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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.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 byBetween 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 × 10We 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.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.For the Chinese translation of the abstract see Supplementary Materials section.

Although several molecular-based studies have demonstrated the involvement of ammonia-oxidizing archaea (AOA) in ammonia oxidation in wastewater treatment plants (WWTPs), factors affecting the persistence and growth of AOA in these engineered systems have not been resolved. Here, we show a seasonal prevalence of AOA in a full-scale WWTP (Shatin, Hong Kong SAR) over a 6-year period of observation, even outnumbering ammonia-oxidizing bacteria in the seasonal peaks in 3 years, which may be due to the high bioavailable copper concentrations. Comparative analysis of three metagenome-assembled genomes of group I.1a AOA obtained from the activated sludge and 16S rRNA gene sequences recovered from marine sediments suggested that the seawater used for toilet flushing was the primary source of the WWTP AOA. A rare AOA population in the estuarine source water became transiently abundant in the WWTP with a metagenome-based relative abundance of up to 1.3% over three seasons of observation. Correlation-based network analysis revealed a robust co-occurrence relationship between these AOA and organisms potentially active in nitrite oxidation. Moreover, a strong correlation between the dominant AOA and an abundant proteobacterial organism suggested that capacity for extracellular polymeric substance production by the proteobacterium could provide a niche for AOA within bioaggregates. Together, the study highlights the importance of long-term observation in identifying biotic and abiotic factors governing population dynamics in open systems such as full-scale WWTPs.

Background Microbial communities in both natural and applied settings reliably carry out myriads of functions, yet how stable these taxonomically diverse assemblages can be and what causes them to transition between states remains poorly understood. We studied monthly activated sludge (AS) samples collected over 9 years from a full-scale wastewater treatment plant to answer how complex AS communities evolve in the long term and how the community functions change when there is a disturbance in operational parameters. Results Here, we show that a microbial community in activated sludge (AS) system fluctuated around a stable average for 3 years but was then abruptly pushed into an alternative stable state by a simple transient disturbance (bleaching). While the taxonomic composition rapidly turned into a new state following the disturbance, the metabolic profile of the community and system performance remained remarkably stable. A total of 920 metagenome-assembled genomes (MAGs), representing approximately 70% of the community in the studied AS ecosystem, were recovered from the 97 monthly AS metagenomes. Comparative genomic analysis revealed an increased ability to aggregate in the cohorts of MAGs with correlated dynamics that are dominant after the bleaching event. Fine-scale analysis of dynamics also revealed cohorts that dominated during different periods and showed successional dynamics on seasonal and longer time scales due to temperature fluctuation and gradual changes in mean residence time in the reactor, respectively. Conclusions Our work highlights that communities can assume different stable states under highly similar environmental conditions and that a specific disturbance threshold may lead to a rapid shift in community composition.

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.

Wastewater treatment plants (WWTPs) are considered reservoirs of viruses, but the diversity and dynamic changes of viruses are not well understood. In this study, we recovered 8478 metagenomic viral contigs (mVCs; 5 kb) from two WWTPs (Shatin, 2806; Shek Wu Hui, 5672) in Hong Kong. Approximately 60% of the mVCs were poorly covered (35% of genes in identified mVCs) by the current NCBI and IMG/VR viral databases. The temporal profile of the newly identified mVCs among 98 Shatin AS samples collected monthly (for approximately 9 years) revealed the presence of periodic dynamics at an interval of approximately one year (341 days). The spatial distribution pattern of the virome in the wastewater treatment systems showed that shared viral clusters (viral populations categorized based on shared gene content and network analysis) can be globally found among similar samples of wastewater treatment systems, indicating the presence of core viral communities among geographically isolated wastewater treatment systems. These results not only supplemented the current virome database of engineered systems but also, to some extent, expanded the understanding of long-term cyclical development and spatial distributions of viral communities in wastewater treatment systems.

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.

