versión impresa ISSN 0038-2353
S. Afr. j. sci. v.104 n.1-2 Pretoria enero/feb. 2008
RESEARCH IN ACTION
C.N. AtebaI, *; M. MbeweI; C.C. BezuidenhoutII
ICentre for Animal Health Studies, North-West University (Mafikeng Campus), Private Bag X2046, Mmabatho, South Africa
IISchool of Environmental Sciences and Development, North-West University (Potchefstroom Campus)
We have determined the prevalence of Escherichia coli O157 strains in cattle, pigs and humans. Eight hundred faecal samples were analysed, but only those isolates that satisfied all primary (oxidase and the triple sugar iron) and secondary identification criteria (API 20E) for E. coli are reported. A total of 294 E. coli isolates were further analysed for E. coli O157 characteristics by the slide agglutination test with E. coli O157-specific monovalent antiserum. Seventy-six E. coli O157 isolates were positively identified. In this group of isolates, the prevalence was higher in pigs (44-50%) than in cattle (5.4-20%) and humans (7.5%). Within the pig isolates, the prevalence was higher in the commercial (51%) than in the communal (44%) pigs. Similarly, the prevalence was higher in faeces from commercial (14-20%) than communal cattle (5.4%). This study highlights the need for correct hygiene, especially with commercial farming of cattle and pigs, and the processing of their products. Through this precaution, prevalence of E. coli O157 in farm animals can be minimized, preventing spread from animals to humans.
Escherichia coli O157:H7 has been identified as an important pathogen that causes disease in humans in developing countries, particularly in the southern hemisphere, including South Africa.1 A causative factor is the faecal contamination of food.2 Undercooked or ready-to-eat food items have been identified as a reason for some outbreaks.3 This pathogen can also be transmitted by person-to-person contact, and by drinking contaminated water.4 The prevalence of E. coli O157 in humans depends on the frequency and severity of exposure to this pathogen.5 E. coli can be the cause of diseases in humans that range from diarrhoea to the more complicated haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC).6 The disease mechanism is through the production of virulent gene products that include shiga toxins (stx1, stx2), enterohaemolysin (hlyA) and the attachment and effacing lesion gene (eae).7,8 There is inadequate information published on the prevalence of E. coli O157 in faeces of animals, especially pigs,1,9 in South Africa. While only three cases of human infection by this pathogen are recorded in South Africa, we ascribe this to the fact that patients rarely report their cases to a hospital.1,10,11 E. coli O157 can contaminate meat as a result of poor animal management practices, transmitting the pathogen to humans. We report on the prevalence of E. coli O157 in human, cattle and pig faecal samples, and discuss the health risk that it may present to South African meat consumers.
Materials and methods
Sample collection and microbiological analysis
Analytical protocol was as previously reported12 but with some modifications. In our study, we used MacConkey broth that is generally recommended for the selection and recovery of Enterobacteriaceae. Human stool samples were collected from a local provincial hospital in the North West province of South Africa. Pig faecal samples were collected from a communal and a commercial farm in Tlapeng and Mareetsane, respectively. Cattle faecal samples were obtained from a communal farm in Mogosane and two commercial farms in Lichtenburg and Rustenburg, respectively. Animal faecal samples were collected directly from the rectum of animals using sterile arm-length gloves and the material placed in sterile sample collection bottles. Samples were immediately transported on ice to the laboratory for analysis. A loopful of the faecal sample was inoculated into MacConkey broth medium (3 ml) [Biolab, Merck (South Africa)] and incubated at 37°C for 18 to 24 hours. After incubation, a tenfold serial dilution with sterile distilled water was performed, and aliquots (100 µl) of each dilution plated onto Sorbitol MacConkey agar (Biolab, Merck). Plates were incubated at 37°C for 18 to 24 hours. Potential E. coli isolates were subcultured onto Sorbitol MacConkey agar and these plates were incubated at 37°C for 18 to 24 hours. Isolates were Gram stained13 and pure Gram-negative rods were selected for biochemical identification.
Primary and secondary identification tests are described in the Appendix.
Experimental results are all recorded in Table 1. In total, 76 E. coli O157 samples were isolated during our study. These comprised 37 and 23 isolates obtained from commercial and communal pigs, respectively, and only three from human stool samples. Similarly, only five and four isolates were identified from the faecal samples of commercial cattle in Rustenburg and Lichtenburg, respectively. Furthermore, only four E. coli O157 samples were isolated from the faecies of cattle from the communal farm at Mogosane.
