The New Scientific Advancement

Bacterial Source Tracking(BST)

Overview

• Section 1- The Current Status

• Section 2 – Scientific Advancement: Bacterial Source Tracking

(BST)

• Conclusion

• Discussion

Section 1• Faecal contamination of water is a

problem throughout the world due to human health and safety concerns

• Impacts caused by harvesting closures are one of the main impediments to shellfish industry development

• For example: Table 1: Number of New Closures and Re-Opened Closure Orders for BC…In 2000, approx 105,000 ha closed to harvesting

Year 199119921993199419951996199719981999Total

No. of NewClosures 7 13 12 30 5 4 15 25 4 105

No. of ClosuresRevoked

(Re-opened)4 5 3 2 2 2 18

Source: EC, 2001.

The New Scientific Advancement

Bacterial Source Tracking(BST)

Overview

• Section 1- The Current Status

• Section 2 – Scientific Advancement: Bacterial Source Tracking

(BST)

• Conclusion

• Discussion

Section 1• Faecal contamination of water is a problem

throughout the world due to human health and safety concerns

• Impacts caused by harvesting closures are one of the main impediments to shellfish industry development

• For example: Table 1: Number of New Closures and Re-Opened Closure Orders for BC…In 2000, approx 105,000 ha closed to harvesting

Year 199119921993199419951996199719981999Total

No. of NewClosures 7 13 12 30 5 4 15 25 4 105

No. of ClosuresRevoked

(Re-opened)4 5 3 2 2 2 18

Source: EC, 2001.

Section 1 Continued• In Canada/US, monitoring programs use

“indicator” species- so called because their presence indicates that faecal contamination may have occurred

• The MTF five tube dilution test has been used for more than 60 yrs as the indicator test…but can/will never address one of the major questions

• What is the source of the contamination?• New methods called BST are under development

to address this question.

– Knowing the source (CAUSE) rather than monitoring the level of microbial pollution (SYMPTOMS) was the driving force behind the development of BST methodologies

Background: Faecal Coliforms

“Indicator” Organisms (Coliforms)

• Difficulties in the detection and identification of viruses and bacteria in the environment and in food samples led to the use of indicator organisms.

What is Faecal Contamination?

• Faecal coliforms: bacteria (such as E. coli ) that live in the digestive tract of all warm-blooded animals and are excreted in the faeces have been chosen as the indicator of the presence of disease-causing (pathogenic) viruses and bacteria.

• Faecal coliforms: (generally)do not pose a danger to people or animals, but indicate the presence of other disease-causing bacteria and viruses, such as those that cause typhoid and Hep A

Facts: Faecal Coliforms

• While E. coli is known to be faecal in origin, others within this group, for example: Klebsiella are found in soils and vegetation

• The non-faecal biotypes are frequently associated with runoff, have a tendency to multiply in nutrient-rich waters and give false-positives when testing for faecal contaminated waters.

• This is a key issue for shellfish farmers as it results in more frequent closures…and are unnecessary

How Do Faecal Coliforms Get into the Water?

• The transport of faecal coliforms to a water body occurs either directly (point source) or indirectly (non-point source)

• Directly = Point Source: refers to a single identifiable source like a pipe

• Indirectly = Non-point sources: typically wet-weather–dominated and diffuse in nature, in that they do not enter water bodies from any single point (e.g. urban litter, contaminated refuse, domestic pet/ wildlife excrement and failing sewer lines).

Limitations of the MTF Method• 1) The MTF technique is not E. coli specific.

E. coli was recommended to be used as the indicator organisms instead of faecal coliforms because it gave a more accurate measurement. So far, this is not a CSSP standard.

• 2)Faecal coliforms have been isolated in pristine areas

• 3) Bacteria other than those that originated in the colon can yield a positive faecal coliform test, for example, Klebsiella

• 4) MTF testing is a slow process. People may become sick before test results are released

• 5) MTF does not give specific information for source identification.

Summary of Section 1

• Goals – 1) Human health and safety issues – this is

imperative– 2) Maximize certainty for growers (e.g.

cashflow, markets)

• But…the current system is only achieving the first goal….and how well?

• Question – are there new tools available?

Section 2:The New Scientific

Advancement

BST

BST Methods: 4 Types• Molecular Methods

(MST)– Fingerprinting/Ribotyping– Pulse Field Gel

Electrophoresis (PFGE) – Randomly Amplified

Polymorphic DNA (RAPD)

• Biochemical Methods (Phenotype)– ß-glactosidase test

• Colilert Test: A Result of the ß –glactosidase method

– Multiple Antibiotic Resistance (MAR)

– Sterols or Fatty Acid Analysis– Coliphages– Bacteroides– Coprostanol

• Species-Specific Indicators (BST tracking) *Note*: Not all are listed – Streptococcus bovis– Clostridium

perfringens– Bacteroides fragilis

group– Rhodococcus

coprophilus

• Chemical BST Methods– Detergents/Optical

Brighteners– Fluorescent Dye

Tracing– Caffeine

Background: Molecular Methods: (Microbial Source Tracking or MST)

• A bacteria’s genetic structure are clonal. That is, all descendants of a common ancestral cell are genetically related to each other.

