Moses Mbewe

Detection of Escherichia coli O157:H7 virulence genes in isolates from beef, pork, water, human and animal species in the northwest province, South Africa: public health implications

The aim of the present study was to isolate and identify Escherichia coli O157:H7 from pigs, cattle, humans, beef, pork and water samples and to determine their putative virulence genes by PCR analysis. A total of 220 samples were analysed; 5600 presumptive E. coli O157:H7 were screened for the presence of rfb(O157) and fliC(H7) gene fragments by PCR and 130 isolates were confirmed. The prevalence of E. coli O157:H7 was higher in pigs and pork 88(67.7%) than in cattle and beef 36(27.7%), water 3(2.3%) or humans 1 (0.77%). Moreover, the pathogen was more frequently isolated from faecal (16.9%-43.1%) than from meat samples (10.8%-24.6%). A large proportion--73 (56.2%)--of the isolates possessed the hlyA gene, while 48 (36.9%) harboured the eaeA gene. Although there were no major differences in the number of isolates harbouring the stx(1) and stx(2) genes, respectively, only a small proportion 13(10%) harboured both shiga toxin genes. Despite this, the proportion of isolates that possessed the stx(1) 29(22.3%) was higher than those possessing the stx(2) gene. None of the E. coli O157:H7 isolates harboured all four shiga-toxin producing E. coli (STEC) virulence genes investigated. When comparing the proportion of isolates obtained from the different sample sources and/or stations, significant positive correlations were observed between isolates from Mafikeng and Lichtenburg (r = 0.981, p < 0.05) and those from Mafikeng and Rustenburg (r = 0.991, p < 0.05). These results therefore indicate that meat and faeces samples obtained from major cities in the northwest province were contaminated with E. coli O157:H7. We suggest that there is a need for improving the sanitary conditions of farms, abattoirs and butcher shops. This could reduce transmission of E. coli O157:H7 to humans.

Isolation of Environmental Bacteria from Surface and Drinking Water in Mafikeng, South Africa, and Characterization Using Their Antibiotic Resistance Profiles

The aim of this study was to isolate and identify environmental bacteria from various raw water sources as well as the drinking water distributions system in Mafikeng, South Africa, and to determine their antibiotic resistance profiles. Water samples from five different sites (raw and drinking water) were analysed for the presence of faecal indicator bacteria as well as Aeromonas and Pseudomonas species. Faecal and total coliforms were detected in summer in the treated water samples from the Modimola dam and in the mixed water samples, with Pseudomonas spp. being the most prevalent organism. The most prevalent multiple antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All organisms tested were resistant to erythromycin, trimethoprim, and amoxicillin. All isolates were susceptible to ciprofloxacin and faecal coliforms and Pseudomonas spp. to neomycin and streptomycin. Cluster analysis based on inhibition zone diameter data suggests that the isolates had similar chemical exposure histories. Isolates were identified using gyrB, toxA, ecfX, aerA, and hylH gene fragments and gyrB, ecfX, and hylH fragments were amplified. These results demonstrate that (i) the drinking water from Mafikeng contains various bacterial species and at times faecal and total coliforms. (ii) The various bacteria are resistant to various classes of antibiotics.

Determination of the genetic similarities of fingerprints from Escherichia coli O157:H7 isolated from different sources in the North West Province, South Africa using ISR, BOXAIR and REP-PCR analysis

The aim of this study was to determine the genetic relationships of Escherichia coli O157:H7 isolated from pigs, cattle, pork, beef, humans and water samples using REP, ISR and BOXAIR PCR analysis. A total of 94 isolates were subjected to the REP-PCR analysis while 95 were screened for ISR and BOXAIR PCR fingerprints. The band sizes for amplicons from the ISR-PCR analysis ranged from 0.173kb to 0.878kb. However, a large proportion of the isolates had four bands ranging from 0.447kb to 0.878kb. Cluster analysis of the BOXAIR PCR profiles based on banding patterns revealed seven main clusters. It was identified in the clusters III, IV and VII in the BOXAIR PCR that 17.9%, 16.8% and 18.9%, of E. coli O157:H7 isolates respectively were present from all the animal species, meat and water samples. REP-PCR analysis produced 9 different patterns with bands ranging from 0 to 12 per isolate. The band sizes ranged from 200bp to 8000bp. Nine major clusters (I-IX) were identified. From the three different species sampled cluster eight was the largest and a mixed cluster with 23.4% (22/94) of the E. coli O157:H7 isolates. These indicate that food products obtained from supermarkets in the study area are contaminated with E. coli O157:H7.