Table 1 indicates that the overall prevalence of E. coli O157 in pig faeces (44-50%) is higher than that in cattle (5.4-20%) and human stool samples (7.5%). Interestingly, the prevalence of E. coli O157 is relatively higher in the faeces samples of animals that have been farmed commercially, rather than communally. The results of our study on the prevalence of E. coli O157 are higher than those previously reported.1,9 In one of these studies, only 10 E. coli O157 isolates were from pigs with haemolytic colitis.9 Similarly, despite the fact that the animals at the Mogosane communal farm were all suffering from diarrhoea, it was surprising that the prevalence of E. coli O157 was lower in these animals than in non-diarrhoea commercial animals (Table 1). Commercial farm animals are kept closer together, facilitating mutual faecal and other forms of contamination. Animals in the communal (free-range) environment can acquire pathogens through rainfall runoff, or soil contamination, such as by human excreta. The prevalence of E. coli O157 in pig faeces contradicts the view that pigs are not potential hosts to these pathogens.16 The greater incidence of E. coli O157 in pig faeces over cattle faeces may be attributable to differences in the hygiene management practices of the respective animals. A high market demand for pork in South Africa amplifies the risk that diseased animals pose to human health.
Cattle, rather than pigs, have been identified as the main reservoir of E. coli O157 strains, due to the higher pH in the gastro-intestinal tract9,17 of cattle, compared with pigs. Our results contradict these findings, as we found that the prevalence in cattle is lower than in pigs. Nonetheless, our results are in agreement with other published data of 7.5-8% in adult cows.18,19 The higher prevalence of E. coli O157 in faecal samples from commercially farmed animals stresses the need for improved hygiene practices on farms. The faecal shedding rates of E. coli O157 by animals, especially cattle, change with season.20 Our study does not take account of this variable.
The prevalence of E. coli O157 in humans can be linked to the degree of exposure to contaminated animal products. Poor animal hygiene has been identified by others as the contributor to human infections.2,7 Our study shows that only three cases of E. coli O157 were diagnosed from isolated stool samples of patients who visited the hospital with diarrhoea. The reason we give is that few diarrhoea patients visit the hospital. Diagnosis of E. coli O157 diarrhoeal cases can also be hampered by the possible presence of other diarrhoea-causing pathogens.21 E. coli O157 was found to be present in a small proportion (7.5%) of the human stool samples analysed and can be the causative agent of diarrhoea in these cases. Our study did not investigate the role that pigs and/or cattle might play in the transmission of E. coli O157 to humans in North West province. The results obtained in this study highlight the need for responsible hygienic practices to control the incidence of E. coli O157 in farm animals and prevent its spread from animals to humans. More research is needed that would involve sampling animal faecal samples concurrently with meat products, water samples and human stool samples, so as to identify clearly the direct roles that animals play in the transmission of these pathogens to humans. This may facilitate the identification of the source of contamination during outbreaks of infections.
This work was supported by funds made available by the Department of Animal Health, North-West University (Mafikeng Campus). We thank the technical staff and postgraduate microbiology students for technical support. We also thank Etienne E. Müller of the Department of Medical Virology, University of Pretoria, for providing the control strains used in this study.
1. WHO (1997). Prevention and control of enterohaemorrhagic Escherichia coli infections. In Report of a WHO Consultation, Geneva, 28 April-1 May. [ Links ]
2. Dunn J.R., Keen J.E., Moreland D. and Thompson R.A. (2004). Prevalence of Escherichia coli O157:H7 in white-tailed deer from Louisiana. J. Wildlife Dis. 40, 361-365. [ Links ]
3. Hilborn E.D., Mshar P.A., Fiorentino T.R., Dembek Z.F., Barrett T.J., Howard R.T. and Carter M.L. (2000). An outbreak of Escherichia coli O157:H7 infections and haemolytic uraemic syndrome associated with consumption of unpasturized apple cider. Epidemiol. Infect. 124, 31-36. [ Links ]
4. Paunio M., Pebody R., Keskimäki M., Kokki M., Ruutu P., Oinonen S., Vuotari V., Siitonen A., Lahti E. and Leinikki P. (1999). Swimming-associated outbreak of Escherichia coli O157:H7. Epidemiol. Infect. 122, 1-5. [ Links ]
5. Galland J.C., Hyatt D.R., Crupper S.S. and Acheson D.W. (2001). Prevalence, antibiotic susceptibility and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots. Appl. Environ. Microbiol. 67, 1619-1627. [ Links ]
