Collins Njie Ateba

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.

Characterisation of Escherichia coli O157 strains from humans, cattle and pigs in the North-West Province, South Africa

Escherichia coli O157 strains cause diseases in humans that result from the consumption of food and water contaminated with faeces of infected animals and/or individuals. The objectives of this study were to isolate and characterise E. coli O157 strains from humans, cattle and pigs and to determine their antibiotic resistant profiles as well as detection of virulence genes by PCR. Eight hundred faecal samples were analysed for typical E. coli O157 and 76 isolates were positively identified as E. coli O157 strains. 16S rRNA sequence data were used to confirm the identity of the isolates. Susceptibility profiles to 9 antibiotics were determined and the multiple antibiotic resistant (MAR) patterns were compiled. A large proportion (52.6%-92.1%) of the isolates from pigs, cattle and humans were resistant to tetracycline, sulphamethoxazole and erythromycin. Thus the phenotype Smx-T-E (sulphamethozaxole-tetracycline-erythromycin) was present in most of the predominant MAR phenotypes obtained. Cluster analysis of antibiotic resistances revealed a closer relationship between isolates from pig and human faeces than cattle and humans. PCR were performed to amplify STEC virulence and tetracycline resistance gene fragments. A tetB gene fragment was amplified among the isolates. Eighteen (60%) of the isolates possessed the hlyA gene and 7(23.3%) the eae gene while only 5(16.7%) possessed both genes. Although shiga toxin genes were detected in the E. coli O157:H7 positive control strain none of the isolates that were screened possessed these genes. In a related study we reported that the prevalence of E. coli O157 was higher in pigs than cattle and humans. A high market demand for pork and beef in South Africa amplifies the risk that diseased animals pose to human health. This highlighted the need for proper hygiene management to reduce the prevalence of E. coli O157 in farm animals and prevent the spread from animals 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.

Detection of Antibiotic Resistant Staphylococcus aureus from Milk: A Public Health Implication

The aim of this study was to investigate the occurrence, antibiotic susceptibility profiles, and virulence genes determinants of S. aureus isolated from milk obtained from retail outlets of the North-West Province, South Africa. To achieve this, 200 samples of raw, bulk and pasteurised milk were obtained randomly from supermarkets, shops and some farms in the North-West Province between May 2012 and April 2013. S. aureus was isolated and positively identified using morphological (Gram staining), biochemical (DNase, catalase, haemolysis and rapid slide agglutination) tests, protein profile analysis (MALDI-TOF mass spectrometry) and molecular (nuc specific PCR) methods. The antimicrobial resistance profiles of the isolates were determined using the phenotypic agar diffusion method. Genes encoding enterotoxins, exfoliative toxins and collagen adhesins were also screened using PCR. Among all the samples examined, 30 of 40 raw milk samples (75%), 25 of 85 bulk milk samples (29%) and 10 of 75 pasteurised milk samples (13%) were positive for S. aureus. One hundred and fifty-six PCR-confirmed S. aureus isolates were obtained from 75 contaminated milk samples. A large proportion (60%–100%) of the isolates was resistant to penicillin G, ampicillin, oxacillin, vancomycin, teicoplanin and erythromycin. On the contrary, low level resistance (8.3%–40%) was observed for gentamicin, kanamycin and sulphamethoxazole. Methicillin resistance was detected in 59% of the multidrug resistant isolates and this was a cause for concern. However, only a small proportion (20.6%) of these isolates possessed PBP2a which codes for Methicillin resistance in S. aureus. In addition, 32.7% of isolates possessed the sec gene whereas the sea, seb sed, see, cna, eta, etb genes were not detected. The findings of this study showed that raw, bulk and pasteurised milk in the North-West Province is contaminated with toxigenic and multi-drug resistant S. aureus strains. There is a need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains and the burden of disease in humans.

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.

