scholarly journals Molecular Method for Detection of Total Coliforms in Drinking Water Samples

2014 ◽  
Vol 80 (14) ◽  
pp. 4074-4084 ◽  
Author(s):  
Andrée F. Maheux ◽  
Dominique K. Boudreau ◽  
Marc-Antoine Bisson ◽  
Vanessa Dion-Dupont ◽  
Sébastien Bouchard ◽  
...  
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
Water Samples ◽  
Potable Water ◽  
Total Coliform ◽  
Rrna Genes ◽  
Molecular Assay ◽  
Content Type ◽  
Total Coliforms ◽  
Pcr Assays

ABSTRACTThis work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting thelacZ,wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species ofEnterobacteriaceaeencountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas thelacZ,wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with anEscherichia colimolecular assay to assess drinking water quality.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

scholarly journals Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

2012 ◽  
Vol 78 (6) ◽  
pp. 1909-1916 ◽  
Author(s):  
Hodon Ryu ◽  
John F. Griffith ◽  
Izhar U. H. Khan ◽  
Stephen Hill ◽  
Thomas A. Edge ◽  
...  
Keyword(s):  
16S Rrna ◽  
Water Samples ◽  
Environmental Water ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Recreational Waters ◽  
Fecal Samples ◽  
Content Type

ABSTRACTTwo novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targetingStreptococcusspp. (gull3) and a hydrolysis TaqMan assay targetingCatellicoccus marimammalium(gull4). The objectives of this study were to compare the host specificity of a previousC. marimammaliumqPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n= 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n= 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n= 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.

scholarly journals Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

2001 ◽  
Vol 67 (9) ◽  
pp. 3985-3993 ◽  
Author(s):  
Nele Wellinghausen ◽  
Cathrin Frost ◽  
Reinhard Marre
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Potable Water ◽  
Water Systems ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACT Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionellaspp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed ofLegionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r= 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitativeL. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developedLegionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.

scholarly journals Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

2013 ◽  
Vol 79 (24) ◽  
pp. 7654-7661 ◽  
Author(s):  
Andrée F. Maheux ◽  
Ève Bérubé ◽  
Dominique K. Boudreau ◽  
Romain Villéger ◽  
Philippe Cantin ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Sample ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Potable Water ◽  
Alpha Toxin ◽  
Pcr Assay ◽  
Content Type ◽  
The Public

ABSTRACTWe first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of aClostridium perfringens-specific real-time PCR (rtPCR) assay based on thecpagene (cpartPCR) by using a bacterial strain panel composed ofC. perfringensand non-C. perfringens Clostridiumstrains. All non-C. perfringens Clostridiumstrains tested negative, whereas allC. perfringensstrains tested positive with thecpartPCR, for an analytical specificity and ubiquity of 100%. ThecpartPCR assay was then used to confirm the identity of 116 putativeC. perfringensisolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar andcpartPCR were identified by sequencing the 16S rRNA andcpagenes. Four mCP−/rtPCR+colonies were identified asC. perfringens, whereas 3 mCP+/rtPCR−colonies were identified as non-C. perfringens. ThecpartPCR was negative with all 51 non-C. perfringensstrains and positive with 64 of 65C. perfringensstrains. Finally, we compared mCP agar and a CRENAME (concentration andrecovery of microbial particles,extraction ofnucleicacids, andmolecularenrichment) procedure pluscpartPCR (CRENAME +cpartPCR) for their abilities to detectC. perfringensspores in drinking water. CRENAME +cpartPCR detected as few as oneC. perfringensCFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +cpartPCR also allows the simultaneous and sensitive detection ofEscherichia coliandC. perfringensfrom the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

scholarly journals Halomicrococcus hydrotolerans gen. nov., sp. nov., an extremely halophilic archaeon isolated from a subterranean salt deposit

