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Water Science & Technology Vol 54 No 3 pp 101–107 © IWA Publishing 2006 doi:10.2166/wst.2006.455

Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques
W.Q. Betancourt and R.S. Fujioka
Water Resources Research Center, University of Hawaii, Honolulu, , HI, 96822 USA (E-mail: roges@hawaii.edu)

ABSTRACT

Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because QPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination.

Keywords: Bacteroides; faecal indicators; molecular markers; sewage contamination





Water Science & Technology Vol 54 No 3 pp 119–126 © IWA Publishing 2006 doi:10.2166/wst.2006.457

Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis
Y. Masago*, K. Oguma*, H. Katayama* and S. Ohgaki*
*Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656 Japan
**Department of Civil Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi, 980-8579 Japan (E-mail: masago@water.civil.tohoku.ac.jp)

ABSTRACT

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean =35%, standard deviation =8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

Keywords: Cryptosporidium; denaturing gradient gel electrophoresis (DGGE); genotyping; quenching probe PCR (QProbe PCR); real time PCR



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Water Science & Technology Vol 54 No 3 pp 127–134 © IWA Publishing 2006 doi:10.2166/wst.2006.458

Analysis of enterococci using portable testing equipment for developing countries – variance of Azide NutriDisk medium under variable time and temperature
S. Godfrey*, J. Watkins**, K. Toop*** and C. Francis**
*Water and Sanitation Project, UNICEF, Madhya Pradesh, India (E-mail: sgodfrey@unicef.org)
**CREH Analytical, Leeds, , UK
***Water, Engineering and Development Centre, Dept of Civil and Building Engineering, Loughborough University, Loughborough, , UK

ABSTRACT

This report compares the enterococci count on samples obtained with Azide NutriDisk (AND) (sterile, dehydrated culture medium) and Slanetz & Bartley (SB) medium when exposed to a variable in incubation time and temperature. Three experiments were performed to examine the recovery of enterococci on AND and SB media using membrane filtration with respect to: (a) incubation time; (b) incubation temperature; and (c) a combination of the two. Presumptive counts were observed at 37, 41, 46 and 47 °C and at 20, 24, 28 and 48 h. These were compared to AWWA standard method 9230 C (44 °C, 44 h). Samples were confirmed using Kanamycin Aesculin Azide (KAA) agar. Friedman's ANOVA and Students t-test analysis indicated higher enumeration of enterococci when grown on AND (p=0.45) than SB (p=<0.001) at all temperatures with a survival threshold at 47 °C. Significant results for AND medium were noted at 20 h (p=0.021), 24 h (p=0.278) and 28 h (p=0.543). The study concluded that the accuracy of the AND medium at a greater time and temperature range provided flexibility in incubator technology making it an appropriate alternative to SB medium for monitoring drinking water using field testing kits in developing countries.

Keywords: Developing countries; enterococci; field testing kits; microbiological methods





Water Science & Technology Vol 54 No 3 pp 135–140 © IWA Publishing 2006 doi:10.2166/wst.2006.459

Direct detection of bacterial faecal indicators in water samples using PCR
K. Horáková*, H. Mlejnková** and P. Mlejnek***
*Department of Microbiology, Faculty of Science, Masaryk Univesity, Brno, , Czech Republic
**T.G. Masaryk Water Research Institute, Brno, , Czech Republic
***Department of Biology, Faculty of Medicine, Palacky University Olomouc, , Czech Republic

ABSTRACT

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (b-d-galactosidase) and uidA (b-d-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.

Keywords: Coliform bacteria; Escherichia coli; faecal indicator; PCR





Water Science & Technology Vol 54 No 3 pp 141–145 © IWA Publishing 2006 doi:10.2166/wst.2006.460

Comparison of three different media for the detection of E. coli and coliforms in water
C. Bernasconi*, G. Volponi and L. Bonadonna**
*European Commission, Joint Research Center, Institute for Environment and Sustainability, Via E. Fermi 1, 21020 Ispra (Varese), , Italy (E-mail: camilla.bernasconi@jrc.it)
**Istituto Superiore di Sanita', Rome, , Italy

ABSTRACT

The European Drinking Water Directive defines reference methods for the enumeration of microbiological parameters in drinking water. The method to be used for Escherichia coli and coliforms is the membrane filtration technique on Lactose TTC agar with Tergitol 7. Many technical drawbacks of the procedure, as well as its limitations regarding the recent taxonomy of coliforms, make it necessary to evaluate alternative methods. Two alternative assays, a chromogenic media (m-ColiBlu24®) and a defined substrate technology-DST test (Colilert 18/Quanty Tray™) were compared with the ISO standard with attention to the phenotypic characteristic of the isolates. Results showed that the ISO method failed to detect an important percentage of coliforms and E. coli while m-ColiBlu24® and Colilert 18 provided results in a shorter time allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.

Keywords: Coliforms; E. coli; microbiological methods; water





Water Science & Technology Vol 54 No 3 pp 153–159 © IWA Publishing 2006 doi:10.2166/wst.2006.463

The association of E. coli and soil particles in overland flow
R.W. Muirhead*,**, R.P. Collins*** and P.J. Bremer*
*Dept of Food Science, Otago University, PO Box 56, Dunedin, , New Zealand
**AgResearch, Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel, , New Zealand (E-mail: richard.muirhead@agresearch.co.nz)
***National Institute of Water and Atmospheric Research (NIWA) Ltd. Gate 10 Silverdale Road, PO Box 11-115, Hillcrest, Hamilton, , New Zealand

ABSTRACT

The removal of E. coli from overland flow under saturation-excess runoff conditions was investigated in experimental field plots that were 1 m wide and 5 m long. Variation in the attenuation of bacteria and distance transported was quantified under contrasting flow conditions. In addition, the impact of soil tillage upon microbial attenuation was examined by comparing results derived from grassed plots (intact) with those subject to tillage with the soil left bare (cultivated). For intact plots subjected to a flow of 2 L/min, 27% of the E. coli in the flow was removed after 5 m with removal following a logarithmic function with respect to distance. For the higher flow rates of 6 L/min and 20 L/min, no attenuation trend was observed over this distance. E. coli removal during flow across the cultivated plots was significantly greater compared to the intact plots. This was attributed to a greater infiltration rate in the cultivated plots (due to the tillage) which promoted a greater volume of flow to pass through the soil matrix, providing the opportunity for filtration and adsorption of microbes. Logarithmic trends with respect to distance were observed for all flow rates tested on the cultivated plots (2, 6 and 20 L/min). Total removal after 5 m at a flow rate of 2 L/min was 41% and again removal efficiency decreased as the flow rate increased. Analysis of the transported state of the E. coli revealed that the bacteria were being transported predominantly in particles less than 20 mm in diameter and were not attached to large (dense) soil particles. The limited removal (<50%) of bacteria from overland flow under saturation-excess runoff conditions in these experiments appeared, therefore, to be primarily due to a lack of settling or deposition. Instead, most bacteria remained entrained within the overland flow down the length of the plots.

Keywords: Attachment; cultivation; grass; riparian; runoff; transport







Murulee Byappanahalli, Ph. D.
Research Microbiologist
U.S. Geological Survey, Great Lakes Science Center
Lake Michigan Ecological Research Station,
1100 N. Mineral Springs Road
Porter, Indiana 46304
Phone: (219) 926-8336 ext. 421
Fax:      (219) 929-5792
E-mail: byappan@usgs.gov