Developments in CSIR's water microbiology laboratory and the introduction of molecular research CSIR NRE

Neil Leat

© CSIR CSIR Beyond 60 process Go beyond routine testing and include more innovative research. Microbiology laboratory will introduce molecular methods in research. Interact with clients to ensure interests are taken care of. -Continue to offer testing service. -Refer clients to competent external service provider. -Involve clients in research.

© CSIR Traditional Pour-Plate membrane filtration method. -A 100 ml volume of a water sample is drawn through a membrane filter (0.45 µm pore size). -Bacterial cells are trapped on the membrane surface. -The filter is placed on a petri dish containing m-FC agar and incubated for 24 hours at 44.5 °C.agar -The elevated temperature suppresses the growth of non-faecal bacteria. -As the faecal coliform colonies grow they produce an acid (through fermenting lactose) that reacts with the aniline dye in the agar thus giving the colonies their blue colour. -For enumeration faecal coliform colony density should be between 60 and 150 per plate. -For enumeration of E.coli transfer membrane from m-FC to NA-MUG media.

© CSIR Colilert tests for monitoring Total coliforms and E.coli. -Colilert-18 uses Defined Substrate Technology® -Coliforms use their β-galactosidase enzyme to metabolize ONPG and change it from colorless to yellow. -E.coli use β-glucuronidase to metabolize MUG and create fluorescence.

© CSIR Step 1. Add reagent to sample. Step 2. Pour into Quanti-Tray®/2000 (counts 1 to 2,419) Step 3. Seal in Quanti-Tray® Sealer and place in 35 °C incubator for 18 hours. Step 4. Read results: Yellow wells = total coliforms Fluorescent wells = E. coli Using Colilert tests

© CSIR Easy Ease of use Unit-dosed packaging eliminates media preparation. No repeat testing due to clogged filters or heterotrophic interference. Rapid Under one minute hands-on time. Detects coliforms and E. coli simultaneously in 18 hours or less. No glassware cleaning or colony counting. Colilert is US EPA-approved and is included in Standard Methods for Examination of Water and Wastewater. Accurate Identifies E. coli specifically. Suppresses up to 2 million heterotrophs per 100 mL. Eliminates the subjective interpretation found in traditional methods. Detects a single viable coliform or E. coli per sample. Economical Minimizes evening and weekend work. Up to 12-month shelf life at room temperature. Flexible A Colilert®-18 Snap Pack can be used for presence/absence (P/A) or quantification testing. Quanti-Tray®/2000 provides counts to 2,419/100 mL without dilutions. Colilert-18 Benefits

© CSIR Key areas include the following: -Direct Pathogen Detection: The rapid identification and quantification of pathogens in water. These approaches are likely to have the greatest impact for pathogens which are difficult to culture or analyse such as viruses and protozoan parasites and whose presence is not closely associated with faecal indicator microbes. -Microbial Source-Tracking of faecal pollution: Tracing sources of faecal contamination and facilitating targeted and cost effective remediation efforts. -Microbial Community Analysis: Providing a greater understanding of microbial communities in wastewater treatment environments by studying rRNA sequence heterogeneity. This provides engineers with information needed to manipulate treatment processes so as to optimize microbial community structure for wastewater processing. -Epidemiological studies: In epidemics involving waterborne diseases, modern molecular techniques provide tools for tracing infectious agents. Molecular Research Powerful because it provides access to the vast amount of information encoded in nucleic acid sequences.

© CSIR Faecal source-tracking aims to identify which species contribute to the pollution and to establish the relative importance of each source. -Most useful in situations where faecal indicator bacteria have been identified but there is uncertainty about the significance of contributing sources (humans, gulls and agricultural runoff etc). -Faecal microbes have genotypic characteristics which reflect their origin. -Analysis of these characteristics can be used to identify which hosts are contributing to the pollution. Emerging source tracking techniques include: [1] Analysis of F+RNA coliphage groups. [2] Analysis of 16S rRNA sequences from the order Bacteroidales. [3] Direct detection of host-specific enteric viruses. Faecal Source Tracking

© CSIR Source-tracking using F+RNA coliphages relies on quantifying the ratio of the four subgroups in contaminated water samples. Classical method relies on plaque formation followed by serotyping or genotyping using specific probes. Modern culture-independent methods use real-time PCR assays for the simultaneous quantification of all four subgroups in a single tube (Kirs and Smith, 2007). Faecal Source Tracking 4 F+RNA coliphages subgroups non-human faeces Subgroups I & IV human faeces Subgroups II & III Analysis of F+RNA coliphage serotypes

© CSIR Bacteroidales 16S rRNA analysis: Bacteroidales represent a major component of human and animal faecal flora (10 9 to per gram of faeces). Specific host-associated 16S rRNA sequences used for source-tracking assays. Assays range from a quantitative assay for the detection of overall faecal pollution to assays which discriminated between pollution from humans, horses, dogs, elk, cattle, pigs and avian sources. Faecal Source Tracking

© CSIR Human Bovine Canine Phylogenetic relationships among Bacteroidales 16S rRNA sequences

© CSIR Direct detection of host specific enteric viruses: Host specificity of enteric viruses has made them potential source tracking targets. Detection of a given group of viruses is a clear indication of contamination by a specific host. An obvious challenge with this approach is that virus concentrations in contaminated waters are likely to be relatively low. Conventional nested PCR assays are available for -25 Human Enteroviruses (HEV) -47 Human adenoviruses (HAdV) - 7 Bovine Enteroviruses (BEV) Faecal Source Tracking

© CSIR Freshwater beach, Hamilton Harbour, Lake Ontario, Canada. E. coli concentrations highest in ankle-depth water (177,000CFU/100ml). Two source tracking techniques used. Faecal Source Tracking: An Example Figure – Two-way assignment of Escherichia coli isolates in Bayfront Park Beach sand and water samples to bird or wastewater fecal sources by antimicrobial resistance (left) and rep-PCR DNA fingerprinting (right) analyses. (Edge and Hill, Water Research: : ) Antimicrobial Resistance Rep-PCR

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