2020 ◽  
Vol 70 (8) ◽  
pp. 4425-4431 ◽  
Author(s):  
Shaoxing Chen ◽  
Yao Xu ◽  
Siqi Sun ◽  
Jingwen Liu ◽  
Feilong Chen
Keyword(s):  
16S Rrna ◽  
In Silico ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Rrna Genes ◽  
Salt Deposit ◽  
Halophilic Archaeon ◽  
Content Type ◽  
Link Type ◽  
The Family

A halophilic archaeon, strain H22T, was isolated from a subterranean salt deposit sampled at Yunnan salt mine, PR China. Colonies of strain H22T were light pink-pigmented. Cells were coccus, non-motile, Gram-stain-negative, and did not lyse in distilled water. The strain was aerobic and grew at 20–55 °C (optimum, 37 °C), in the presence of 10–30 % (w/v) NaCl (20 %) and at pH 6.5–9.0 (pH 7.0). Mg2+ was required for growth (optimum, 0.005 M). Major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated mannosyl-glucosyl-glycerol diether-1. Sequence similarity search based on the multiple 16S rRNA genes (rrnA, rrnB and rrnC) of strain H22T revealed that it was most closely related to species of the genera Haloarchaeobius , Haladaptatus , Halorussus and Halorubellus with relative low sequence similarities (91.9–93.7 %). The strain, however, shared highest rpoB′ gene sequence identities with Halorussus rarus TBN4T (90.8 % rpoB′ gene sequence similarity). Phylogenetic trees based on 16S rRNA and rpoB′ gene sequences revealed a robust lineage of the strain H22T with members of related genera of the family Halobacteriaceae . The DNA G+C content of strain H22T was 62.9 mol%. Genome-based analysis of average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) between strains H22T and its closest relative were equal or lower than 77.7 and 22.4 %, respectively, which were far below the threshold for delineation of a new species. Based on ANI values, in silico DDH, and distinct morphological and physiological differences from the previously described taxa, we suggest that strain H22T represents a novel species of a new genus within the family Halobacteriaceae , for which the name Halomicrococcus hydrotolerans gen. nov., sp. nov. is proposed. The type strain is H22T (=CGMCC 1.16291T=NBRC 113231T).

scholarly journals Physiochemical and Microbiological Analysis of Drinking Water in Chattogram City, Bangladesh

2020 ◽  
pp. 42-50
Author(s):  
Kanij Fatema Nishan ◽  
Nilufa Yeasmin ◽  
Urmi Rani Devi ◽  
Sumiya Akter ◽  
Md. Abu Bakar ◽  
...  
Keyword(s):  
Water Quality ◽  
Drinking Water ◽  
Water Samples ◽  
Total Coliform ◽  
Drinking Water Quality ◽  
Sampling Location ◽  
Microbiological Analysis ◽  
Alternative Source ◽  
Safe Alternative ◽  
Total Dissolved Solid

Chattogram is the second most populated city in Bangladesh. This port city faces a serious threat mainly due to the lack of safe drinking water. This study was conducted for determining drinking water quality of groundwater sources in Chattogram city. The study was performed in the BCSIR laboratory, Chattogram. It was carried out for a period of six months from 1st July, 2018 to 31th December, 2018. Total six water samples were collected from three different locations (Baluchora, C&B colony and Khulshi area). Each sampling location consists of two separate sampling points. Physicochemical parameters of the collected samples like Temperature, pH, Electrical Conductivity (EC), Total dissolved solid (TDS), Hardness, Turbidity and concentration of Cl, As, Mn, Fe, Pb, Cr and Cd were examined. Microbial parameters like Total Coliform (TC) were also measured. All the analyzed parameters compared with BSTI and WHO drinking water quality standards to understand the overall ground water quality status of the study area. The results reveal that water samples in almost all locations were contaminated with microbial contamination and that the range of physico-chemical parameters was not adequate for consumption. Preliminary treatments like boiling, filtering etc are required before using groundwater for drinking and the necessary measures must be taken for a safe alternative source of drinking water.

