REPORT NO: 1068/1/03

February 2003


1. Background and Motivation

Escherichia coli is a typical member of the bacterial flora of the gastrointestinal tract of humans and other warm-blooded animals. Generally they are not associated with adverse health effects. For these and related reasons they are widely used as indicators of faecal pollution in water quality assessment. However, under circumstances E coli may cause serious disease. Wild type E coli is notorious for infections with severe implications when it ends up in places where it does not belong, of which the urinary tract is a typical example.

E coli may also cause severe disease in the gastrointestinal tract. This happens when wild type E coli bacteria are genetically converted by the integration of genes into the host genome which code for the production of virulence factors. These virulence factors turn the normally harmless bacteria into pathogens. A wide variety of virulence factors has been described for E coli. Depending on the virulence factors concerned, pathogenic strains of E coli have been classified into a number of groups.

The origin and purpose of the genetic elements (short sections of nucleic acid which carry one or more genes responsible for the production of the virulence factors) is not clear. It is known that the genetic elements concerned are carried by specific phages which transfer the genetic elements from one E coli bacterium to another. Infection of an E coli bacterium by one of these phages will convert the bacterium into a pathogen. The phages are known to occur in the environment and they seem to serve as a reservoir for the genetic elements which code for the production of the virulence factors. Some of the genetic elements concerned may have been transferred form pathogenic bacteria to E coli by these phages. This theory is supported by the close similarity of the Shiga toxins produced by pathogenic E coli strains to the Shiga toxins of Shigella dysenteriae which causes dysentery with clinical symptoms resembling those caused by E coli 0157:H7. This would seem to suggest that the genetic elements which code for the production of Shiga toxins in E coli were transmitted to E coli from S dysenteriae by phages. The phenomenon of phages carrying genetic elements which convert hosts into pathogens is not uncommon among bacteria, and a number of examples are well known.

The above suggests that eventually research should actually focus on the key to the problem, namely the phages which convert harmless E coli bacteria into life-threatening pathogens. However, this project does not cover research on the phages which carry the genetic elements concerned. This project is restricted to the incidence of E coli pathogens in selected water resources.

Enterotoxigenic strains of E coli (ETEC) are associated with two major clinical syndromes: weanling diarrhoea among children in the developing world, and traveller's diarrhoea typical among people of all ages visiting parts of the world with inadequate hygiene. Epidemiological investigations have implicated contaminated food and water as the most common vehicles for ETEC infection.

Enteropathogenic strains of E coli (EPEC) are an import group of pathogens typically associated with infant diarrhoea in the developing world. Transmission seems to be predominantly by direct faecal-oral transfer via contaminated hands, contaminated weaning foods or contaminated fomites. Water and food have been implicated as the mode of transmission in outbreaks among adults.

Enterohaemorrhagic strains of E coli (EHEC) cause life threatening haemorrhagic colitis and haemolytic uraemic syndrome. These strains of E coli produce potent cytotoxins similar to Shiga toxin produced by Shigella species responsible for dysentery. EHEC infections have also been associated with transmission by water and food.

Waterborne outbreaks of diseases caused by pathogenic strains of E coli have been described in many parts of the world. One such outbreak which received world-wide publicity occurred in the small community of Walkerton in Canada during May 2000. The borehole drinking water supply got contaminated by rainwater runoff which contained cattle faeces. More than 2000 cases of severe illness and 6 deaths are on record. The outbreak serves as an example not only of the health risks constituted by pathogenic E coli in water sources and supplies, but also of the legal and financial implications to all parties concerned. The ensuing court cases involved the responsible water supply utility as well as public health and water affairs authorities concerned. Ministers of Parliament appeared in court to stand trial on charges of negligence and to take responsibility for the outbreak and claims by victims for large sums of money for compensation. Due to world-wide attention and in the interest of transparency, proceedings of the court case were presented on the internet.

The pathogen involved in the Walkerton outbreak was an EHEC strain designated E coli 0157 :H7. This particular strain has a history of outbreaks in many parts of the world resulting in severe illness with a high mortality rate. It is therefore one of the most feared strains of E coli pathogens.

The incidence of waterborne outbreaks of diseases associated with pathogenic strains of E coli would appear to increase. Contamination of water sources is possible in all parts of the world. One reason is that domestic animals, notable cattle and pigs, serve as reservoir for the pathogens. Practical methods for detecting and typing the pathogens are therefore essential. The techniques are required to control the diseases by monitoring the quality of raw and treated water sources, and to monitor the efficiency of treatment and disinfection processes. Efficient control would also require a better understanding of the epidemiology of the E coli pathogens, and the origin and transmission of the genetic elements which turn harmless wild type E coli bacteria into pathogens with serious health and related implications.

