MICROBIAL SOURCE TRACKING - FAQs

BioVir Laboratories, Inc. - 1-800-GIARDIA (442-7342) www.biovir.com Rev 3.18.10

What is Microbial Source Tracking?

Microbial Source Tracking (MST) is the attempt to determine the source of fecal pollution within a

watershed.

Different tools can be used in applying a Microbial Source Tracking strategy. Detection of antibiotic

resistance profiles, genetic fingerprinting of isolates and examination of W hole W ater Extracts are all

MST techniques.

BioVir focuses on examination of W hole W ater Extracts using genetic markers relating to the

Bacteroidales group of bacteria.

Why would I want to Microbial Source Track?

Microbial Source Tracking within your watershed can provide important information regarding the Point

Source (structures, pipes, sewage outfalls) and Non-Point Source (runoff, animals) contamination present

in your service area. Determination of human, ruminant and other fecal inputs into surface source or

ground waters can provide a scientific basis for watershed management decisions and can assist in the

efficient use of Utility dollars.

I have one Sample that I want to have tested for Microbial Source Tracking. What will it tell me?

Unfortunately, not a lot. Microbial Source Tracking is effective when a history is developed from samples

taken over time at a particular sampling location. A minimum 3-Month, sampling every other week,

sampling regimen per site is recommended.

What is the best strategy for Microbial Source Tracking?

BioVir believes that the best strategy for Microbial Source Tracking is by examination of W hole W ater

Extracts for the Bacteroidales group of bacteria. Employing this strategy is currently the most time and

cost effective way to generate useful watershed data sets .

Source tracking strategies rely on multiple samples from many sites within a watershed taken over a

period of time. This builds a profile and history within the watershed for the target organisms. Newer

testing strategies (like testing for Bacteroidales) have dramatically reduced the number of samples

required and, as a result, the cost of obtaining useful data sets.

Are there different Approaches to Source Tracking?

Yes.

Among the tools available to Microbial Source Tracking is the technique of checking W hole W ater nucleic

acid extracts by PCR for the presence of source-specific pathogens including the Bacteroidales group of

bacteria. This is called a Library Independent Method of testing. Another technique checks bacterial

isolates (ie. grown in petri dishes) by PCR to determine their genotype. This is called a Library Dependent

Method of testing.

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MICROBIAL SOURCE TRACKING - FAQs

BioVir Laboratories, Inc. 1-800-GIARDIA (442-7342) Rev.3.18.10

Library Dependent vs. Library Independent Approach

Library Independent

The newest and least expensive approach to Microbial Source Tracking is what is known as the library-

independent method. Simply described, the testing targets PCR detection of an extremely common

bacterial group, Bacteroidales, with known genetic markers for general Bacteroides, humans, deer, cattle,

hogs and non-ruminants. No culturing or isolation of individual bacteria is required.

Library Dependent Method

The more expensive approach to Microbial Source Tracking is what is known as a library-dependent

method. W ith library dependent methods a single bacteria type (for example E. coli) is used to track

distribution within the watershed. It is believed that E. coli can be differentiated by source animal/human,

but this will require sampling human and animal fecal pollution (feces) within the watershed. The E. coli

are isolated from the feces, and then E. coli found in watershed water samples are matched with those

found associated directly with the feces to confirm their origin. Obviously, one will need to compile many

E. coli from feces within a watershed for an accurate picture of the source of E. coli in water. The

recommended study plan for this approach is a six-month study with bi-weekly samples and at least 25 E.

coli isolate per sampling site. Finally, the comparison of E. coli can be made to known libraries from the

literature, however, it is strongly recommended that a local library be constructed as this will more

accurately reflect the presence of local E. coli types. In fact the scientific literature has stated that in order

to have an accurate usable library, a minimum of 2000 3000 isolates are desirable.

What are Bacteroidales?

The order Bacteroidales are made up of four families: Bacteroidaceae, Rikinellaceae;Porphyromonadaceae; and Prevotellaceae. In general, these are a group of anaerobic, gramnegative, non-spore forming rod-shaped bacteria that are commonly found in the feces ofhumans and other warm-blooded animals. Although these organisms can be persistent in theenvironment, the presence of Bacteroidales in water is an indication of fecal pollution and thepossible presence of enteric pathogens.

Does Microbial Source Tracking tell me how many of each type of Bacteroidales are inmy sample?

Samples can be tested using a Presence/Absence approach (PCR) or a Semi-QuantitativeApproach (qPCR). Using the qPCR approach a quantitative ratio is developed between thevarious Bacteroidales genotypes present in the sample. You dont get a hard number result butyou do get data regarding the relative quantity of each genotype.

What is a genetic Marker?

A genetic Marker is a short, specific location of a gene or DNA Sequence. Genetic Markers arethe target that PCR uses to determine the presence of genotypes of interest.

MICROBIAL SOURCE TRACKING - FAQs

BioVir Laboratories, Inc. 1-800-GIARDIA (442-7342) Rev.3.18.10

What is PCR?

PCR stands for polymerase chain reaction. This is an analytical technique whereby definedsequence of DNA (or RNA) can be amplified from a very few copies to a detectable level.

What is Real-Time PCR?

Real-Time PCR (sometimes denoted as RT-PCR, note the upper-case in the RT) describesan analytical technique that relies upon an instrument to measure the increase in concentrationof DNA during the PCR. As the DNA sequence target increases during the PCR over time,there is a chemical fluorescent signal that also increases. The instrument follows this increasein real-time. Unfortunately, this does not mean that complete sample analysis is real-time,only this one step of the total procedure. The total procedure consists of many additional stepsthat can add several hours to the completion of the test.

What is reverse-transcriptase PCR?

Reverse-transcriptase PCR (sometimes denoted as rt-PCR, note the lower case in the rt)refers to a PCR technique whereby RNA is first converted to DNA so that the DNA (also knownas complementary or cDNA) can be detected by the PCR method. This method is used todetect RNA-viruses that can cause gastroenteritis. It is unfortunate that there are twoprocedures with similar abbreviations, and sometimes these are confused.

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