The discovery of complete ammonia oxidizer (comammox) has fundamentally upended our perception of the global nitrogen cycle. Here, we reported four metagenome assembled genomes (MAGs) of comammox Nitrospira that were retrieved from metagenome datasets of tap water in Singapore (SG-bin1 and SG-bin2), Hainan province, China (HN-bin3) and Stanford, CA, USA (ST-bin4). Genes of phylogenetically distinct ammonia monooxygenase subunit A (amoA) and hydroxylamine dehydrogenase (hao) were identified in these four MAGs. Phylogenetic analysis based on ribosomal proteins, AmoA, hao and nitrite oxidoreductase (subunits nxrA and nxrB) sequences indicated their close relationships with published comammox Nitrospira. Canonical ammonia-oxidizing microbes (AOM) were also identified in the three tap water samples, ammonia-oxidizing bacteria (AOB) in Singapore's and Stanford's samples and ammonia-oxidizing archaea (AOA) in Hainan's sample. The comammox amoA-like sequences were also detected from some other drinking water systems, and even outnumbered the AOA and AOB amoA-like sequences. The findings of MAGs and the occurrences of AOM in different drinking water systems provided a significant clue that comammox are widely distributed in drinking water systems.

Few studies on orthosilicate cathodes co-doped with two cations have been reported until now. Here, we report the synthesis of Mn and Al co-doped Li2Fe0 .8− x Mn0.2Al x SiO4 (x = 0.05 and 0.1) by a solid-state reaction route and characterized by X-ray diffraction (XRD), particle size analysis, scanning electron microscopy (SEM), galvanostatic charge/discharge tests, and capacity intermittent titration technique (CITT), as compared to the single-doped Li2Fe0.8Mn0.2SiO4. Though the co-doping leads to a slight decreased capacity owing to the increased impurity and Al3+ inertia, a better cycling performance is obtained as expected. Especially when x is 0.05, the modified sample (Li2Fe0.75Mn0.2Al0.05SiO4) shows an initial discharge capacity of 159.3 mAh/g and high capacity retention of 78% after 50 charge/discharge cycles. The present work indicates that a synergistic effect of Mn and Al co-substitution at the Fe site could partly make up the disadvantage of single Mn doping, and might provide an effective guide for the dopant incorporation to Li2FeSiO4 systems.

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.

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.

The electrochemical behaviors of natural graphite (NG198), artificial graphite (AG360) and carbon microbeads (CMB) in an ionic liquid based electrolyte are investigated by cyclic voltammetry (CV). The surface and structure of three graphite materials are characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD) before and after cycling. It is found that solid electrolyte interface (SEI) is closely related to graphite structure. Benefiting from larger specific surface area and more dispersed Li + insertion points, CMB shows a better Li + insertion/de-insertion behavior than NG198 and AG360. Furthermore, electrochemical impedance spectra (EIS) prove that the SEI of different graphite electrodes has different intrinsic resistance and Li + penetrability. By comparison, CMB behaves better cell performances than AG360, while the narrow edge plane makes NG198 uncompetitive as a potential anode for the ionic liquids (ILs)-type Li-ion battery.

Background Antimicrobial agents have been widely used in animal farms to prevent and treat animal diseases and to promote growth. Antimicrobial agents may change the bacterial community and enhance the resistome in animal feces. We used metagenome-wide analysis to investigate the changes in bacterial community, variations in antibiotic resistance genes (ARGs), and their bacterial hosts in the feces of broiler chickens over a full-treatment course of chlortetracycline at low and therapeutic dose levels. Results The effects of chlortetracycline on resistome were dependent on the specific ARG subtypes and not simply the overall community-level ARGs. Therapeutic dose of chlortetracycline promoted the abundance of tetracycline resistance genes (tetA and tetW) and inhibited multidrug resistance genes (mdtA, mdtC, mdtK, ompR, and TolC). The therapeutic dose of chlortetracycline led to loss of Proteobacteria mainly due to the decrease of Escherichia/Shigella (from 72 to 58%). Inhibition of Escherichia by chlortetracycline was the primary reason for the decrease of genes resistant to multiple drugs in the therapeutic dose group. The ARG host Bifidobacterium were enriched due to tetW harbored by Bifidobacterium under chlortetracycline treatment. Escherichia was always the major host for multidrug resistance genes, whereas the primary host was changed from Escherichia to Klebsiella for aminoglycoside resistance genes with the treatment of therapeutic dose of chlortetracycline. Conclusions We provided the first metagenomic insights into antibiotic-mediated alteration of ARG-harboring bacterial hosts at community-wide level in chicken feces. These results indicated that the changes in the structure of antibiotic-induced feces microbial communities accompany changes in the abundance of bacterial hosts carrying specific ARGs in the feces microbiota. These findings will help to optimize therapeutic schemes for the effective treatment of antibiotic resistant pathogens in poultry farms. Graphical abstract Resistome variations in faecal microbiome of chickens exposed to chlortetracycline Electronic supplementary material The online version of this article (10.1186/s40168-018-0419-2) contains supplementary material, which is available to authorized users.

