in the EAML14 group and 54·9 years (12·3) in the BQ10 group. Eradication of H pylori was achieved in 246 (88%) of 280 participants in the EAML14 group and 245 (88%) of 280 in the BQ10 group according to ITT analysis (risk difference -0·4%, 95% CI -5·8 to 5·1; p=0·90). In the per-protocol analysis, 246 (90%) of 273 participants in the EAML14 group and 245 (93%) of 264 participants in the BQ10 group achieved H pylori eradication (risk difference 2·7%, 95% CI -0·2 to 7·4; p=0·27). Transient perturbation of faecal microbiota diversity at week 2 was largely restored to basal state 1 year after EAML14 or BQ10. Diversity recovery was slower with BQ10, and recovery in species abundance was partial after both therapies. On shotgun sequencing, we observed significant increases in total resistome after EAML14 (p=0·0002) and BQ10 (p=4·3 × 10-10) at week 2, which were restored to pretreatment level by week 8. The resistance rates of Escherichia coli and Klebsiella pneumonia to levofloxacin, ciprofloxacin, ampicillin (ampicillin-sulbactam for K pneumonia), and various cephalosporins were significantly increased in the EAML14 group compared with in the BQ10 group at week 2, which were restored to pretreatment levels and showed no significant differences at week 8 and 1 year. The frequency of any adverse effects was significantly higher after BQ10 therapy (211 [77%] of 273 participants) than after EAML14 therapy (134 [48%] of 277; p<0·0001). INTERPRETATION: We found no evidence of superiority between levofloxacin-based quadruple therapy and bismuth-based quadruple therapy in the second-line treatment of H pylori infection. The transient increase in the antibiotic resistome and perturbation of faecal microbiota diversity were largely restored to pretreatment state from 2 months to 1 year after eradication therapy. FUNDING: The Ministry of Science and Technology of Taiwan, the Ministry of Health and Welfare of Taiwan, National Taiwan University Hospital, Taipei Veteran General Hospital, and the Australian Federal Government through the St George and Sutherland Medical Research Foundation. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

High serum IL-21 levels after 12 weeks of antiviral therapy predict HBeAg seroconversion in chronic hepatitis B.

BACKGROUND & AIMS: Interleukin-21 (IL-21) stimulates T cell and B cell responses and plays a role in control of chronic viral infections. The role of IL-21 in chronic hepatitis B virus (HBV) infection is not understood. METHODS: Serum IL-21 levels were measured by enzyme immunoassay in 75 HBeAg-positive chronic hepatitis B (CHB) patients undergoing telbivudine treatment. The findings were validated in 103 patients from a separate clinical trial of telbivudine. A complete response to telbivudine was defined as having both HBeAg seroconversion and serum HBV-DNA level <300 copies/ml by treatment week 52. The proportions of T-cells producing IL-21 and/or expressing programmed death 1 (PD-1) in peripheral blood mononuclear cells were assessed longitudinally during treatment by intracellular cytokine staining and flow cytometry. RESULTS: Median serum IL-21 levels at treatment week 12 were significantly higher in patients who did achieve vs. patients who did not achieve a complete response in both the initial (128.4 vs. 69.2 pg/ml, p=0.003) and the validation (142.2 vs. 89.9 pg/ml, p=0.004) trials. Serum levels of IL-21 (p=0.005) or HBV-DNA (p=0.003) levels at treatment week 12 independently predicted HBeAg seroconversion in the first year of treatment. The decrease in PD-1 expression on CD4(+) and CD8(+) T cells during the first 12 weeks on telbivudine treatment was not correlated with changes in IL-21 concentrations. CONCLUSIONS: Serum IL-21 levels may be a biomarker for HBeAg seroconversion, and may contribute to individualization of antiviral therapy in HBeAg-positive CHB. IL-21 may also have a role in immunotherapy for CHB.

Nationwide biogeography and health implications of bacterial communities in household drinking water.

