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Sajs.stagingcopy.co.zaAntibiotic resistAnce profiles of EschErichia coli isolAted from
different wAter sources in the mmAbAtho locAlity, north-west
province, south AfricA
Constance N. Wose Kinge1
The antibiotic resistance profiles of Escherichia coli (E. coli), isolated from different water sources in D. Tonderai Kawadza1 the Mmabatho locality were evaluated. Water samples were collected from the local wastewater- and water-treatment plants, the Modimola Dam and homes in the area, and then analysed for the presence of E. coli, using standard methods. Presumptive isolates obtained were confirmed 1Department of Biological by the analytical profile index test. Antibiotic susceptibility testing was performed by the disc Sciences, North-West diffusion method. Of the 230 E. coli isolates tested, marked antibiotic resistances (over 70%) were University, Mafikeng observed for erythromycin, tetracycline, ampicillin, chloramphenicol and norfloxacin. Multiple Campus, South Africa antibiotic resistance patterns were also compiled. Overall, the phenotype T-Ap-E was frequent for E. coli isolated from the local wastewater and water-treatment plants, Modimola Dam and tap water. Cluster analysis performed showed a unique antibiotic resistance pattern which suggested Constance Wose Kinge a link between isolates from all sampling points. The findings indicated that improper wastewater treatment may have a potential impact on the dissemination and survival of E. coli, as well as other pathogenic bacteria in water for human and animal consumption. This may result in water- and food-borne disease outbreaks with a negative effect on antibiotic therapy. Postal address:
Department of Biological
Sciences, North-West University (Mafikeng Escherichia coli (E. coli) is an organism that occurs universally in sewage and, because it is a faecal coliform, Campus), Private Bag it plays an important role in the sanitary analysis of water.1 Its presence in water indicates the presence X2046, Mmabatho 2735, of faecal contamination and the likelihood of other pathogenic microbes.1 Five pathogenic strains of E. coli are frequently isolated from humans and animals suffering from diarrhoea.2 These differ from other commensals in that they express virulence factors, which are molecules directly involved in pathogenesis, African Journal of Science Keywords:
but which are also important for normal metabolic functions.3 These pathogenic strains include: E. coli; antibiotic resistance; • The enterotoxigenic E. coli strain, which causes traveller and infantile diarrhoea and is the main multiple antibiotic cause of haemolytic-uraemic syndrome associated with food-borne infections.4 resistance (MAR) • The enteroinvasive E. coli strain, which produces shigellosis-like diseases in children and adults. phenotypes; MAR indices; • The enteropathogenic E. coli strain, which is the major cause of acute infantile diarrhoea in water-borne bacteria • The enteroaggressive E. coli strain, which produces persistent gastroenteritis and diarrhoea in infants and children,5,6 and is prevalent in developing countries.
Received: 18 June 2009 • The enterohemorrhagic E. coli strain, which is the major cause of sporadic outbreaks of haemorrhagic Accepted: 05 Nov. 2009 Published: 11 Mar. 2010 Antibiotic resistance in E. coli has been globally identified in isolates from environmental, animal and human sources.10 This is a consequence of the use of antimicrobials in medicine and their application How to cite this article:
in animal husbandry, which have brought about phenotypic changes, often due to chromosomal Wose Kinge CN, Ateba mutations.11 Studies have shown that many pathogenic organisms have developed some degree of resistance to antimicrobials and they confer resistance through different mechanisms, with a negative Antibiotic resistance impact on veterinary and human medicine.10,12,13 These mechanisms of resistance include the alteration profiles of Escherichia coli of receptor-binding sites of drugs, a decreased intake of drugs by altering the entry or active efflux of the isolated from different drug, the destruction or inactivation of the drug, and development of resistant metabolic pathways. 13 water sources in the Mmabatho locality, The surfacing of antibiotic resistance usually results from the misuse of antibiotics as growth-promoters in North-west Province, animal production, for therapy and prophylaxis.14 Because humans consume these animal products, there South Africa. S Afr J Sci. is a probability of the spread of resistant strains from animals to humans and thus healthy individuals can 2010;106(1/2), Art. #14, 6 become carrier hosts for multiple antibiotic-resistant bacteria.15 This may enhance the risk of developing pages. DOI: 10.4102/sajs.
haemolytic-uraemic syndrome, a disease more severe in children infected with E. coli O157:H7.16 Several studies have revealed that E. coli is resistant to a number of antibiotics. 17,18,19,20 Adding to the consequences of microbial resistance to antibiotics on human health, contamination of surface water bodies with resistant This article is available
bacterial strains from human activities and livestock operations has also been reported.21 The objective of this study was to isolate E. coli organisms from water collected from different water sources in the Mmabatho locality in order to test their resistance to commonly used antibiotics.