scholarly journals Drinking Water Security in the Mid-hill Region of Nepal

2020 ◽  
Vol 24 (1) ◽  
pp. 44-48
Author(s):  
Durga D. Poudel ◽  
Timothy W. Duex ◽  
Roshan Poudel
Keyword(s):  
Land Use ◽  
Drinking Water ◽  
Water Samples ◽  
Water Security ◽  
Total Coliform ◽  
Spring Water ◽  
Land Use Type ◽  
Hill Region ◽  
Land Use Types ◽  
Spring Type

Drinking water security is increasingly becoming a global concern in recent decades. The mid-hill region of Nepal is also experiencing serious water shortages in recent years. In order to assess the availability of drinking water in the mid-hill regions of Nepal, we studied hydrogeology, land use types and collected water samples from 30 springs in Kavre, Kahmandu Valley, Nuwakot and Tanahu in Nepal between July 17-September 12, 2017. For each sampling spring, while surrounding land use type (mixed, agriculture, urban, vegetation) and spring type (fracture, depression, contact) were determined through field observation, the field pH, conductivity and temperature was determined using relevant probes and thermometers. Water samples were collected in 1L and 165mL plastic bottles for chemical and total coliform determination, respectively, in the lab. Bottles were rinsed twice using spring water before filling them with sample water, then stored in an ice chest, and brought to the lab. In the laboratory, turbidity, conductivity, Ca, Mg, HCO3, SO4, Na, NO3, Cl, Fe, As, and total coliform were determined using standard methods. Spring water in agricultural areas showed significantly higher suspended solids compared to other land use types whereas spring water in urban areas showed significantly higher dissolved substances. By spring type, turbidity and conductivity values and the concentration of dissolved constituents (Ca, Mg, HCO3, SO4, NO3, and Cl) were ranked in the order of fracture < contact < depression. Na and Fe concentration were in the order of fracture = contact < depression. By land-use type, conductivity and dissolved constituents (Ca, Mg, HCO3) were in the order of agriculture < vegetation < mixed < urban. Whereas urban land use had the highest values for SO4, Na, NO3, and Cl, other land use types showed variable order. Fe concentration was ranked in the order of urban < mixed < vegetation < agriculture. Total coliform was in the order of mixed < agriculture < urban < vegetation. These results indicate that land use type and surface condition, which is possibly associated with human activities, heavily affect spring water properties in the region. These results suggest that drinking water security of mid-hill region of Nepal is threatened heavily due to poor spring water quality. Protection of drinking water sources should be specific to land use type and activities around the springs. Index Terms— three to six pertinent, specific to the paper, keywords added after the abstract, separated by commas.

scholarly journals Genome-based classification of Calidifontibacillus erzurumensis gen. nov., sp. nov., isolated from a hot spring in Turkey, with reclassification of Bacillus azotoformans as Calidifontibacillus azotoformans comb. nov. and Bacillus oryziterrae as Calidifontibacillus oryziterrae comb. nov.

2020 ◽  
Vol 70 (12) ◽  
pp. 6418-6427
Author(s):  
Ahmet Adiguzel ◽  
Hilal Ay ◽  
Mustafa Ozkan Baltaci ◽  
Sumeyya Akbulut ◽  
Seyda Albayrak ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Hot Spring ◽  
Rrna Genes ◽  
Sequence Comparisons ◽  
Novel Genus ◽  
Content Type ◽  
Sequence Identity ◽  
Link Type ◽  
Bacillus Azotoformans

