Extended Spectrum Beta-Lactamase-Resistant Determinants among Carbapenem-Resistant Enterobacteriaceae from Beef Cattle in the North West Province, South Africa: A Critical Assessment of Their Possible Public Health Implications.

Carbapenems are considered to be the last resort antibiotics for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing strains. The purpose of this study was to assess antimicrobial resistance profile of Carbapenem-resistant Enterobacteriaceae (CRE) isolated from cattle faeces and determine the presence of carbapenemase and ESBL encoding genes. A total of 233 faecal samples were collected from cattle and analysed for the presence of CRE. The CRE isolates revealed resistance phenotypes against imipenem (42%), ertapenem (35%), doripenem (30%), meropenem (28%), cefotaxime, (59.6%) aztreonam (54.3%) and cefuroxime (47.7%). Multidrug resistance phenotypes ranged from 1.4 to 27% while multi antibiotic resistance (MAR) index value ranged from 0.23 to 0.69, with an average of 0.40. Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabilis) and Salmonella (34.4, 43.7, 1.3 and 4.6%, respectively) were the most frequented detected species through genus specific PCR analysis. Detection of genes encoding carbapenemase ranged from 3.3% to 35% (blaKPC, blaNDM, blaGES, blaOXA-48, blaVIM and blaOXA-23). Furthermore, CRE isolates harboured ESBL genes (blaSHV (33.1%), blaTEM (22.5%), blaCTX-M (20.5%) and blaOXA (11.3%)). In conclusion, these findings indicate that cattle harbour CRE carrying ESBL determinants and thus, proper hygiene measures must be enforced to mitigate the spread of CRE strains to food products.

Analysis of physico-chemical and bacteriological quality of drinking water in Mafikeng, South Africa.

Mafikeng, the capital of the North West Province, receives water from two sources, namely the Molopo eye and the Modimola dam. Once treated, the potable water is mixed and supplied to the city via distribution systems. This study was designed to assess the quality of drinking water in Mafikeng and also to determine whether the water from the two sources has an impact on the mixed water quality. Physico-chemical parameters and bacteriological quality (faecal coliforms (FCs), total coliforms (TCs), heterotrophic bacteria and Peudomonas spp.) was monitored at three drinking water sites weekly for 4 months. The results revealed that the physico-chemical quality of the water was generally acceptable. The pH ranged from 5.7 ± 0.18 to 8.6 ± 0.14, the temperature ranged from 18.3 ± 0.69 to 25.1 ± 0.69 °C and the total dissolved solids (TDS) ranged from 159.9 ± 22.44 to 364.4 ± 12.44 mg/l. These values are within the target water quality range for drinking water as prescribed by WHO, Department of Water Affairs and SANS 241. What is of concern was the microbial quality of the water. FCs, TCs, heterotrophic bacteria and Pseudomonas spp. were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas spp. (>100/100 ml).

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.

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.

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.

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.

Antibiotic-Resistant Staphylococcus aureus Isolated from Milk in the Mafikeng Area; North West Province; South Africa

The aim of this study was to isolate Staphylococcus aureus from samples of cow's milk obtained from different farm settings and to determine their antibiotic susceptibility patterns. Gram staining; oxidase; catalase; DNase; haemolysis and the MASTASTAPHTM rapid agglutination tests were employed for bacterial identification. A total of 28 milk samples were collected and screened for the presence of S. aureus. All the samples were contaminated with S. aureus. A total of 240 S. aureus isolates were obtained during this study. The levels of contamination with S. aureus were higher in milk obtained from the communal farms in Lokaleng and Mogosane (24.6and 35.4; respectively)compared to the commercial farms in Rooigrond and Molelwane (17.9and 22.1; respectively). A large percentage of the S. aureus isolates (39-100) from both communal farms was resistant to methicillin (MT); ampicillin (AP); penicillin G (PG); sulphamethoxazole (Smx); oxytetracycline (OT); erythromycin (E); nitrofurantoin (NI) and streptomycin (S); but not vancomycin (V). An even higher percentage (64.2- 100) of the isolates from both commercial farms was resistant to sulphamethoxazole and nitrofurantoin. A comparably smaller percentage (3.4- 4.7) of the isolates from both communal farms was resistant to vancomycin; but all isolates from commercial farm milk were susceptible to this drug. The predominant multiple antibiotic resistant phenotypes for isolates from the commercial farms were AP-Smx-NI and MT-AP-PG-OT-Smx-NI for Rooigrond and Molelwane farms; respectively; while those for isolates from the communal farms were MT-AP-PG-Smx-E-NI-S and MT-AP-PG-OT-Smx-NI-S for Lokaleng and Mogosane; respectively. When comparing the percentage of antibiotic resistance; a significant positive correlation was observed between the isolates from the commercial farms (r = 0.966; p 0.01). S. aureus is normally resident in humans; therefore; the S. aureus present in the cows may have resulted from transmission between the two species; emphasising the need to improve sanitary conditions in the milking environment