Collection of samples
Sampled sites were the inlet, primary, secondary, tertiary digesters and effluent from the local wastewater-
treatment plant; the local water-treatment plant inlet and outlet; inlet, midpoint and outlet of the Modimola Dam; and tap water from a few homes in Units 8, 10 and 12 in the Mmabatho locality of the Mafikeng 2010. The Authors.
Licensee: OpenJournals Publishing. This work Water samples were collected weekly over a period of two months (July to September 2006). Samples is licensed under the were collected aseptically in sterile 500 mL Schott Duran bottles, transported on ice to the Microbiology Laboratory at the Department of Biological Sciences, University of the North-West (South Africa) and plated out within 24 h. Vol. 106 No. 1/2 Page 1 of 6 S Afr J Sci 44
Wose Kinge, Ateba & Kawadza Identification of E. coli isolates
and aliquots of 100 µL plated out on Mueller Hinton agar Analyses of water samples were performed according to the (Merck, Johannesburg, South Africa). Antimicrobial discs (Mast standard method22 for total and faecal coliform counts on Diagnostics, Sefton, UK) impregnated with kanamycin (30 µg), m-Endo (Merck , Johannesburg, South Africa) and m-FC (Merck, streptomycin (300 µg), erythromycin (15 µg), tetracycline (30 Johannesburg, South Africa) agar plates incubated at 37 °C and µg), ampicillin (10 µg), norfloxacin (10 µg) and chloramphenicol 44.5 °C for 24 h, respectively. Escherichia coli ATCC® 25922 was (30 µg) were placed on the Mueller Hinton agar plates and used as a positive control.23 Characteristic metallic-sheen and incubated at 37 °C for 24 h. After incubation, the inhibition zone blue-coloured colonies on m-Endo and m-FC agar plates were diameters were measured and classified using reference values.26 selected and purified by streaking on nutrient agar (Biolab, Multiple antibiotic resistant (MAR) phenotypes were generated Johannesburg, South Africa) plates. Plates were incubated at for isolates that showed resistance to three or more antibiotics. 27 37 °C for 24 h and stored for further use. Isolates were Gram- MAR indices were evaluated as previously described.28 stained according to standard methods24 and all Gram-negative isolates were subjected to primary and secondary biochemical identification. The primary biochemical tests performed were the Susceptibility data for E. coli isolates from the different samples triple sugar iron (TSI) agar, Simmons citrate agar, and oxidase were determined using Ward's method and Euclidean distances tests, while the secondary biochemical test performed was the on Statistica Software (version 7.0). analytical profile index (API) 20E test. All tests were performed according to manufacturer's instructions (BioMérieux, France).
Antibiotic susceptibility test
Antibiotic resistance data
Antibiotic susceptibility tests were performed on all E. coli A total of 230 E. coli isolates were obtained following biochemical positive isolates by the disc diffusion method, as previously characterisation (Table 1). Antibiogram results of E. coli isolates described.25 Bacterial suspensions of isolates were prepared (Table 2) revealed resistance to more than one antibiotic, similar The proportion of isolates obtained for the various biochemical tests Oxidase test
African Journal of Science TSI: triple sugar iron agar test, SCT: Simmons citrate agar test, API: analytical profile index, +ve: positive, -ve: negative.
The percentage antibiotic resistance of E. coli isolated from the different sampling sites Wastewater (Inlet) Wastewater (Primary) Wastewater (Secondary) Wastewater (Tertiary) Wastewater (Effluent) Modimola Dam (Inlet) Modimola Dam (Midpoint) Modimola Dam (Outlet) Tap water (Unit 8) Tap water (Unit 10) Tap water (Unit 12) 45 S Afr J Sci
Vol. 106 No. 1/2 Page 2 of 6 Antibiotic resistance profiles of E. coli in the water sources of the Mmabatho locality TABLE 3 (CONTINUES.)
The predominant multiple antibiotic resistant (MAR) phenotypes for E. coli isolated The predominant multiple antibiotic resistant (MAR) phenotypes for E. coli isolated from the different sampling sites from the different sampling sites Wastewater inlet isolates (N = 30)
Dam inlet isolates (N = 20)
Dam midpoint isolates (N = 20)
Wastewater primary digester isolates (N = 20)
Wastewater secondary digester isolates (N = 20)
Dam outlet isolates (N = 20)
Inlet water isolates (N = 20)
African Journal of Science Wastewater tertiary digester isolates (N = 20)
Outlet water isolates (N = 20)
Tap water (Unit 8) isolates (N = 20)
TSI: triple sugar iron agar test, SCT: Simmons citrate agar test, API: analytical profile index, +ve: positive, -ve: negative.
