Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

People living in rural areas still rely on the use of environmental water that is contaminated by human and animal activities. This study assessed the occurrence of human enteric pathogens in rivers that are used by rural communities Vhembe District of South Africa as a source of drinking water covering two seasons (winter and summer) over a one-year period. Water quality was assessed using physico characteristics and indicator organisms (total coliforms, E. coli, Clostridium perfringens). Pathogens tested included bacteria (Pathogenic E. coli, Salmonella-, Shigella- and Vibrio spp.), protozoa (Cryptosporidium- and Giardia spp.), and enteric viruses (Rota-, Noro-, Entero-, and Adenoviruses) while using published molecular protocols. The results showed that the indicator bacteria counts exceeded South African drinking water quality guideline limits and pathogenic E. coli was detected in the samples. No Shigella spp. were isolated, while Vibrio spp. and Salmonella spp. were present; parasites were detected in four rivers and Enteric viruses were predominantly detected in the winter season. The results indicated the poor condition of water and the potential health risks to consumers highlighting the need for implementing river catchment management strategies for continued sustainability in these rivers.

Infectious diarrhoea contributes to high morbidity and mortality in young children from sub-Saharan Africa. The aim of this study was to assess the prevalence of single and multiple diarrhoeal-causing pathogen combinations in children suffering from diarrhoea from rural and peri-urban communities in South Africa. A total of 275 diarrhoea stool specimens were collected between 2014 and 2016 from Hospitals and Primary Health Care clinics. The BioFire^® FilmArray^® Gastrointestinal panel was used to simultaneously detect 22 diarrhoea pathogens (viruses, bacteria, parasites) known to cause diarrhoea. A total of 82% (226/275) enteric pathogens were detected in the stool specimens. The two most detected bacterial, viral and parasitic pathogens each included: EAEC (42%), EPEC (32%), Adenovirus F40/41 (19%), Norovirus (15%), Giardia (8%) and Cryptosporidium (6%), respectively. Single enteric pathogen infections were recorded in 24% (65/275) specimens with EAEC, and Norovirus was found in 26% (17/65) and 14% (9/65) of the specimens, respectively. Multiple enteric pathogen combinations were recorded in 59% (161/275) of the stool specimens with 53% (85/161) containing two pathogens, 22% (35/161) containing three pathogens and 25% (41/161) containing four or more pathogens. The results from this study demonstrated the complex nature of pathogen co-infections in diarrhoeal episodes which could have an impact on treatment effectiveness.

Antibacterial silver nanoparticles (AgNPs) were synthesised via an environmentally benign thermally-assisted one-pot and microwave-assisted experimental set-up using apple extract as a reducing agent. The formation of these AgNPs was confirmed by UV-Vis, XRD and EDS while their size distribution was determined by TEM micrographs. The AgNPs were uniformly distributed with diameters of 28.24 ± 1.15 nm and 22.05 ± 1.05 nm for the thermally-assisted one-pot and microwave-assisted reduction methods respectively. The rate of reduction was faster in a microwave-assisted reduction method compared to that of a thermally-assisted one-pot synthesis method. The antibacterial AgNPs were embedded in polyvinylidene fluoride (PVDF) nanofibre membranes and their antibacterial activity against Gram-positive Geobacillus stearothermophilus and Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae was studied. The antibacterial nanofibres successfully inhibited the growth of these mesophilic and thermophilic bacteria. Therefore, AgNP-embedded PVDF nanofibre membranes showed a high potential for use in water purification systems that are subject to contamination by mesophilic or thermophilic bacteria without compromising the rate of water recovery.

Background: Water quality testing is dictated by microbial agents found at the time of sampling in reference to their acceptable risk levels. Human activities might contaminate valuable water resources and add to the microbial load present in water bodies. Therefore, the effects of human activities on the microbial quality of rivers collected from twelve catchments in the Vhembe District in South Africa were investigated, with samples analyzed for total coliform (TC) and Eschericha coli (E. coli) contents. Methods: Physical parameters and various human activities were recorded for each sampling site. The Quanti-Tray^® method was adopted for the assessment of TC and E. coli contents in the rivers over a two-year period. A multiplex polymerase chain (PCR) method was used to characterize the strains of E. coli found. Results: The microbial quality of the rivers was poor with both TC and E. coli contents found to be over acceptable limits set by the South African Department of Water and Sanitation (DWS). No significant difference (p > 0.05) was detected between TC and E. coli risks in dry and wet seasons. All six pathogenic E. coli strains were identified and Enteroaggregative E. coli (EAEC), atypical Enteropathogenic E. coli (a-EPEC) and Enterotoxigenic E. coli (ETEC) were the most prevalent E. coli strains detected (respectively, 87%, 86% and 83%). Conclusions: The study indicated that contamination in the majority of sampling sites, due to human activities such as car wash, animal grazing and farming, poses health risks to communities using the rivers for various domestic chores. It is therefore recommended that more education by the respective departments is done to avert pollution of rivers and prevent health risks to the communities in the Vhembe District.

This study was undertaken to determine the virulence and antibiotic resistance profiles of diarrhoeagenic Escherichia coli (DEC) in environmental waters of Johannesburg, South Africa. Samples were collected and cultured on selective media. An 11-plex PCR assay was used to differentiate five DEC, namely: enteroaggregative (EAEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The antibiotic resistance profile of isolates was determined using the VITEK^®-2 automated system. The virulence profiles of 170 E. coli tested showed that 40% (68/170) were commensals and 60% (102/170) were pathogenic. EPEC had a prevalence of 19.2% (32/170), followed by ETEC 11.4% (19/170), EAEC 6% (10/170) and EHEC 3% (5/170). Hybrid DEC carrying a combination of simultaneously two and three pathogenic types was detected in twenty-eight and nine isolates, respectively. The antibiotic susceptibility testing showed isolates with multidrug resistance, including cefuroxime (100%), ceftazidime (86%), cefotaxime (81%) and cefepime (79%). This study highlighted the widespread occurrence of DEC and antibiotic resistance strains in the aquatic ecosystem of Johannesburg. The presence of hybrid pathotypes detected in this study is alarming and might lead to more severe diseases. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant strains in this area.