Assessment of microbial levels in the Plankenburg and Eerste Rivers and subsequent carry-over to fresh produce using source tracking as indicator

Thesis (MSc Food Sc)--Stellenbosch University, 2012.ENGLISH ABSTRACT: The agricultural sector of South Africa is currently facing a serious water crisis. The decreased availability of water as a result of climate change and the constantly growing population has left many farmers increasingly dependant on surface water as primary source of irrigation. Urbanisation along with out-dated and insufficient wastewater treatment works have all contributed to polluting large volumes of these resources. Consequently, many farmers have been forced to use irrigation water, not only of poor quality, but often water which has been polluted with untreated sewage. As a result, this project aimed at investigating the link between the quality of irrigation water and the impact on the safety of fresh produce. A base-line of the microbial load at three sites along the Plankenburg and Eerste Rivers was established using standard microbial methods for the detection of indicator organisms such as total and faecal coliforms, Escherichia coli and Enterococci as well as potential pathogens that included Salmonella, Listeria, Staphylococcus, endosporeformers and aerobic colony counts. Chemical parameters such as pH, alkalinity, conductivity and chemical oxygen demand (COD) were also monitored, but were not correlated to microbial pollution levels in the rivers. High faecal coliform and E. coli concentrations, ranging from 310 to 7 x 106 cfu.100 mL-1 and 230 to 7 x 106 cfu.100 mL-1, respectively, were detected. The recommended irrigation water guidelines of ≤1 000 (WHO, 1989) and ≤4 000 cfu.100 mL-1 (DWAF, 2008) for faecal coliforms and E. coli were exceeded, indicating faecal pollution and thus a high health risk. This health risk was confirmed when potential pathogens such as Aerococcus viridans, Klebsiella, Listeria monocytogenes and Salmonella typhimurium were detected at all three sites. The carryover of organisms from rivers to produce (green beans and grapes) was investigated by comparing the microbial population of the Plankenburg and Eerste Rivers to the population recovered from irrigation water and the surface of fresh produce. Faecal coliforms, E. coli, Aerococcus viridans, Enterobacter aerogenes, Klebsiella, L. innocua, L. grayi, L. monocytogenes and Staphylococcus aureus were detected in all three sample types, indicating a similarity between the microbial populations found in the river, the irrigation water and produce. Thus, the transfer of potential pathogens from the rivers to produce is a strong possibility. The build-up of organisms on the surface of green beans as a result of multiple irrigations was also confirmed by an increase in faecal coliform concentrations from initial concentrations of none detected to 44 000 cfu.100 mL-1 over a 10 day irrigation period. Finally, microbial source-tracking techniques including multi-antibiotic resistance (MAR) profiling, and the API 20E classification system were used to determine genotypic and phenotypic characteristics of 92 faecal isolates (from irrigation water and produce) and 13 reference strains. Numerical classification systems was used to classify the 105 faecal isolates according to the degree of similarity between the genotypic and phenotypic characteristics of the 105 isolates. A high degree of similarity indicates a high probability that isolates originate from the same strain and therefore from the same source, thereby confirming the transfer of organisms Faecal isolates (93 and 98%, respectively) were found to be resistant to Vancomycin at both the 5 and 30 μg concentrations. The majority of isolates presented some resistance to Erythromycin (15 μg) and Ampicillin (25 μg), with 82% of isolates presenting an inhibition zone ≤4 mm. Isolates were sensitive towards Ciprofloxacin (1 and 5 μg), Ofloxacin (15 μg), Ceftriaxone (30 μg) and Cefotaxime (5 μg), which were able to inhibit the growth of 79.8, 93.3, 79.8, 88.5 and 71.2% of the isolates, respectively. The 13 medical reference strains all presented different genotypic and phenotypic characteristics and thereby indicated a high degree of variability between isolates from the same species. Finally, 35% of the isolates could be grouped together based on similar genotypic and phenotypic characteristics, therefore, more than a third of the faecal isolates obtained from the surface of the fresh produce was as a result of faecal contaminants in the irrigation water. It could therefore be concluded that a health risk is associated with the water from the Plankenburg and to a lesser extent, Eerste River when used as source of irrigation, thereby risking the transfer of potentially harmful organisms, present in the rivers as result of faecal