Pathogenic potentials of Aeromonas species isolated from aquaculture and abattoir environments

The present study elucidated the presence of antibiotics resistance, virulence genes and biofilm potentials among Aeromonas species isolated from abattoir and aquaculture environments in Benin City, Nigeria. A total of 144 wastewater samples were obtained from two independent aquaculture and abattoir environments between May and October 2016. Aeromonas species were isolated on Glutamate Starch Phenol Red (GSP) agar and confirmed using API 20NE kits. Antimicrobial susceptibility profile of the isolates was carried out using standard disc diffusion assay while biofilm potentials were detected by the microtitre plate method and PCR technique was used to detect antibiotics resistance and virulence gene markers. Overall, 32 and 26 Aeromonas species were isolated from the abattoir and aquaculture environments respectively. Isolates from both environments were completely resistant (100%) to penicillin G, ertapenem and tetracycline; whereas aquaculture isolates exhibited absolute sensitivity (100%) towards cefepime. All the virulence gene markers (aerA, hlyA, alt, ast, laf, ascF-G, fla, lip, stx1, and stx2) investigated in this study (except laf) were detected in isolates from both environments. The laf genes were only detected in isolates from abattoir environments. Antibiotics resistant genes including pse, blaTEM and class 1 integron were detected in isolates from both environments. Majority of the isolates (53/58 91.4%) from both environments; demonstrated capacity for biofilm potential. The detection of antibiotic resistance and virulence gene markers as well as biofilm forming ability in Aeromonas species isolated from aquaculture and abattoir environments raise serious public health concern worthy of further investigation.

Detection of vanA and vanB genes in vancomycin-resistant enterococci (VRE) from groundwater using multiplex PCR analysis.

A total of 22 groundwater samples were randomly collected from three rural communities in the Mafikeng area. Bile esculin agar was used for selective isolation of enterococci. Standard preliminary tests (Gram staining, oxidase test, catalase test) and confirmatory tests (Prolex™ Streptococcal Grouping Rapid Latex Agglutination test kit) were used to determine the identities of presumptive enterococci. The antibiotic sensitivity test was performed on all positively identified enterococci; percentage resistance and multiple antibiotic resistance (MAR) phenotypes were generated. Multiplex polymerase chain reaction (PCR) was performed to detect vanA and vanB genes vancomycin-resistant enterococci (VRE). A total of 179 enterococci were positively identified and the proportion of isolates from Dibate (62.5%) was higher compared to those from Majemantsho and Motlhabeng (22.3 and 15.0, respectively). A large proportion (81.5 to 100%) of the isolates from Dibate, Motlhabeng and Majemantsho were resistant to ampicillin, vancomycin and penicillin G. Two main MAR phenotypes, PG-VA-Ap-A-OX and PG-VA-Ap-OX, were identified. Multiplex PCR analysis of 50 VRE indicated that 17 (34%) were positive for vanA and vanB genes. This highlights the need to determine the cause of vancomycin resistance in enterococci in the sampled sites and suggests that sequence analysis be used to confirm the identities of these amplicons.

Detection of E. coli Isolates in Water from Setumo Dam Mmabatho Area - North West Province, South Africa Using mdh Specific PCR Analysis

Abstract Most countries including South Africa, process surface water from lakes, rivers, dams and ponds by filtration, chlorination, alum treatment before use for drinking purposes. In South Africa, the North West Province in particular, residents of most rural communities do not have access to treated water. This exposes them to contamination with pathogens such as Escherichia coli. The aim of the study was, therefore, to isolate, identify and characterize E. coli strains from water collected from Setumo Dam, located at the Modimola village, in the periphery of the Mmabatho suburban area using conventional microbiological methods and PCR analysis. Water samples were collected during the summer season from three different sites of the dam, viz; upstream, middle stream and the downstream of the dam. Tap water was used as a control during analysis. Bacterial loads were determined by spread plating on mFC and EMBA and incubating the plates aerobically at 45oC and 37oC, respectively for 24 hours. Blue colonies on mFC agar were counted and recorded while pure metallic sheen colonies on EMBA were identified using preliminary (oxidase test, TSI) and confirmatory (API 20E and mdh specific PCR) assays. The results obtained indicated that all the water samples were contaminated with faecal coliform bacteria. Samples from upstream had high colony counts (320 to 1624 cfu/50ml) than those from the mid-stream (56 to 90 cfu/50ml) and the downstream (26 to 44 cfu/50ml). There was no contamination from tap water. Of the 57 randomly selected E. coli colonies that satisfied the preliminary identification tests and API 20E, 46 (80.7%) were positively identified as E. coli since they possessed the mdh gene fragment. Water from the dam contained high E. coli load and it is recommended that the Local municipality makes informed decisions pertaining to the control and management of the pathogen. Improvement of the sanitary and water systems in the area is therefore necessary.