Genotypic Characterization of Escherichia coli O157:H7 Isolates from Different Sources in the North-West Province, South Africa, Using Enterobacterial Repetitive Intergenic Consensus PCR Analysis

In many developing countries, proper hygiene is not strictly implemented when animals are slaughtered and meat products become contaminated. Contaminated meat may contain Escherichia coli (E. coli) O157:H7 that could cause diseases in humans if these food products are consumed undercooked. In the present study, a total of 94 confirmed E. coli O157:H7 isolates were subjected to the enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) typing to generate genetic fingerprints. The ERIC fragments were resolved by electrophoresis on 2% (w/v) agarose gels. The presence, absence and intensity of band data were obtained, exported to Microsoft Excel (Microsoft Office 2003) and used to generate a data matrix. The unweighted pair group method with arithmetic mean (UPGMA) and complete linkage algorithms were used to analyze the percentage of similarity and matrix data. Relationships between the various profiles and/or lanes were expressed as dendrograms. Data from groups of related lanes were compiled and reported on cluster tables. ERIC fragments ranged from one to 15 per isolate, and their sizes varied from 0.25 to 0.771 kb. A large proportion of the isolates produced an ERIC banding pattern with three duplets ranging in sizes from 0.408 to 0.628 kb. Eight major clusters (I–VIII) were identified. Overall, the remarkable similarities (72% to 91%) between the ERIC profiles for the isolate from animal species and their corresponding food products indicated some form of contamination, which may not exclude those at the level of the abattoirs. These results reveal that ERIC PCR analysis can be reliable in comparing the genetic profiles of E. coli O157:H7 from different sources in the North-West Province of South Africa.

Prevalence of Campylobacter Contamination in Fresh Chicken Meat and Milk Obtained from Markets in the North-West Province, South Africa

Abstract Campylobacter species are food-borne enteropathogens that are usually transmitted to humans through the consumption of contaminated food products such as milk, beef and chicken. The aim of the study was to isolate and indentify Campylobacter species in chicken meat and milk obtained from some supermarkets in the North-west Province, South Africa. Sixteen chicken meat and milk samples were collected from seven regions in the Northwest Province. In chicken meat samples, the prevalence of C. coli contamination was 60 percent, 100 percent, 44.4 percent, 100 percent and 80 percent in Mafikeng, Rustenburg, Vryburg region 1, Vryburg region 2 and Dellareyville, respectively. On the other hand, C. jejuni was detected at 20 percent, 40 percent and 11.1 percent in Dellareyville, Mafikeng and Vryburg region 1, respectively. Only Vryburg region 1 had 44.4 percent prevalence of C. lari among chicken samples. In milk samples, the prevalence of C. coli contamination was 86 percent, 65 percent, 75 percent, 67 percent and 41 percent in Rustenburg, Vryburg region 1, Lichtenburg, Zeerust and Vryburg region 2, while 96 percent and 100 percent C. jejuni were obtained from Koster and Dellareyville, respectively. C. jejuni and C. coli were the most frequently (44.4 percent-100 percent) isolated contaminant among chicken samples when compared to C. lari(44.4 percent). The result of this study indicated a high prevalence of contamination with C. jejuni and C. coli, both of which are pathogens. This raises questions of food safety in South African markets.