Methods used in the past to identify and type pathogenic strains of E coli were labour intensive, expensive and time consuming. Some of the tests even required infection of laboratory animals. More recently molecular techniques have been developed which offer practical approaches to the identification and accurate typing of E coli pathogens. These techniques are based on the genetic identification of the genes which code for the virulence factors responsible for the pathogenicity of E coli. The detection techniques are based on either hybridisation of host nucleic acid with gene probes specific for the pathogenicity genes, or on physical detection of the virulence genes after amplification by specific primers in tests based on the polymerase chain reaction (PCR).

There is no meaningful information on the incidence of pathogenic E coli in water resources in South Africa. To the best of our knowledge there is currently no other laboratory in the country engaged in comparable work. The pathogens are known to occur in South Africa among humans and animals like they do in the rest of the world. In other parts of the world the pathogens have been isolated from rivers as well as swimming pools and drinking water supplies.

2. Objectives

A study of this kind has not previously been carried out in South Africa. The results were expected to have meaningful benefits for practical technology and expertise on water quality assessment with regard to potentially important pathogens.

3. Literature Review

A literature review is presented in the Project Report. Further details have been recorded by Muller (2002) and also appear in publications which emanated from this project.

4. Results

Two multiplex (triplex) PCR procedures were successfully established for the detection of the virulence factors of E coli pathogens in water. The new techniques were applied in a survey of water at 26 sites in the Vaal Barrage Reservoir drainage basin. Another multiplex PCR was established for the detection of the Stx1 and -2 toxin genes of EHEC E coli strains, notably E coli 0157, after preliminary selection of these bacteria on a selective agar growth medium.

The two triplex PCR procedures were designed to detect the E coli virulence genes ST and LT (ETEC strains), BFP (Bundle Forming Pilus gene of EPEC strains), eaeA, CNF (Cytotoxic Necrotising Factor gene) and the EIEC invasiveness plasmid gene. One or more of these genes were detected in all but 47 (26 %) of 180 E coli isolates from the Vaal Barrage. The eaeA gene was detected in 101 (56 %) of the isolates, the CNF gene in 96 (53 %), the BFP gene in 25 (13.8 %), the ST gene in 23 (12.7 %), the EIEC gene in 7 (3.8 %) and the LT gene in 3 (1.7%).

A total of 204 samples was analysed for the presence of the Stx1 and Stx2 genes, the haemolysin plasmid gene and E coli 0157 specific genes. The haemolysin plasmid gene was detected in 7 (3.4 %), the Stx1 gene in 2 (0.98 %) and the Stx2 in 1 (0.49 %). One of the Stx1 isolates also carried the haemolysin plasmid. No E coli 0157 specific genes were detected in any of the isolates.

The multiplex PCR procedure established in this project for the detection and typing of E coli pathogens in water proved reliable, rapid (colonies are typed within 4-6 hours) and cost effective since two PCR reactions screen for six genes simultaneously.

Since no E coli O157:H7 pathogens were detected in the survey, it was decided to improve the isolation method. The new procedure is based on preliminary enrichment of test samples by means of peptone-saline containing three antibiotics to suppress background growth. The commercial immuno-magnetic (IMS) procedure is then used to specifically recover E coli 0157 :H7 from the enrichment cultures. The technique is based on magnetic beads covered with specific monoclonal antibodies which bind E coli 0157:H7. The beads are then plated on growth media designed for the selective cultivation of E coli 0157:H7 colonies. Suspect colonies are picked from these plates and tested for E coli 0157:H7 by means of biochemical tests, agglutination with E coli 0157 and H7 antisera, and molecular techniques to detect genes coding for toxicity factors.

An assessment of the sensitivity of the new enrichment-IMS-selective agar procedure revealed that it was capable of detecting one E coli 0157:H7 per ml of seeded samples of sewage, river water, grounded beef and milk. The results confirmed that this procedure was more sensitive than any method previously used for the isolation of E coli O157:H7 from water.

The new enrichment-IMS-selective agar procedure was then applied in a survey for E coli 0157 :H7 in selected samples of sewage, river water, grounded beef and milk. At least one E coli O157:H7 organism was successfully isolated from a sewage sample. This is the first time these pathogens have been isolated from sewage in South Africa.

This project confirms for the first time in South Africa the presence of a variety of E coli pathogens in sewage-contaminated river water, and E coli 0157 :H7 in sewage.

The incidence of E coli 0157:H7 in the water environments concerned, appeared lower than might be expected. The apparent low incidence of E coli 0157 :H7 may at least in part be due to observed shortcomings of the final selective cultivation procedure of the isolation procedure used. This warrants attempts to further improve the isolation procedure for E coli 0157 :H7 . Unfortunately it was not possible to assess the findings by comparison to data elsewhere because no meaningful studies of this kind have as. yet been reported from other parts of the world.

If indeed the incidence of E coli 0157 :H7 is as low in sewage and river water as indicated by the results of this study, these pathogens would appear not to constitute a meaningful risk to consumers at least for purposes such as recreation and possibly even domestic use. However, results obtained and experience gained suggest the findings may under-estimate the situation (see Chapter 4).

