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      Profiling Bacterial Diversity and Potential Pathogens in Wastewater Treatment Plants Using High-Throughput Sequencing Analysis

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          Abstract

          Next-generation sequencing provides new insights into the diversity and structure of bacterial communities, as well as the fate of pathogens in wastewater treatment systems. In this study, the bacterial community structure and the presence of pathogenic bacteria in three wastewater treatment plants across Gauteng province in South Africa were studied. The physicochemical results indicated that the quality of wastewater varies considerably from one plant to the others. Proteobacteria, Actinobacteria, Firmicutes, and Chloroflexi were the dominant phyla across the three wastewater treatment plants, while Alphaproteobacteria, Actinobacteria, Bacilli, and Clostridia were the dominant classes. The dominant bacterial functions were highly associated with carbohydrate, energy, and amino acid metabolism. In addition, potential pathogenic bacterial members identified from the influent/effluent samples included Roseomonas, Bacillus, Pseudomonas, Clostridium, Mycobacterium, Methylobacterium, and Aeromonas. The results of linear discriminant analysis (LDA) effect size analysis also confirmed that these bacterial pathogens were significantly abundant in the wastewater treatment systems. Further, the results of this study highlighted that the presence of bacterial pathogens in treated effluent pose a potential contamination risk, transmitted through soil, agriculture, water, or sediments. There is thus a need for continuous monitoring of potential pathogens in wastewater treatment plants (WWTPs) in order to minimize public health risk.

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          Metagenomics - a guide from sampling to data analysis

          Metagenomics applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity over the past 5 to 10 years, and many research laboratories are actively engaged in it now. With the growing numbers of activities also comes a plethora of methodological knowledge and expertise that should guide future developments in the field. This review summarizes the current opinions in metagenomics, and provides practical guidance and advice on sample processing, sequencing technology, assembly, binning, annotation, experimental design, statistical analysis, data storage, and data sharing. As more metagenomic datasets are generated, the availability of standardized procedures and shared data storage and analysis becomes increasingly important to ensure that output of individual projects can be assessed and compared.
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            454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants.

            Activated sludge (AS) contains highly complex microbial communities. In this study, PCR-based 454 pyrosequencing was applied to investigate the bacterial communities of AS samples from 14 sewage treatment plants of Asia (mainland China, Hong Kong, and Singapore), and North America (Canada and the United States). A total of 259 K effective sequences of 16S rRNA gene V4 region were obtained from these AS samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in AS, that is, 1183-3567 OTUs in a sludge sample, at 3% cutoff level and sequencing depth of 16,489 sequences. Clear geographical differences among the AS samples from Asia and North America were revealed by (1) cluster analyses based on abundances of OTUs or the genus/family/order assigned by Ribosomal Database Project (RDP) and (2) the principal coordinate analyses based on OTUs abundances, RDP taxa abundances and UniFrac of OTUs and their distances. In addition to certain unique bacterial populations in each AS sample, some genera were dominant, and core populations shared by multiple samples, including two commonly reported genera of Zoogloea and Dechloromonas, three genera not frequently reported (i.e., Prosthecobacter, Caldilinea and Tricoccus) and three genera not well described so far (i.e., Gp4 and Gp6 in Acidobacteria and Subdivision3 genera incertae sedis of Verrucomicrobia). Pyrosequencing analyses of multiple AS samples in this study also revealed the minority populations that are hard to be explored by traditional molecular methods and showed that a large proportion of sequences could not be assigned to taxonomic affiliations even at the phylum/class levels.
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              Strategies for culture of 'unculturable' bacteria.

              Molecular ecology methods are now well established for the culture-independent characterization of complex bacterial communities associated with various environmental and animal habitats and are revealing the extent of their diversity. By comparison, it has become clear that only a small minority of microorganisms are readily cultivated in vitro, with the majority of all bacteria remaining 'unculturable' using standard methods. Yet, it is only through the isolation of bacterial species in pure culture that they may be fully characterized, both for their physiological and pathological properties. Hence, the endeavour to devise novel cultivation methods for microorganisms that appear to be inherently resistant to artificial culture is a most important one. This minireview discusses the possible reasons for 'unculturability' and evaluates advances in the cultivation of previously unculturable bacteria from complex bacterial communities. Methods include the use of dilute nutrient media particularly suited for the growth of bacteria adapted to oligotrophic conditions, and the provision of simulated natural environmental conditions for bacterial culture. This has led to the recovery of 'unculturables' from soil and aquatic environments, likely to be due to the inclusion of essential nutrients and/or signalling molecules from the native environment.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                29 October 2019
                November 2019
                : 7
                : 11
                : 506
                Affiliations
                [1 ]Nanotechnology and Water Sustainability Research Unit, College of Science Engineering and Technology, University of South Africa, Florida Science Campus, Florida 1710, South Africa; coadebo@ 123456yahoo.com (C.O.O.); Mambabb@ 123456unisa.ac.za (B.B.M.)
                [2 ]College of Agriculture and Environmental Sciences, Department of Environmental Sciences, University of South Africa, Florida Science Campus, Florida 1710, South Africa; ramganesh.presidency@ 123456gmail.com
                Author notes
                [* ]Correspondence: msagatam@ 123456unisa.ac.za ; Tel.: +27-636-895-964
                Author information
                https://orcid.org/0000-0003-2930-2179
                https://orcid.org/0000-0002-7104-3599
                https://orcid.org/0000-0002-9621-5051
                Article
                microorganisms-07-00506
                10.3390/microorganisms7110506
                6921039
                31671809
                c834ed2c-45a2-469b-8f5e-7e74f73389af
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 26 August 2019
                : 09 October 2019
                Categories
                Article

                high-throughput sequencing,bacteria,biodiversity,pathogens,wastewater treatment plant

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