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      Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview

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          Abstract

          Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture‐based methods are laborious, time‐consuming, and yield false‐positive results, whereas viable but nonculturable ( VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid‐based, immunology‐based, and biosensor‐based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction ( PCR), digital droplet PCR, real‐time PCR, multiplex PCR, DNA microarray, Next‐generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid‐based methods. Enzyme‐linked immunosorbent assay ( ELISA) and immunofluorescence are classified into immunology‐based methods. Optical, electrochemical, and mass‐based biosensors are grouped into biosensor‐based methods. Overall, these methods are sensitive, specific, time‐effective, and important in prevention and diagnosis of waterborne bacterial diseases.

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          Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform

          With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.
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            The importance of the viable but non-culturable state in human bacterial pathogens

            Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.
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              Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

              The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.
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                Author and article information

                Contributors
                utpalroy@gmail.com
                Journal
                Microbiologyopen
                Microbiologyopen
                10.1002/(ISSN)2045-8827
                MBO3
                MicrobiologyOpen
                John Wiley and Sons Inc. (Hoboken )
                2045-8827
                10 July 2016
                December 2016
                : 5
                : 6 ( doiID: 10.1002/mbo3.2016.5.issue-6 )
                : 901-922
                Affiliations
                [ 1 ] Department of Biological SciencesBirla Institute of Technology and Science, Pilani‐K.K. Birla Goa Campus NH17B Bypass Zuarinagar Goa 403726India
                [ 2 ] Biosensor Lab Department of ChemistryBirla Institute of Technology and Science, Pilani‐K.K. Birla Goa Campus NH17B Bypass Zuarinagar Goa 403726India
                Author notes
                [*] [* ] Correspondence

                Utpal Roy, Department of Biological Sciences, Birla Institute of Technology and Science, Pilani‐ K.K. Birla Goa Campus, NH17B Bypass, Zuari Nagar, Goa 403726, India. Tel: +91‐8322580393; E‐mail: utpalroy@ 123456gmail.com

                Article
                MBO3383
                10.1002/mbo3.383
                5221461
                27397728
                9726f6f9-e579-461b-ba28-efced9016e94
                © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 16 December 2015
                : 20 April 2016
                : 02 May 2016
                Page count
                Figures: 2, Tables: 3, Pages: 22, Words: 16443
                Funding
                Funded by: Birla Institute of Technology and Science, Pilani‐KK Birla Goa campus for Centre of Research Excellence in Water, Wastewater and Energy Management (CORE WWEM)
                Categories
                Review
                Reviews
                Custom metadata
                2.0
                mbo3383
                December 2016
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.0.0 mode:remove_FC converted:09.01.2017

                Microbiology & Virology
                bacteria,biosensor,detection,ngs,pcr,rapid,waterborne
                Microbiology & Virology
                bacteria, biosensor, detection, ngs, pcr, rapid, waterborne

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