Drinking water at the point of use harbors microorganisms that may pose potential risks to human health. However, the microbial diversity and health impacts of household drinking water are poorly understood, since culture-based methods only target on specific microorganisms and low biomass of drinking water hinders a high-throughput profiling. Here, we used an optimized workflow to efficiently collect microorganisms from low-biomass drinking water and performed deep sequencing of 16S rRNA genes to profile the bacterial diversity and biogeography of 110 household drinking water samples covering 38 cities of 29 provinces/regions in China, and further explored environmental drivers and potential health implications. Our analyses revealed a diverse drinking water community comprising a total of 22,771 operational taxonomic units (OTUs). The spatial turnover of drinking water communities is scale-dependent and appears to be driven largely by rainfall and water source river. The identified potential pathogenic species may have the possibility of causing health risks. Our novel insights enhance the current understanding of the diversity and biogeography of drinking water bacterial communities within a theoretical ecological framework and have further important implications for safe drinking water management and public health protection.

Corrigendum: The P4 Study: Postpartum Maternal and Infant Faecal Microbiome 6 Months After Hypertensive Versus Normotensive Pregnancy.

[This corrects the article DOI: 10.3389/fcimb.2022.646165.].

Fecal DNA Virome Is Associated with the Development of Colorectal Neoplasia in a Murine Model of Colorectal Cancer.

Alteration of the gut virome has been associated with colorectal cancer (CRC); however, when and how the alteration takes place has not been studied. Here, we employ a longitudinal study in mice to characterize the gut virome alteration in azoxymethane (AOM)-induced colorectal neoplasia and identify important viruses associated with tumor growth. The number and size of the tumors increased as the mice aged in the AOM treated group, as compared to the control group. Tumors were first observed in the AOM group at week 12. We observed a significantly lower alpha diversity and shift in viral profile when tumors first appeared. In addition, we identified novel viruses from the genera Brunovirus, Hpunavirus that are positively associated with tumor growth and enriched at a late time point in AOM group, whereas members from Lubbockvirus show a negative correlation with tumor growth. Moreover, network analysis revealed two clusters of viruses in the AOM virome, a group that is positively correlated with tumor growth and another that is negatively correlated with tumor growth, all of which are bacteriophages. Our findings suggest that the gut virome changes along with tumor formation and provides strong evidence of a potential role for bacteriophage in the development of colorectal neoplasia.

The P4 Study: Postpartum Maternal and Infant Faecal Microbiome 6 Months After Hypertensive Versus Normotensive Pregnancy.

OBJECTIVE/HYPOTHESIS: To explore potential differences in faecal microbiome between women, and their infants, who had normotensive pregnancies (NP) and those who had a hypertensive pregnancy (HP), either gestational hypertension (GH) or preeclampsia (PE). METHODS: This is a sub study of P4 (Postpartum Physiology, Psychology, and Paediatrics Study) and includes 18 mother-infant pairs: 10 NP and 8 HP (HP as defined by blood pressure > 140/90mmHg; of which 6 had PE, and 2 GH), six months postpartum. The participating mothers collected stool samples from themselves and their infants. 16S rRNA V3-V4 amplicons were used to study the faecal microbiome. RESULTS: The sample of women and their infants were mostly primiparous (n =16) with vaginal birth (n = 14). At the time of faecal sampling 8 women were using hormonal contraception, and one HP woman remained on an antihypertensive. All women had blood pressure < 130/80mmHg, and 10 had high BMI (> 30). All infants had started solids, 8 were exclusively breastfed, 1 exclusively formula fed and 9 both. Three infants had been exposed to a course of antibiotics. Six months postpartum, there were no significant differences in alpha or beta diversity between the gut microbiota of HP and NP women (P > 0.05). However, a statistically significant difference was detected in alpha diversity between infants following HP and NP, with lower diversity levels in HP infants (P < 0.05). It was also found that at a genus and species level, the gut microbiota of HP women was enriched with Bifidobacterium and Bifididobacterium sp. and depleted in Barnisiella and Barnesiella intestinihominis when compared to NP women (P < 0.05). Similarly, the gut microbiota of infants born from HP was enriched in Streptococcus infantis and depleted in Sutterella, Sutterella sp., Bacteroides sp. and Clostridium aldenense compared to infants born from NP (P < 0.05). DISCUSSION: While our findings are at best preliminary, due to the very small sample size, they do suggest that the presence of hypertension in pregnancy may adversely affect the maternal microbiota postpartum, and that of their infants. Further analysis of postpartum microbiome data from future studies will be important to validate these early findings and provide further evidence about the changes in the microbiota in the offspring of women following hypertensive disorders of pregnancy (HDP), including possible links to the causes of long-term cardiovascular disease, the prevalence of which is increased in women who have experienced HDP.