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR

The aquatic environments receive a significant number of human microbial pathogens from point and non-point sources of pollution. Point-source pollution enters the environment at different locations, through a direct route of discharge of treated or untreated domestic sewage, industrial effluent and acid mine drainage (State of the Environment Report [SER], 2002). Non-point (or diffuse) sources of pollution comprises up to 80 % of the pollution entering major river systems thus are of significant concern with respect to the dissemination of pathogens and their indicators in water systems. They may be attributable to the run-off from urban and agricultural areas, leakage from sewers and septic systems, insecticides and herbicides from agricultural land, and sewer overflows (Stewart et al., 2008). Although majority of pathogenic microbes can be eliminated by sewage treatment, many end up in the effluent which is then discharged into receiving bodies of water. These pathogenic microbes have been implicated in human diseases linked with the use of contaminated water and food. Adequate sanitation and clean water, being two critical factors in ensuring human health, protects against a wide range of water-related diseases. These include diarrhoea, cholera, trachoma, dysentery, typhoid, hepatitis, polio, malaria, and filariasis (United Nations Department of Public Information [UNDPI], 2005).

Phenotypic and Genetic Characterization of Sorbitol-Fermenting Escherichia coli O157: H7 Isolated from Retail Beef and Mince Beef

Abstract Escherichia colioccur as normal flora in the gastrointestinal tract of humans and animals. However, shiga-toxin producing E. coli and most especially serotype O157:H7 are a major group of food-borne pathogens. The consumption of undercooked contaminated food products of animal origin particularly beef has identified as a potential source for the transmission of these pathogens to humans. The aim of this paper was to determine the level of E. coli O157:H7 contamination in beef and mince beef samples obtained from some supermarkets and evaluate the virulence gene profiles of the isolates. A total of 128 beef and mince beef samples were collected from selected supermarkets and butcheries in the North West Province of South Africa. Samples were analysed for heterotrophic bacterial counts on PCA and potential E. coli O157:H7 counts on SMAC. Preliminary (oxidase test, TSI agar test) and confirmatory biochemical (API 20E, O157 and H7 serological assays, rfb O157 and fliC H7 PCR analysis) tests were used to identify E. coli O157:H7 isolates while the virulence profiles of the isolates were determine through amplification of the stx1, stx2 , eaeA and hlyA virulence genes. Despite the fact that all the beef samples were contaminated with heterotrophic bacteria none of those from Ventersdorp and Rustenburg were contaminated with potential E. coli O157:H7. Beef samples from Swaartruggens had highest heterotrophic microbial count (5.3x106 CFU/mL) while those from Garankwa had the lowest level of contamination (1.3x104 CFU/mL). All (100 percent) the mince beef samples collected from the different sampling stations were contaminated with both heterotrophic and potential E. coli O157 bacteria. Mince beef samples from Potchefstroom, Rustenburg, Klerksdorp, Ventersdorp, Zeerust, Mafikeng, Swaartruggens, Vryburg, Bloemhof and Taung had very high levels (1.8x103 - 4.1x106) of potential E. coli O157:H7 contamination. All the isolates from beef except for those isolated from Rustenburg and Ventersdorp as well as those from mince beef were Gram-negative rods. A large proportion (71.4 percent to 100 percent) of the isolates from both beef and mince beef were oxidase negative. Large proportions (92.9 percent to 100 percent) of isolates from beef as well as small proportions (14.3 percent to 42.9 percent) of those from mince beef fermented the sugars in the TSI medium. Small proportions (7.1 percent to 42.9 percent) of the isolates from both beef and mince beef produced gas and hydrogen sulphide from the TSI agar test. A total of 39 and 38 E. coli isolates were obtained from beef and mince beef samples respectively while a large proportion 27 (70.1 percent) of these isolates were positively identified as E. coli O157:H7 using a serological assay. Only 8 E. coli O157:H7 isolates were positively identified in the beef samples while large proportions (23) of the isolates from mince beef were positive for the fliC H7 and rfb O157 gene fragments. Among the isolates from beef, the stx 2 gene was present in all the 8 isolates while only 3 isolates possessed the stx 1 gene. Similarly, the stx 2 gene was frequently (13) detected among the isolates from mince beef when compared to the stx 1 gene. Putative virulence genes hlyA and eaeA were detected in 12 and 6 isolates respectively from mince beef. Six isolates from beef possessed both the hlyAand eaeA gene fragments. The findings of this study revealed that beef and mince beef samples obtained from some supermarkets in the area may pose severe health risks to consumers if they are not properly cooked before consumption.