pollution, to the surface of fresh produce.AFRIKAANSE OPSOMMING: Suid-Afrika stuur tans af op 'n dreigende water krisis. Klimaatsverandering tesame met 'n spoedig groeiende bevolking het gelei tot 'n aansienlike vermindering in die land se varswaterbronne terwyl veranderende reënvalpatrone daartoe bygedra het dat talle boere al hoe meer afhanklik geword het van oppervlakvarswaterbronne as hul hoof-besproeïngsbron. Verstedeliking, armoede asook verouderde en onvoldoende infrastrukture het egter bygedra tot die besoedeling van baie van hierdie oppervlakvarswaterbronne. Gevolglik is meeste boere genoodsaak om klaar te kom met besproeïngswater van, nie net onaanvaarbare mikrobiese kwaliteit nie, maar dikwels water wat gekontamineer is met onbehandelde riool. Hierdie studie was gevolglik daarop gemik om die impak van die mikrobiologiese kwaliteit van besproeïngswater op die veiligheid van vars groente en vrugte te bepaal. Standaard mikrobiologiese metodes vir die bepaling van indikator organismes soos totale en fekale kolivorms, E. coli en enterococci asook potensiële patogene wat Salmonella, Listeria en Staphylococcus insluit, was gebruik om die mikrobiese lading by drie verskillende punte (P1, P2 en P3) in die Plankenburg en Eerste Rivier te bepaal. Chemiese parameters soos pH, alkaliniteit, konduktiwiteit en Chemiese Suurstof Behoefte was ook bepaal maar geen korrelasie kon tussen hierdie eienskappe en die mikrobiese besoedelingsvlakke getref word nie. Hoë konsentrasies fekale kolivorms en E. coli wat onderskeidelik vanaf 3.1 x 102 tot 7 x 106 kve.100 mL-1 en 2.3 x 102 tot 7 x 106 kve.100 mL-1 gestrek het en gereeld die voorgeskrewe riglyne van onderskeidelik ≤1 000 (WHO, 1989) en ≤4 000 kve.100 mL-1 (DWAF, 2008) oorskry het, was by al drie punte gevind. Hierdie resultate het gedui op fekale besoedeling wat gevolglik met 'n hoë gesondheidsrisiko geassosieer kon word. Hierdie risiko was bevestig deur die teenwoordigheid van talle potensiële patogene, soos Aerococcus viridans, Klebsiella, Listeria monocytogenes en Salmonella typhimurium, wat vanaf al drie punte geïsoleer was. Die oordrag van organismes vanaf die besoedelde riviere na vars vrugte en groente (groen bone en druiwe) was bepaal deur die mikrobiese lading in die Plankenburg en Eerste Rivier te vergelyk met dié verkry vanuit die besproeïngswater en vanaf groen bone wat besproei was met hierdie water. Fekale kolivorms, E. coli, Aerococcus viridans, Enterobacter aerogenes, Klebsiella, L. innocua, L. grayi, L. monocytogenes en Staphylococcus aureus was vanaf al drie die monster tipes geïsoleer. Hierdie resultate het gedui op eenderse mikrobiese populasies in al drie bronne en het daarom die moontlike oordrag van patogene bevestig. Die opbou van organismes as gevolg van veelvuldige besproeïngsessies aan die oppervlak van groen bone was bevestig deur die toename in fekale kolivorm konsentrasie vanaf 'n begin telling van nul tot 'n maksimum konsentrasie van 44 000 kve.100 mL-1. Laastens was mikrobiologiese bron naspeurbaarheidstegnieke soos multi-antibiotika weerstandbiedende profiele en die API 20E klassifikasie sisteem gebruik om individuele genotipe en fenotipe profiele van die 105 fekale isolate saam te stel. Numeriese klassifikasie sisteme was gebruik om die isolate op grond van ooreenkomste tussen hul individuele fenotipiese en genotipiese karaktereienskappe te groeppeer. 'n Hoë mate van ooreenkomstigheid sal dan daarop dui dat isolate van dieselfde besoedlingsbron afkomstig is en gevolglik die oordrag van organismes vanaf besproeïngswater na vrugte en groente bevestig. Onderskeidelik 93 en 98% van die fekale isolate het daarop gedui om weerstandbiedend te wees teen beide 5 en 30 μg Vancomycin. Die meerderheid isolate (82%) het ook 'n mate van weerstand teenoor Erythromycin (15 μg) en Ampicillin (25 μg) getoon met inhibisie sones ≤4 mm. Isolate was ook sensitief teenoor Ciprofloxacin (1 en 5 μg), Ofloxacin (15 μg), Ceftriaxone (30 μg) en Cefotaxime (5 μg). Hierdie antibiotikums was in staat om die groei van onderskeidelik 79.8, 93.3, 79.8, 88.5 en 71.2 % van die isolate te inhibeer. Alhoewel resultate 'n hoë mate van variasie tussen isolate van dieselfde spesie getoon het was dit nogtans moontlik om 35% van die isolate saam te groeppeer op grond van ooreenstemmende genotipe en fenotipe profiele. Meer as 'n derde van die fekale isolate wat vanaf die oppervlakte van die groente en vrugte geïsoleer was, was afkomstig vanaf fekale besmetting in die besproeïngswater. Die oordrag van potensieël patogene organismes vanaf besoedelde riviere na vars vrugte en groente tydens besproeïng was sodoende bevestig. 'n Hoë gesondheidsrisiko was gevolglik gekoppel aan die gebruik van water vanaf die Plankenburg Rivier, en in 'n minder mate die Eerste Rivier, as bron van besproeïngswater.Water Research CommissionNational Research Foundatio