Critical Assessment of Whole Genome and Viral Enrichment Shotgun Metagenome on the Characterization of Stool Total Virome in Hepatocellular Carcinoma Patients.

Viruses are the most abundant form of life on earth and play important roles in a broad range of ecosystems. Currently, two methods, whole genome shotgun metagenome (WGSM) and viral-like particle enriched metagenome (VLPM) sequencing, are widely applied to compare viruses in various environments. However, there is no critical assessment of their performance in recovering viruses and biological interpretation in comparative viral metagenomic studies. To fill this gap, we applied the two methods to investigate the stool virome in hepatocellular carcinoma (HCC) patients and healthy controls. Both WGSM and VLPM methods can capture the major diversity patterns of alpha and beta diversities and identify the altered viral profiles in the HCC stool samples compared with healthy controls. Viral signatures identified by both methods showed reductions of Faecalibacterium virus Taranis in HCC patients' stool. Ultra-deep sequencing recovered more viruses in both methods, however, generally, 3 or 5 Gb were sufficient to capture the non-fragmented long viral contigs. More lytic viruses were detected than lysogenetic viruses in both methods, and the VLPM can detect the RNA viruses. Using both methods would identify shared and specific viral signatures and would capture different parts of the total virome.

The Role of the Microbiome in the Metabolic Health of People with Schizophrenia and Related Psychoses: Cross-Sectional and Pre-Post Lifestyle Intervention Analyses.

The microbiome has been implicated in the development of metabolic conditions which occur at high rates in people with schizophrenia and related psychoses. This exploratory proof-of-concept study aimed to: (i) characterize the gut microbiota in antipsychotic naïve or quasi-naïve people with first-episode psychosis, and people with established schizophrenia receiving clozapine therapy; (ii) test for microbiome changes following a lifestyle intervention which included diet and exercise education and physical activity. Participants were recruited from the Eastern Suburbs Mental Health Service, Sydney, Australia. Anthropometric, lifestyle and gut microbiota data were collected at baseline and following a 12-week lifestyle intervention. Stool samples underwent 16S rRNA sequencing to analyse microbiota diversity and composition. Seventeen people with established schizophrenia and five people with first-episode psychosis were recruited and matched with 22 age-sex, BMI and ethnicity matched controls from a concurrent study for baseline comparisons. There was no difference in α-diversity between groups at baseline, but microbial composition differed by 21 taxa between the established schizophrenia group and controls. In people with established illness pre-post comparison of α-diversity showed significant increases after the 12-week lifestyle intervention. This pilot study adds to the current literature that detail compositional differences in the gut microbiota of people with schizophrenia compared to those without mental illness and suggests that lifestyle interventions may increase gut microbial diversity in patients with established illness. These results show that microbiome studies are feasible in patients with established schizophrenia and larger studies are warranted to validate microbial signatures and understand the relevance of lifestyle change in the development of metabolic conditions in this population.

Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma.

The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method.

BACKGROUND: The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform. RESULTS: The PfuUltra II Fusion HS DNA Polymerase (Stratagene) with higher fidelity showed lower amount of PCR artifacts and determined lower taxa richness than the Ex Taq (Takara). More importantly, the two polymerases showed different efficiencies for amplifying some of very abundant sequences, and determined significantly different community structures. As expected, the dilution of the DNA template resulted in a reduced estimation of taxa richness, particularly at the 200 fold dilution level, but the community structures were similar for all dilution levels. The 30 cycle group increased the PCR artifacts while comparing to the 25 cycle group, but the determined taxa richness was lower than that of the 25 cycle group. The PCR cycle number did not changed the microbial community structure significantly. CONCLUSIONS: These results highlight the PCR conditions, particularly the polymerase, have significant effect on the analysis of microbial diversity with next generation sequencing methods.

Metagenomic and Resistome Analysis of a Full-Scale Municipal Wastewater Treatment Plant in Singapore Containing Membrane Bioreactors.