Background Bacterial communities are essential to the biogeochemical cycle in riverine ecosystems. However, little is presently known about the integrated biogeography of planktonic and sedimentary bacterial communities in large rivers. Results This study provides the first spatiotemporal pattern of bacterial communities in the Yangtze River, the largest river in Asia with a catchment area of 1,800,000 km2. We find that sedimentary bacteria made larger contributions than planktonic bacteria to the bacterial diversity of the Yangzte River ecosystem with the sediment subgroup providing 98.8% of 38,906 operational taxonomic units (OTUs) observed in 280 samples of synchronous flowing water and sediment at 50 national monitoring stations covering a 4300 km reach. OTUs within the same phylum displayed uniform seasonal variations, and many phyla demonstrated autumn preference throughout the length of the river. Seasonal differences in bacterial communities were statistically significant in water, whereas bacterial communities in both water and sediment were geographically clustered according to five types of landforms: mountain, foothill, basin, foothill-mountain, and plain. Interestingly, the presence of two huge dams resulted in a drastic fall of bacterial taxa in sediment immediately downstream due to severe riverbed scouring. The integrity of the biogeography is satisfactorily interpreted by the combination of neutral and species sorting perspectives in meta-community theory for bacterial communities in flowing water and sediment. Conclusions Our study fills a gap in understanding of bacterial communities in one of the world’s largest river and highlights the importance of both planktonic and sedimentary communities to the integrity of bacterial biogeographic patterns in a river subject to varying natural and anthropogenic impacts. Electronic supplementary material The online version of this article (10.1186/s40168-017-0388-x) contains supplementary material, which is available to authorized users.

Metatranscriptome has become increasingly important along with the application of next generation sequencing in the studies of microbial functional gene activity in environmental samples. However, the quantification of target active gene is hindered by the current relative quantification methods, especially when tracking the sharp environmental change. Great needs are here for an easy-to-perform method to obtain the absolute quantification. By borrowing information from the parallel metagenome, an absolute quantification method for both metagenomic and metatranscriptomic data to per gene/cell/volume/gram level was developed. The effectiveness of AQMM was validated by simulated experiments and was demonstrated with a real experimental design of comparing activated sludge with and without foaming. Our method provides a novel bioinformatic approach to fast and accurately conduct absolute quantification of metagenome and metatranscriptome in environmental samples. The AQMM can be accessed from https://github.com/biofuture/aqmm.

The human gut microbiota is an important reservoir of antibiotic resistance genes (ARGs). A metagenomic approach and network analysis were used to establish a comprehensive antibiotic resistome catalog and to obtain co-occurrence patterns between ARGs and microbial taxa in fecal samples from 180 healthy individuals from 11 different countries. In total, 507 ARG subtypes belonging to 20 ARG types were detected with abundances ranging from 7.12 × 10-7 to 2.72 × 10-1 copy of ARG/copy of 16S-rRNA gene. Tetracycline, multidrug, macrolide-lincosamide-streptogramin, bacitracin, vancomycin, beta-lactam and aminoglycoside resistance genes were the top seven most abundant ARG types. The multidrug ABC transporter, aadE, bacA, acrB, tetM, tetW, vanR and vanS were shared by all 180 individuals, suggesting their common occurrence in the human gut. Compared to populations from the other 10 countries, the Chinese population harboured the most abundant ARGs. Moreover, LEfSe analysis suggested that the MLS resistance type and its subtype 'ermF' were representative ARGs of the Chinese population. Antibiotic inactivation, antibiotic target alteration and antibiotic efflux were the dominant resistance mechanism categories in all populations. Procrustes analysis revealed that microbial phylogeny structured the antibiotic resistome. Co-occurrence patterns obtained via network analysis implied that 12 species might be potential hosts of 58 ARG subtypes.

