Metagenomic 16S rDNA Targeted PCR-DGGE in Determining Bacterial Diversity in Aquatic Ecosystem

Hasan, Nur A.; Chowdhury, Wasimul B.; Rahim, Niaz; Sultana, Marzia; Shabnam, Syeda Antara; Mai, Volker; Ali, Afsar; Morris, Glen J; Sack, R. Bradley; Huq, Anwar; Colwell, Rita R.; Endtz, Hubert P.; Cravioto, Alejandro; Alam, Munirul

doi:10.3329/bjm.v27i2.9171

Cited by 5 publications

(4 citation statements)

References 27 publications

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“…Next generation sequencing is a high-throughput and more quantitative technique for analyzing microbial diversity in comparison with DGGE. The PCR-DGGE approach is cost effective and has been successfully employed previously for the analysis of bacterial communities in surface and ground water systems in Bangladesh (57, 58) and hence the reasons behind application of this technique in the current study for analysis of seasonal variations on bacterial communities. However, through the PCR-DGGE approach seasonal variations on bacterial community structure were only observed in between the dominant bacterial groups.…”

Section: Discussionmentioning

confidence: 99%

Bacterial Community Profiling of Tropical Freshwaters in Bangladesh

Azmuda

Fakruddin

Khan

et al. 2019

Front. Public Health

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Seasonal and spatial variations in the bacterial communities of two tropical freshwater sources in Bangladesh, Lake Dhanmondi in central Dhaka, and a pond in the outskirts of Dhaka, were assessed and compared using PCR-DGGE and deep sequencing of 16S rRNA genes, as well as heterotrophic enrichments using water samples collected at nine different time points during 1 year. Temporal and spatial variations of common aquatic bacterial genera were observed, but no clear seasonal variations could be depicted. The major bacterial genera identified from these two sites were members of the Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Verrucomicrobia , and Firmicutes . Among the proteobacterial groups, members of the α - , β - , and γ - Proteobacteria predominated. γ - Proteobacteria belonging to the Escherichia coli/Shigella group even the diarrheagenic pathotypes of E. coli e.g., EPEC and ETEC were detected in most samples throughout the year, with no apparent correlations with other microbial groups. The other pathotypes, EHEC, EAEC, and EIEC/ Shigella spp. were also detected occasionally. This study represents the first thorough analysis of the microbial diversity of tropical freshwater systems in Bangladesh.

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Section: Discussionmentioning

confidence: 99%

Bacterial Community Profiling of Tropical Freshwaters in Bangladesh

Azmuda

Fakruddin

Khan

et al. 2019

Front. Public Health

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“…To ensure quality of PCR test, positive samples and those that were equivocal were re-run to give the final test results. The samples that were negative by both multiplex reactions were in addition tested for 16S rRNA gene by PCR to confirm bacterial genetic material presence in the extract ( Hasan et al, 2011 ).…”

Section: Methodsmentioning

confidence: 99%

Environmental Surveillance of Vibrio cholerae O1/O139 in the Five African Great Lakes and Other Major Surface Water Sources in Uganda

et al. 2018

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Cholera is a major public health problem in the African Great Lakes basin. Two hypotheses might explain this observation, namely the lakes are reservoirs of toxigenic Vibrio cholerae O1 and O139 bacteria, or cholera outbreaks are a result of repeated pathogen introduction from the neighboring communities/countries but the lakes facilitate the introductions. A prospective study was conducted in Uganda between February 2015 and January 2016 in which 28 selected surface water sources were tested for the presence of V. cholerae species using cholera rapid test and multiplex polymerase chain reaction. Of 322 water samples tested, 35 (10.8%) were positive for V. cholerae non O1/non O139 and two samples tested positive for non-toxigenic atypical V. cholerae O139. None of the samples tested had toxigenic V. cholerae O1 or O139 that are responsible for cholera epidemics. The Lake Albert region registered the highest number of positive tests for V. cholerae non O1/non O139 at 47% (9/19). The peak period for V. cholerae non O1/non O139 positive tests was in March–July 2015 which coincided with the first rainy season in Uganda. This study showed that the surface water sources, including the African Great Lakes in Uganda, are less likely to be reservoirs for the observed V. cholerae O1 or O139 epidemics, though they are natural habitats for V. cholerae non O1/non O139 and atypical non-toxigenic V. cholerae O139. Further studies by WGS tests of non-toxigenic atypical V. cholerae O139 and physicochemical tests of surface water sources that supports V. cholerae should be done to provide more information. Since V. cholerae non O1/non O139 may cause other human infections, their continued surveillance is needed to understand their potential pathogenicity.

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“…All negative samples were further tested for the presence of 16S rDNA to confirm DNA preservation techniques as described by Hasan et al . [20]. PCR was performed according to methods previously described [14].…”

Section: Methodsmentioning

confidence: 99%

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

Ontweka

Deng²,

Rauzier

et al. 2016

PLoS ONE

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Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited.

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Metagenomic 16S rDNA Targeted PCR-DGGE in Determining Bacterial Diversity in Aquatic Ecosystem

Cited by 5 publications

References 27 publications

Bacterial Community Profiling of Tropical Freshwaters in Bangladesh

Bacterial Community Profiling of Tropical Freshwaters in Bangladesh

Environmental Surveillance of Vibrio cholerae O1/O139 in the Five African Great Lakes and Other Major Surface Water Sources in Uganda

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

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