• Over time members of a clone may accumulate genetic changes: diverge from the main group to form one or several new clonal groups.

– The E. coli strain for example, that inhabits the intestines of one species (e.g. humans) is genetically different from the strain that might inhabit another (e.g. cows, dogs, deer or bear).

• MST makes use of this, in order to classify organisms based on their genetic fingerprints into groups of clonal descent.

• When bacteria with an identical genetic fingerprint are isolated from both a polluted site and a suspected animal source, the species can be proven as a contributor

Background: Biochemical Methods

• Most of biochemical methods offer certain advantages over molecular methods:

• less training for lab personnel

• lower/isolate cost

• potential to perform the methods on hundreds of isolates per week (versus a few dozen isolates per week, which is typical for molecular methods).

• Biochemical methods: based on the fact that an organism’s genes actively produce a biochemical substance

• The biochemical substances produced is what is being measured.

Background: Species-specific Indicators

• The extent to which faecal coliforms settle, grow, and are re-suspended after they are released into receiving waters remains controversial, leaving most indicator testing/accuracy in question.

• Testing whether there are better suited alternate indicators than the feacal coliforms though...remains relevant

Background: Chemical BST Methods

• Chemical BST methods do not detect faecal bacteria, but chemical compounds that are associated with humans.

• These chemicals are often found in wastewater such as septic tank effluent…if found in a water body, then it is likely from a human source

$$$ Costs $$$

All US Funds Ribotyping analysis 5-8 isolates: $320.00 / sample

MAR analysis of 20 E. coli isolates$200.00 / sample

Pulse field gel electrophoresis:Genetic fingerprinting analysis of bacterial species. 2 restriction enzymes $180.00 /bacterial isolate

Conclusion

• There is no evidence that any one BST method will emerge as the single best method for all situations

• Comparative test b/t the BST methods with the same collection of isolates has yet to be done on a sufficiently large scale.

• Obtaining similar results with different BST methods may also improve the chances that the source identifications are correct.

• A “toolbox” approach may seem warranted. 1st.. one could save money using Biochemical or Chemical methods. Then verify with MST afterward

• Therefore...a corroboration of results using different methods

Conclusion• Regulations are currently aimed at preventing

faecal contamination from any mammalian source

• BUT...Despite efforts to minimize faecal input into coastal waterways a problem remains: – The inability to identify the source

• It is also known that:

– the MTF technique will never address one of the major questions that have perplexed water quality managers for years…what is the source of the contamination?

• Better-suited testing alternatives may be used to refine closures while still protecting consumers

Conclusion

• BST Could be used as a tool for the ID and remediation of upland sources of contamination in sanitary surveys…. in advance of the validation of techniques

• BST could be used as a preventative measure instead of a mechanism to minimize or reduce water contamination.

• BST = False positive prevention…e.g. Klebsiella

• In the meantime, as the costs of BST declines, their use as a tool for prevention of contamination may prove the best application

Conclusion

• The advantages provided by BST will only be captured if there is a willingness to adopt these new techniques into regulation as they are validated

The End

Section 1 Continued• In Canada/US, monitoring programs use

“indicator” species- so called because their presence indicates that faecal contamination may have occurred

• The MTF five tube dilution test has been used for more than 60 yrs as the indicator test…but can/will never address one of the major questions

• What is the source of the contamination?• New methods called BST are under development

to address this question.

– Knowing the source (CAUSE) rather than monitoring the level of microbial pollution (SYMPTOMS) was the driving force behind the development of BST methodologies

Background: Faecal Coliforms

“Indicator” Organisms (Coliforms)

• Difficulties in the detection and identification of viruses and bacteria in the environment and in food samples led to the use of indicator organisms.

What is Faecal Contamination?

• Faecal coliforms: bacteria (such as E. coli ) that live in the digestive tract of all warm-blooded animals and are excreted in the faeces have been chosen as the indicator of the presence of disease-causing (pathogenic) viruses and bacteria.

• Faecal coliforms: (generally)do not pose a danger to people or animals, but indicate the presence of other disease-causing bacteria and viruses, such as those that cause typhoid and Hep A

Facts: Faecal Coliforms

• While E. coli is known to be faecal in origin, others within this group, for example: Klebsiella are found in soils and vegetation

• The non-faecal biotypes are frequently associated with runoff, have a tendency to multiply in nutrient-rich waters and give false-positives when testing for faecal contaminated waters.

• This is a key issue for shellfish farmers as it results in more frequent closures…and are unnecessary

How Do Faecal Coliforms Get into the Water?

• The transport of faecal coliforms to a water body occurs either directly (point source) or indirectly (non-point source)

• Directly = Point Source: refers to a single identifiable source like a pipe

• Indirectly = Non-point sources: typically wet-weather–dominated and diffuse in nature, in that they do not enter water bodies from any single point (e.g. urban litter, contaminated refuse, domestic pet/ wildlife excrement and failing sewer lines).