A novel Gram-stain-positive, rod-shaped, endospore-forming, motile, aerobic bacterium, designated as P2T, was isolated from a hot spring water sample collected from Ilica-Erzurum, Turkey. Phylogenetic analyses based on 16S rRNA gene sequence comparisons affiliated strain P2T with the genus Bacillus , and the strain showed the highest sequence identity to Bacillus azotoformans NBRC 15712T (96.7 %). However, the pairwise sequence comparisons of the 16S rRNA genes revealed that strain P2T shared only 94.7 % sequence identity with Bacillus subtilis subsp. subtilis NCIB 3610T, indicating that strain P2T might not be a member of the genus Bacillus . The digital DNA–DNA hybridization and average nucleotide identity values between strain P2T and B. azotoformans NBRC 15712T were 19.8 and 74.2 %, respectively. The cell-wall peptidoglycan of strain P2T contained meso-diaminopimelic acid. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminophospholipid, five unidentified phospholipids and two unidentified lipids while the predominant isoprenoid quinone was MK-7. The major fatty acids were iso-C15 : 0 and iso-C16 : 0. The draft genome of strain P2T was composed of 82 contigs and found to be 3.5 Mb with 36.1 mol% G+C content. The results of phylogenomic and phenotypic analyses revealed that strain P2T represents a novel genus in the family Bacillaceae , for which the name Calidifontibacillus erzurumensis gen. nov., sp. nov. is proposed. The type strain of Calidifontibacillus erzurumensis is P2T (=CECT 9886T=DSM 107530T=NCCB 100675T). Based on the results of the present study, it is also suggested that Bacillus azotoformans and Bacillus oryziterrae should be transferred to this novel genus as Calidifontibacillus azotoformans comb. nov. and Calidifontibacillus oryziterrae comb. nov., respectively.

scholarly journals Borehole water: a potential health risk to rural communities in South Africa

2018 ◽  
Vol 19 (1) ◽  
pp. 128-136 ◽  
Author(s):  
S. Taonameso ◽  
L. S. Mudau ◽  
A. N. Traoré ◽  
N. Potgieter
Keyword(s):  
Drinking Water ◽  
Rural Communities ◽  
Water Samples ◽  
Rural Areas ◽  
Total Coliform ◽  
Water Sources ◽  
Potential Health ◽  
Point Mapping ◽  
E Coli ◽  
Mapping Tool

Abstract Sporadic outbreaks of diarrhoea in children in the Vhembe rural areas could be an indication of contamination in drinking water sources. In areas where improved water sources are used, not all rural households experience the benefits of these improved water sources. Water samples were collected from boreholes in three wards in the Vhembe District to determine microbiological risks over a 5-month period. A Water Point Mapping tool was used to indicate the borehole distribution. Water samples were taken from each functional borehole and analysed for total coliform and Escherichia coli counts, electrical conductivity, pH and temperature. A multiplex PCR protocol was used for identification of pathogenic E. coli. A total of 125 boreholes were identified of which only 12 were functional. Seven boreholes tested positive for total coliforms and E. coli counts. Four boreholes (33.3%) tested positive for diarrhoeagenic E. coli. Fifty-eight percent (58%) of water samples were without health risks, 17% were low risk and 25% could cause infection according to the South African water quality standards. This study indicated the importance of the role of the Municipalities and the maintenance plans that need to ensure that all boreholes are functional and provide safe drinking water to the rural communities.

scholarly journals Temporal Variability of Coastal Planctomycetes Clades at Kabeltonne Station, North Sea

2011 ◽  
Vol 77 (14) ◽  
pp. 5009-5017 ◽  
Author(s):  
Ilaria Pizzetti ◽  
Bernhard M. Fuchs ◽  
Gunnar Gerdts ◽  
Antje Wichels ◽  
Karen H. Wiltshire ◽  
...  
Keyword(s):  
16S Rrna ◽  
North Sea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Group A ◽  
Related Group ◽  
Group B ◽  
Card Fish ◽  
Group D

ABSTRACTMembers of the bacterial phylumPlanctomycetesare reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution ofPlanctomycetesin surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH). GenerallyPlanctomycetesare more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed thatPlanctomycetesabundance was correlated to theCentralesdiatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ∼90% of thePlanctomycetesreside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, namedPlanctomyces-related group A, unculturedPlanctomycetesgroup B, andPirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. UnculturedPlanctomycetesgroup B was most abundant in autumn samples, whilePlanctomyces-related group A was present in high numbers only during late autumn and winter. The levels ofPirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of thePlanctomycetesis correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

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