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 β-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc.

The diversity and composition of bacterial communities in mudflat and mangrove sediments were investigated under the stresses of endocrine-disrupting chemicals (EDCs) using high-throughput sequencing technique. Bacterial diversity in the original sediments was highest among all samples, followed by non-sterilized and sterilized sediments after 84-day incubation. Proteobacteria were the predominant phylum in both mangrove and mudflat sediments, which accounted for 40-60% of the total tags, followed by Cyanobacteria, Chloroflexi, Planctomycetes and Acidobacteria. Although the total population of bacteria was not significantly declined due to the addition of EDCs, bacterial community structures were considerably altered. The number of bacterial genera promoted or inhibited by EDCs was 288 and 324, respectively. Bacterial genera affected by EDCs varied greatly with the types of sediments and the initial status of bacterial communities. Overall, our results suggested that bacterial community structure in mangrove sediments were closely related to their re-development and responses to EDC contamination.

The omnipresence of filterable bacteria that can pass through 0.22-μm membrane filters demands a change in the sterile filtration practice. In this study, we identified that filterable bacteria enriched from a surface water are members of the Bacteroidetes, Proteobacteria, Spirochaetae, Firmicutes, and Actinobacteria. Filterable bacteria displayed superior filterability during the entire bacterial growth phase, especially at the exponential phase. Maximal passage percentages were comparable at different cell densities, and achieved earlier at high cell density. Furthermore, filter retention for the investigated bacteria is independent of liquid temperature. However, cultivation temperature could affect the growth of some specific filterable bacteria and lead to variability in the passage percentage. Additionally, membrane materials, pore size and filtering flux greatly affected the passage of filterable bacteria. The majority of filterable Hylemonella and SAR324 could pass through 0.1-μm polyvinylidene fluoride and polyethersulfone filters but could not pass through 0.1-μm polycarbonate and mixed cellulose esters filters. Taken together, our results demonstrated that the ultra-small size of filterable bacteria, membrane characteristics and filtration operational conditions could challenge the validity of the 0.22/0.1-μm sterilizing grade filters in providing bio-safety barriers. Keywords: Filterable bacteria, Microfiltration, Filterability, Bacterial community composition, Flow cytometry, High-throughput sequencing

A sequencing batch reactor fed mainly by acetate was operated to perform enhanced biological phosphorus removal (EBPR). A short-term pH shock from 7.0 to 6.0 led to a complete loss of phosphate-removing capability and a drastic change of microbial communities. 16S rRNA gene pyrosequencing showed that large proportions of glycogen accumulating organisms (GAOs) (accounted for 16% of bacteria) bloomed, including Candidatus Competibacter phosphatis and Defluviicoccus-related tetrad-forming organism, causing deteriorated EBPR performance. The EBPR performance recovered with time and the dominant Candidatus Accumulibacter (Accumulibacter) clades shifted from Clade IIC to IIA while GAOs populations shrank significantly. The Accumulibacter population variation provided a good opportunity for genome binning using a bi-dimensional coverage method, and a genome of Accumulibacter Clade IIC was well retrieved with over 90% completeness. Comparative genomic analysis demonstrated that Accumulibacter clades had different abilities in nitrogen metabolism and carbon fixation, which shed light on enriching different Accumulibacter populations selectively.

The human delta-globin chain, unique to the hemoglobin A2 (HbA2) heterotetramer, is important for the evaluation of hemoglobinopathy. However, there are no well-defined antibodies specific for the delta-globin chain, a fact that is attributed a striking similarity (93%) in amino acid sequence between delta-globin and beta-globin of the hemoglobin A (HbA). In this study, two monoclonal antibodies (mAbs) against the delta-globin chain were generated and designated as 2H4 and 1H11. These antibodies were specific to HbA2 and do not cross-react with HbA and HbF (fetal hemoglobin). Moreover, the expression of HbA2 in fetal liver and mature erythrocytes was determined using these two mAbs. In addition to being useful tools for research or diagnosis, these antibodies could be valuable for development of rapid and effective antibody-based immunoassays of HbA2 expression in erythroid cells and non-erythroid tissue.