6. Bell C. (2002). Approach to the control of entero-haemorrhagic Escherichia coli (EHEC). Int. J. Food Microbiol. 78, 197-216. [ Links ]
7. Riley L.W., Remis R.S., Helgerson S.D., McGee H.B., Wells J.G., Davis B.R., Hebert R.J., Olcott E.S., Johnson L.M, Hargrett N.T., Blake P.A. and Cohen M.L. (1983). Haemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308, 681-685. [ Links ]
8. Beutin L., Geier D., Steinruck H., Zimmermann S. and Scheutz F. (1993). Prevalence and some properties of verotoxin (shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J. Clin. Microbiol. 31, 2483-2488. [ Links ]
9. Müller E.E., Taylor M.B., Grabow W.O.K. and Ehlers M.M. (2001). Isolation and characterisation of Escherichia coli O157:H7 and shiga toxin-converting bacteriophages from strains of human, bovine and porcine origin. Wat. Supp. 2, 29-38. [ Links ]
10. Browning N.G., Botha J.R. and Sacho et al. (1990). Escherichia coli O157:H7 haemorrhagic colitis. Report of the first South African case. S. Afr. J. Surg. 28, 28-29. [ Links ]
11. Müller E.E., Ehlers M.M. and Grabow W.O.K. (2001). The occurrence of E. coli O157:H7 in Southern African water sources intended for direct and indirect human consumption. Wat. Res. 35, 3085-3088. [ Links ]
12. Dodd C.C., Sanderson M.W., Sargeant J.M., Nagaraja T.G., Oberst R.D., Smith R.A. and Griffin D.D. (2003). Prevalence of Escherichia coli O157 in cattle feeds in Midwestern Feedlots. Appl. Environ. Microbiol. 69, 5243-5247. [ Links ]
13. Cruikshank R., Duguid J.P., Marmoin B.P. and Swain R.H. (1975). In Medical Microbiology, 12th edn, chap 2, pp 3-4. Longman, New York. [ Links ]
14. United States Pharmacopeial Convention, Inc. (2001). The United States Pharmacopeia 25. Rockville, MD. [ Links ]
15. Forbes A.B. and Weissfeld A.S. (1998). Bailey and Scott's Diagnostic Microbiology, 10th edn. Mosby, St Louis, MO. [ Links ]
16. Booher S.L., Cornick N.A. and Moon H.W. (2002). Persistence of Escherichia coli O157:H7 in experimentally infected swine. Vet. Microbiol. 89, 69-81. [ Links ]
17. Schouten J.M., Bouwknegt M., van de Giessen A.W., Frankena K., De Jong M.C. and Graat E.A.M. (2004). Prevalence estimation and risk factors for Escherichia coli O157 on Dutch dairy farms. Prevent. Vet. Med. 64, 49-61. [ Links ]
18. Van-Donkersgoed J., Graham T. and Gannon V. (1999). The prevalence of verotoxins: Escherichia coli O157:H7 and Salmonella in the faeces and rumen of cattle at processing. Can. Vet. J. 40, 332-338. [ Links ]
19. Wells J.G., Shipman L.D., Greene K.D., Sowers E.G., Green J.H., Cameron D.N., Downes F.P., Martin M.L., Griffin P.M., Ostroff S.M., Potter M.E., Tauxe R.V., Wachsmuth. I. and Wachsmuth K. (1991). Isolation of Escherichia coli serotype O157:H7 and other shiga-like toxin-producing E. coli from dairy Cattle. J. Clin. Microbiol. 29, 985-989. [ Links ]
20. Elder R.O., Keen J.E., Siragusa G.R., Barkocy-Gallagher G.A., Koohmaraie M. and Laegreid W.W. (2000). Correlation of enterohaemorrhagic Escherichia coli O157 prevalence in faeces, hides and carcasses of beef cattle during processing. Proc. Natl Acad. Sci. USA 97, 2999-3003. [ Links ]
21. Abu-Elyazeed R.R., Wierzba T.F., Frenck R.W., Putnam S.D., Rao M.R., Savarino S.J., Kamal K.A., Peruski L.F., JR., Abd-El Messih I.A., El-Alkamy S.A., Abdollah B.N. and Clemens J.D. (2004). Epidemiology of Shigella-associated diarrhoea in rural Egyptian children. Am. J. Trop. Med. Hyg. 71, 367-372. [ Links ]
Primary identification tests.
This test was performed using the TestOxidaseTM reagent (PL.390) from Mast Diagnostics (Neston, Wirral, U.K.) in accordance with the manufacturer's published protocol. A well-isolated pure colony was placed on a filter paper using a sterile wire loop. A drop of the TestOxidaseTM reagent was added onto it and mixed. After 30 seconds, the filter paper was observed for colour change. Isolates that produced a purple colour were presumptively considered to be E. coli.
Triple sugar iron test
Triple sugar iron (TSI) agar obtained from Biolab, Merck (South Africa) was used to assay E. coli content,14 with the substrates glucose, sucrose and lactose at sample concentrations of 0.1, 1.0 and 1.0%, respectively. Results are interpreted as before.15
Secondary identification tests.
API 20E test
The API 20E test was performed in accordance with the manufacturer's protocol (BioMérieux, Marcy l'Etoile, France).
Escherichia coli isolates were further analysed for characteristics of E. coli O157 by slide agglutination test with E. coli O157-specific monovalent antiserum (Mast Diagnostics, Neston, Wirral). E. coli O157:H7 (ATCC 43889 and ATCC 43888) strains were used as positive controls.
Haemolysis on blood agar
Haemolytic activity was determined by culturing on blood agar, supplemented with 5% (v/v) sheep blood.8