Reclaimed water provides a water supply alternative to address problems of scarcity in urbanized cities with high living densities and limited natural water resources. In this study, wastewater metagenomes from 6 stages of a wastewater treatment plant (WWTP) integrating conventional and membrane bioreactor (MBR) treatment were evaluated for diversity of antibiotic resistance genes (ARGs) and bacteria, and relative abundance of class 1 integron integrases (intl1). ARGs confering resistance to 12 classes of antibiotics (ARG types) persisted through the treatment stages, which included genes that confer resistance to aminoglycoside [aadA, aph(6)-I, aph(3')-I, aac(6')-I, aac(6')-II, ant(2″)-I], beta-lactams [class A, class C, class D beta-lactamases (bla OXA)], chloramphenicol (acetyltransferase, exporters, floR, cmIA), fosmidomycin (rosAB), macrolide-lincosamide-streptogramin (macAB, ereA, ermFB), multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide (sul1, sul2), and tetracycline (tetM, tetG, tetE, tet36, tet39, tetR, tet43, tetQ, tetX). Although the ARG subtypes in sludge and MBR effluents reduced in diversity relative to the influent, clinically relevant beta lactamases (i.e., bla KPC, bla OXA) were detected, casting light on other potential point sources of ARG dissemination within the wastewater treatment process. To gain a deeper insight into the types of bacteria that may survive the MBR removal process, genome bins were recovered from metagenomic data of MBR effluents. A total of 101 close to complete draft genomes were assembled and annotated to reveal a variety of bacteria bearing metal resistance genes and ARGs in the MBR effluent. Three bins in particular were affiliated to Mycobacterium smegmatis, Acinetobacter Iwoffii, and Flavobacterium psychrophila, and carried aquired ARGs aac(2')-Ib, bla OXA-278, and tet36 respectively. In terms of indicator organisms, cumulative log removal values (LRV) of Escherichia coli, Enterococci, and P. aeruginosa from influent to conventional treated effluent was lower (0-2.4), compared to MBR effluent (5.3-7.4). We conclude that MBR is an effective treatment method for reducing fecal indicators and ARGs; however, incomplete removal of P. aeruginosa in MBR treated effluents (<8 MPN/100 mL) and the presence of ARGs and intl1 underscores the need to establish if further treatment should be applied prior to reuse.

The bacterial community significantly promotes cast iron corrosion in reclaimed wastewater distribution systems.

BACKGROUND: Currently, the effect of the bacterial community on cast iron corrosion process does not reach consensus. Moreover, some studies have produced contrasting results, suggesting that bacteria can either accelerate or inhibit corrosion. RESULTS: The long-term effects of the bacterial community on cast iron corrosion in reclaimed wastewater distribution systems were investigated from both spatial (yellow layer vs. black layer) and temporal (1-year dynamic process) dimensions of the iron coupon-reclaimed wastewater microcosm using high-throughput sequencing and flow cytometry approaches. Cast iron coupons in the NONdisinfection and UVdisinfection reactors suffered more severe corrosion than did those in the NaClOdisinfection reactor. The bacterial community significantly promoted cast iron corrosion, which was quantified for the first time in the practical reclaimed wastewater and found to account for at least 30.5% ± 9.7% of the total weight loss. The partition of yellow and black layers of cast iron corrosion provided more accurate information on morphology and crystal structures for corrosion scales. The black layer was dense, and the particles looked fusiform, while the yellow layer was loose, and the particles were ellipse or spherical. Goethite was the predominant crystalline phase in black layers, while corrosion products mainly existed as an amorphous phase in yellow layers. The bacterial community compositions of black layers were distinctly separated from yellow layers regardless of disinfection methods. The NONdisinfection and UVdisinfection reactors had a more similar microbial composition and variation tendency for the same layer type than did the NaClOdisinfection reactor. Biofilm development can be divided into the initial start-up stage, mid-term development stage, and terminal stable stage. In total, 12 potential functional genera were selected to establish a cycle model for Fe, N, and S metabolism. Desulfovibrio was considered to accelerate the transfer of Fe0 to Fe2+ and speed up weight loss. CONCLUSION: The long-term effect of disinfection processes on corrosion behaviors of cast iron in reclaimed wastewater distribution systems and the hidden mechanisms were deciphered for the first time. This study established a cycle model for Fe, N, and S metabolism that involved 12 functional genera and discovered the significant contribution of Desulfovibrio in promoting corrosion.