Lipopolysaccharide (LPS) is located on the cell wall of gram-negative bacteria and plays an important role in the pathogenesis of sepsis, which continues to be a leading cause of death in the intensive care units. There are many strains and serotypes of gram-negative bacteria and each individual has a unique kind of LPS. In addition, LPS belongs to thymus-independent (TI) antigen, making it difficult to produce high-affinity, cross-reactive monoclonal antibodies (MAb) against LPS. Here we report a novel method to produce cross-reactive murine MAbs against LPS by mixed immunization with whole cell bacteria, commercial LPS, and synthetic peptide, which simulates the structure of LPS. Using this approach, an MAb designated SMU-3A8 was generated, which can react with four commercial LPSs and seven gram-negative bacteria with high affinity suited for ELISA, dot-ELISA, Western blotting, immunofluorescence, and flow cytometry. Our results provide a new strategy for the generation of high-affinity, cross-reactive MAbs against LPS and other TI antigens.

Motivation: Environmental dissemination of antibiotic resistance genes (ARGs) has become an increasing concern for public health. Metagenomics approaches can effectively detect broad profiles of ARGs in environmental samples; however, the detection and subsequent classification of ARG-like sequences are time consuming and have been severe obstacles in employing metagenomic methods. We sought to accelerate quantification of ARGs in metagenomic data from environmental samples. Results: A Structured ARG reference database (SARG) was constructed by integrating ARDB and CARD, the two most commonly used databases. SARG was curated to remove redundant sequences and optimized to facilitate query sequence identification by similarity. A database with a hierarchical structure (type-subtype-reference sequence) was then constructed to facilitate classification (assigning ARG-like sequence to type, subtype and reference sequence) of sequences identified through similarity search. Utilizing SARG and a previously proposed hybrid functional gene annotation pipeline, we developed an online pipeline called ARGs-OAP for fast annotation and classification of ARG-like sequences from metagenomic data. We also evaluated and proposed a set of criteria important for efficiently conducting metagenomic analysis of ARGs using ARGs-OAP. Availability and Implementation: Perl script for ARGs-OAP can be downloaded from https://github.com/biofuture/Ublastx_stageone. ARGs-OAP can be accessed through http://smile.hku.hk/SARGs. Contact: zhangt@hku.hk or tiedjej@msu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Bulking and foaming are two notorious problems in activated sludge wastewater treatment plants (WWTPs), which are mainly associated with the excessive growth of bulking and foaming bacteria (BFB). However, studies on affecting factors of BFB in full-scale WWTPs are still limited. In this study, data sets of high-throughput sequencing (HTS) of 16S V3–V4 amplicons of 58 monthly activated sludge samples from a municipal WWTP was re-analyzed to investigate the BFB dynamics and further to study the determinative factors. The population of BFB occupied 0.6~36% (averagely 8.5% ± 7.3%) of the total bacteria and showed seasonal variations with higher abundance in winter-spring than summer-autumn. Pair-wise correlation analysis and canonical correlation analysis (CCA) showed that Gordonia sp. was positively correlated with NO2-N and negatively correlated with NO3-N and Nostocodia limicola II Tetraspharea sp. was negatively correlated with temperature and positively correlated with NH3-N in activated sludge. Bacteria species correlated with BFB could be clustered into two negatively related modules. Moreover, with intensive time series sampling, the dominant BFB could be accurately modeled with environmental interaction network, i.e. environmental parameters and biotic interactions between BFB and related bacteria, indicating that abiotic and biotic factors were both crucial to the dynamics of BFB.

The immune mechanisms underlying failure to achieve hepatitis B e antigen (HBeAg) seroconversion associated with viral control in chronic hepatitis B (CHB) remain unclear. Here we investigated the role of CD4(-)CD8(-) T (double-negative T; DNT) cells including TCRαβ(+) DNT (αβ DNT) and TCRγδ(+) DNT (γδ DNT) cells. Frequencies of circulating DNT cell subsets were measured by flow cytometry in a retrospective cohort of 51 telbivudine-treated HBeAg-positive CHB patients, 25 immune tolerant carriers (IT), 33 inactive carriers (IC), and 37 healthy controls (HC). We found that γδ DNT cell frequencies did not significantly change during treatment, being lower at baseline (P = 0.019) in patients with HBeAg seroconversion after 52 weeks of antiviral therapy (n = 20) than in those without (n = 31), and higher in the total CHB and IT than IC and HC groups (P

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 β-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.