Limitations of the MTF Method• 1) The MTF technique is not E. coli specific.

E. coli was recommended to be used as the indicator organisms instead of faecal coliforms because it gave a more accurate measurement. So far, this is not a CSSP standard.

• 2)Faecal coliforms have been isolated in pristine areas

• 3) Bacteria other than those that originated in the colon can yield a positive faecal coliform test, for example, Klebsiella

• 4) MTF testing is a slow process. People may become sick before test results are released

• 5) MTF does not give specific information for source identification.

Summary of Section 1

• Goals – 1) Human health and safety issues – this is

imperative– 2) Maximize certainty for growers (e.g.

cashflow, markets)

• But…the current system is only achieving the first goal….and how well?

• Question – are there new tools available?

Section 2:The New Scientific

Advancement

BST

BST Methods: 4 Types• Molecular Methods (MST)

– Fingerprinting/Ribotyping– Pulse Field Gel Electrophoresis

(PFGE) – Randomly Amplified

Polymorphic DNA (RAPD)

• Biochemical Methods (Phenotype)– ß-glactosidase test

• Colilert Test: A Result of the ß –glactosidase method

– Multiple Antibiotic Resistance (MAR)

– Sterols or Fatty Acid Analysis– Coliphages– Bacteroides– Coprostanol

• Species-Specific Indicators (BST tracking) *Note*: Not all are listed – Streptococcus bovis– Clostridium

perfringens– Bacteroides fragilis

group– Rhodococcus

coprophilus

• Chemical BST Methods– Detergents/Optical

Brighteners– Fluorescent Dye

Tracing– Caffeine

Background: Molecular Methods: (Microbial Source Tracking or MST)

• A bacteria’s genetic structure are clonal. That is, all descendants of a common ancestral cell are genetically related to each other.

• Over time members of a clone may accumulate genetic changes: diverge from the main group to form one or several new clonal groups.

– The E. coli strain for example, that inhabits the intestines of one species (e.g. humans) is genetically different from the strain that might inhabit another (e.g. cows, dogs, deer or bear).

• MST makes use of this, in order to classify organisms based on their genetic fingerprints into groups of clonal descent.

• When bacteria with an identical genetic fingerprint are isolated from both a polluted site and a suspected animal

source, the species can be proven as a contributor

Background: Biochemical Methods

• Most of biochemical methods offer certain advantages over molecular methods:

• less training for lab personnel

• lower/isolate cost

• potential to perform the methods on hundreds of isolates per week (versus a few dozen isolates per week, which is typical for molecular methods).

• Biochemical methods: based on the fact that an organism’s genes actively produce a biochemical substance

• The biochemical substances produced is what is being measured.

Background: Species-specific Indicators

• The extent to which faecal coliforms settle, grow, and are re-suspended after they are released into receiving waters remains controversial, leaving most indicator testing/accuracy in question.

• Testing whether there are better suited alternate indicators than the feacal coliforms though...remains relevant

Background: Chemical BST Methods

• Chemical BST methods do not detect faecal bacteria, but chemical compounds that are associated with humans.

• These chemicals are often found in wastewater such as septic tank effluent…if found in a water body, then it is likely from a human source

$$$ Costs $$$

All US Funds Ribotyping analysis 5-8 isolates: $320.00 / sample

MAR analysis of 20 E. coli isolates$200.00 / sample

Pulse field gel electrophoresis:Genetic fingerprinting analysis of bacterial species. 2 restriction enzymes $180.00 /bacterial isolate

Conclusion

• There is no evidence that any one BST method will emerge as the single best method for all situations

• Comparative test b/t the BST methods with the same collection of isolates has yet to be done on a sufficiently large scale.

• Obtaining similar results with different BST methods may also improve the chances that the source identifications are correct.

• A “toolbox” approach may seem warranted. 1st.. one could save money using Biochemical or Chemical methods. Then verify with MST afterward

• Therefore...a corroboration of results using different methods

Conclusion• Regulations are currently aimed at preventing

faecal contamination from any mammalian source

• BUT...Despite efforts to minimize faecal input into coastal waterways a problem remains: – The inability to identify the source

• It is also known that:

– the MTF technique will never address one of the major questions that have perplexed water quality managers for years…what is the source of the contamination?

• Better-suited testing alternatives may be used to refine closures while still protecting consumers

Conclusion

• BST Could be used as a tool for the ID and remediation of upland sources of contamination in sanitary surveys…. in advance of the validation of techniques

• BST could be used as a preventative measure instead of a mechanism to minimize or reduce water contamination.

• BST = False positive prevention…e.g. Klebsiella

• In the meantime, as the costs of BST declines, their use as a tool for prevention of contamination may prove the best application

Conclusion

• The advantages provided by BST will only be captured if there is a willingness to adopt these new techniques into regulation as they are validated

The End