Wastewater effluent isolates (N = 20)
Tap water (Unit 10) isolates (N = 20)
Tap water (Unit 12) isolates (N = 20)
Dam inlet isolates (N = 20)
Vol. 106 No. 1/2 Page 3 of 6 S Afr J Sci 46
Wose Kinge, Ateba & Kawadza The percentage representation of E. coli isolated from different sampling areas within the various clusters Cluster I
Wastewater (Inlet) Wastewater (Primary) Wastewater (Secondary) Wastewater (Tertiary) Wastewater (Effluent) Tap water (Unit 8) Tap water (Unit 10) Tap water (Unit 12) Tree Diagram for 65 Cases to reports by other researchers.15,29,30 Marked multiple antibiotic resistances (over 70%) were observed for erythromycin, tetracycline and ampicillin, chloramphenicol and norfloxacin. Euclidean distances Multiple antibiotic resistance refers to the resistance of two or more classes of antibiotics. A large proportion (70%−95%) of E. coli isolated from wastewater samples obtained from the different sampling sites was resistant to chloramphenicol, norfloxacin, tetracycline, ampicillin and erythromycin. Similarly, a large proportion (80%−100%) of E. coli isolated from the Modimola Dam was resistant to chloramphenicol, tetracycline, and erythromycin. Furthermore, a large proportion (65%−100%) of E. coli isolated from the local water-treatment plant was resistant to tetracycline and erythromycin. Lastly, a 50%−90% resistance to chloramphenicol, tetracycline, ampicillin and erythromycin was observed for E. coli isolated from tap water. However, all tap African Journal of Science water isolates were susceptible to norfloxacin. Susceptibility of a few isolates to streptomycin and kanamycin was also observed.
MAR phenotypes were compiled for all isolates obtained (Table 3). The predominant phenotypes from wastewater sites were T-Ap-E (20%, inlet), K-C-Nor-T-Ap-E (30%, primary), Nor-T-Ap-E and K-C-Nor-T-Ap-E (both 10%, secondary), K-C-Nor-T-Ap-E (50%, tertiary), and C-Nor-T-E (50%, effluent). Similarly, the predominant phenotypes obtained for the local water-treatment plant were C-Nor-T-E and C-T-E at 20%, from the inlet and outlet, respectively. Also, predominant phenotypes from the Modimola Dam inlet, midpoint and outlet were Nor-T- Ap-E at 30%, C-Nor-T-E and C-T-Ap-E both at 20%, and T-Ap-E at 30%, respectively. C-Ap-E, T-Ap-E and C-T-Ap-E were the predominant phenotypes in tap water at 30%, 25% and 10% for Units 8, 10 and 12, respectively. Overall, T-Ap-E was a common phenotype observed for E. coli isolated from the local wastewater- and water-treatment plants, Modimola Dam and tap water. A total of 65 E. coli isolates were randomly selected from all sampling sites and subjected to cluster analysis using the antibiotic inhibition zone diameter data. Two major clusters were generated, each subdivided into two minor clusters (IA, IB and IIA, IIB) as shown in Figure 1. Further analysis of the clusters Bacterial designation prefixes are based on the type of sample and area of collection. The was performed for patterns of associations of the isolates from tree was constructed using Ward's method and Euclidean distances in Statistica, version 7. the different sources as shown in Table 4. The analysis obtained Designation: IS = Inlet wastewater, PS = Primary wastewater, SS = Secondary wastewater, TS = Tertiary wastewater, ES = Effluent wastewater, ID = Inlet dam, MD = Midpoint dam, OD = was used as a tool in determining the uniqueness between the Outlet dam, IW = Inlet water, OW = Outlet water, TW 8 = Tap water Unit 8, TW 10 = Tap water antibiotic resistance patterns of E. coli isolates from different Unit 10, TW 12 = Tap water Unit 12.
areas. The largest cluster (Cluster IB) showed E. coli isolated from all sampled areas. The second largest (Cluster IA) represented Dendogram showing the relationship between E. coli isolated from water samples obtained from the Mmabatho E. coli isolated from wastewater, the local water-treatment plant locality based on inhibition zone (outlet) and tap water (Unit 10). Cluster IIA (the third largest diameter (IZD) data cluster) represented E. coli isolated from the Modimola Dam 47 S Afr J Sci
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Amtsblatt der Europäischen Union VERORDNUNG (EG) Nr. 392/2009 DES EUROPÄISCHEN PARLAMENTS UND DES RATES vom 23. April 2009 über die Unfallhaftung von Beförderern von Reisenden auf See (Text von Bedeutung für den EWR) DAS EUROPÄISCHE PARLAMENT UND DER RAT DER EUROPÄ rung unterschieden, weshalb es angebracht ist, Umfang
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