However, the finding that some 74 % of E coli bacteria in water of the Vaal River Barrage drainage basin carry one or more toxicity factors indicates reason for concern (see Chapter 3). The available information is inadequate for a meaningful assessment of the public health risk constituted by these pathogens. However, it is known that E coli which carry one or more of these toxicity factors are capable of causing gastro-intestinal disease in humans. The clinical intensity of the illness depends on a variety of factors. Even if the disease was not as severe as potentially caused by E coli 0157:H7, it cannot be ignored. In an increasing component of consumers, notably the very young and very old as well as immunocompromised and undernourished individuals, these E coli pathogens may cause serious health implications. The high risk component of consumers referred to tends to increase in most parts of the world. South Africa is no exception. On the contrary, the immunocompromised component of consumers in many communities is exceptionally high due to the incidence of AIDS.

A major benefit of the project is that expertise and technology have been established in an area of water quality analysis of particular public health importance. An infrastructure is in place for further research on the incidence of these pathogens in water resources and supplies. Further research is essential for meaningful assessment of the public health implications of these pathogens. This information is required to formulate control strategies and define quality specifications.

5. Cost estimates

The isolation of E coli pathogens from water is a complex process which consists of a number of steps and tests. Not all steps and tests are required for the confirmation and typing of all E coli pathogens. It is therefore not possible to quote a fixed tariff for the isolation, confirmation and typing of E coli pathogens in water. On average the cost may amount to approximately R 700 for an E coli 0157 :H7 isolate. In addition, the running cost of the tests is probably not the most expensive part of the work. The tests require special laboratory facilities, particularly for the molecular work. The tests are complicated and require staff with high levels of training in advanced technology and expertise.

This implies the tests are relatively expensive and not feasible for all laboratories engaged in routine water quality analysis. The tests are therefore best intended for specialist laboratories which render the work as a service to smaller laboratories as and when required. Large scale testing also reduces the cost of the tests. This would place specialist laboratories in a position to conduct research on aspects such as the incidence of the pathogens and their epidemiology, as well as improvement of techniques for the isolation of the pathogens from water and the typing of isolates.

6. Capacity building

7. Technology transfer

8. Conclusions

All the objectives of the project have been accomplished.
New techniques for the detection and typing of pathogenic strains of E coli have been established and successfully applied in practice.
Results of the first survey of its kind in South Africa revealed the presence of a substantial number of E coli strains carrying one or more virulence factors in water sources used for recreational purposes and the production of drinking water supplies.

9. Future Research

10. Publications and presentations emanating from the Project

10.1. Conference presentations

Müller, E.E., Clay, C.G. and Grabow, W.O.K. (2000) Detection and isolation Escherichia coli 0157:H7 from sewage and environmental waters using immunomagnetic separation. Water Institute of Southern Africa. (WISA 2000 Conference). Sun City, South Africa 31 May-2 June 2000. Poster presentation.

Müller, E.E., Clay, C.G. and Grabow, W.O.K. (2000) Improvement of the immunomagnetic separation method to detect Escherichia coli 0157 :H7 in sewage and environmental waters. The 1st World Congress of the International Water Association (IWA). Conference Preprints nr 7 (HRMP-A40). Health-Related Water Microbiology. Paris, France 3-7 July 2000. Poster presentation.

Müuller, E.E., Taylor, M.B., Grabow, W.O.K. and Ehlers, M.M. (2001) Isolation and Characterization of Escherichia coli 0 157:H7 and Shiga Toxin -converting Bacteriophages from Strains of Human, Bovine and Porcine Origin. The 2nd World Congress of the International Water Association (IWA). (B0308). Health-Related Water Microbiology. Berlin, Germany 15-19 October 2001. Oral presentation

10.2. Publications

Müller, E.E., Ehlers, M.M. and Grabow, W.O.K. (2001) The Occurrence of E. coli 0157:H7 in South African Water Sources Intended for Direct and Indirect Human Consumption. Water Research. 35, 3085-3088

Müller, E.E., Grabow W.O.K., and Ehlers, M.M. (2001) Application of the Immunomagnetic Separation Method for the Detection and Isolation of Escherichia coli 0157:H7 from Grounded Beef, Milk, Sewage and Environmental Waters. Submitted for publication to: Journal of Medical Microbiology

Müller, E.E., Taylor, M.B., Grabow, W.O.K. and Ehlers, M.M. (2001) Isolation and Characterization of Escherichia coli 0157:H7 and Shiga Toxin -converting Bacteriophages from Strains of Human, Bovine and Porcine Origin. Submitted for publication to: Water, Science and Technology.

Müller, E.E., Grabow W.O.K., and Ehlers, M.M. (2001) Host range susceptibility of toxin- converting bacteriophages infecting Escherichia coli 0157:H7. Submitted for publication to: Journal of Medical Microbiology.