Clustering 16S/18S rRNA amplicon sequences into operational taxonomic units (OTUs) is a critical step for the bioinformatic analysis of microbial diversity. Here, we report a pipeline for selecting OTUs with a relatively low computational demand and a high degree of accuracy. This pipeline is referred to as two-stage clustering (TSC) because it divides tags into two groups according to their abundance and clusters them sequentially. The more abundant group is clustered using a hierarchical algorithm similar to that in ESPRIT, which has a high degree of accuracy but is computationally costly for large datasets. The rarer group, which includes the majority of tags, is then heuristically clustered to improve efficiency. To further improve the computational efficiency and accuracy, two preclustering steps are implemented. To maintain clustering accuracy, all tags are grouped into an OTU depending on their pairwise Needleman-Wunsch distance. This method not only improved the computational efficiency but also mitigated the spurious OTU estimation from 'noise' sequences. In addition, OTUs clustered using TSC showed comparable or improved performance in beta-diversity comparisons compared to existing OTU selection methods. This study suggests that the distribution of sequencing datasets is a useful property for improving the computational efficiency and increasing the clustering accuracy of the high-throughput sequencing of PCR amplicons. The software and user guide are freely available at http://hwzhoulab.smu.edu.cn/paperdata/.

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from approximately 99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3' end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40-70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20-50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes., link_to_OA_fulltext

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.

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, β-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 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.

Invariant NKT (iNKT) cells are involved in the pathogenesis of various infectious diseases. However, their role in hepatitis B virus (HBV) infection is not fully understood, especially in human species. In this study, 35 chronic hepatitis B (CHB) patients, 25 inactive carriers (IC) and 36 healthy controls (HC) were enrolled and the proportions of circulating iNKT cells in fresh isolated peripheral blood mononuclear cells (PBMC) were detected by flow cytometry. A longitudinal analysis was also conducted in 19 CHB patients who received antiviral therapy with telbivudine. Thereafter, the immune functions of iNKT cells were evaluated by cytokine secretion and a two-chamber technique. The median frequency of circulating iNKT cells in CHB patients (0.13%) was lower than that in HC (0.24%, P = 0.01) and IC (0.19%, P = 0.02), and increased significantly during antiviral therapy with telbivudine (P = 0.0176). The expressions of CC chemokine receptor 5 (CCR5) and CCR6 were dramatically higher on iNKT cells (82.83%±9.87%, 67.67%±16.83% respectively) than on conventional T cells (30.5%±5.65%, 14.02%±5.92%, both P

Background The operational taxonomic unit (OTU) is widely used in microbial ecology. Reproducibility in microbial ecology research depends on the reliability of OTU-based 16S ribosomal subunit RNA (rRNA) analyses. Results Here, we report that many hierarchical and greedy clustering methods produce unstable OTUs, with membership that depends on the number of sequences clustered. If OTUs are regenerated with additional sequences or samples, sequences originally assigned to a given OTU can be split into different OTUs. Alternatively, sequences assigned to different OTUs can be merged into a single OTU. This OTU instability affects alpha-diversity analyses such as rarefaction curves, beta-diversity analyses such as distance-based ordination (for example, Principal Coordinate Analysis (PCoA)), and the identification of differentially represented OTUs. Our results show that the proportion of unstable OTUs varies for different clustering methods. We found that the closed-reference method is the only one that produces completely stable OTUs, with the caveat that sequences that do not match a pre-existing reference sequence collection are discarded. Conclusions As a compromise to the factors listed above, we propose using an open-reference method to enhance OTU stability. This type of method clusters sequences against a database and includes unmatched sequences by clustering them via a relatively stable de novo clustering method. OTU stability is an important consideration when analyzing microbial diversity and is a feature that should be taken into account during the development of novel OTU clustering methods. Electronic supplementary material The online version of this article (doi:10.1186/s40168-015-0081-x) contains supplementary material, which is available to authorized users.