Thesis (M. Tech. (Environmental Health)) - Central University of Technology, Free State, 2014 / In the battle to sustain and produce quality food that is safe and affordable, the limited legislative and regulatory environment continues to allow opportunities for food to become contaminated during processing. The degree of contamination distributed over the final food product (including meat products) depends upon several factors that include knowledge and behaviour of the food handlers, equipment, the hygiene habits of personnel, and the monitoring that takes place at food processing plants (including butcheries). The current study was conducted in five selected butcheries (forming 15% of the registered butcheries at the time the study was conducted) in the Mangaung Metropolitan municipal area, purposely targeting the ones registered with the municipality. The hygiene practices of meat handlers were assessed (through self-administered questionnaires) because meat is a perishable product that requires labour intensive processing for production of quality products. Thus, mishandling by food handlers may create and maintain conditions favourable to microbial contamination. Furthermore, the study assessed and characterised microbial contamination on working surfaces and utensils through swabs as well as bioluminescence instrument [Adenosine Tri-phosphate (ATP) Hygiena] for cleanness of the working environment. Concomitant to the above, meat handlers’ hands and aprons were also assessed for possible microbial contamination as well as their characterisation. Lastly, aerosolised microbes [through an air sampler (Surface Air System) SAS Super 90] were also collected for iv quantification and identification during working hours as airborne microbes can settle on working surfaces and/or utensils as a result of movement of workers and other related working processes. Statistical points such as correlations, standard deviations, group standard deviations as well as significant differences were captured per respective chapter where necessary. Data reported in this study is over 3 month period with two weeks intervals during sampling and thus reported as either weekly or rounds between sampling periods. The results of the current study indicate that the food safety objectives are negligibly achieved, indicating a need for proper food safety training which is audit based. On administration of a questionnaire, food handlers showed poor knowledge of food safety awareness coupled with poor attitude and behaviour in terms of food safety. The five butchery premises were further examined regarding the airborne and surface microbial loads, as well as that of the food handlers’ hands, during processing. The microbial loads in the air appeared to comply with the suggested limits at all the sampled butcheries. Microbial loads on meat contact surfaces showed levels conforming to the South African standard or guideline of 1 × 102 cfu.m-2. Total Coliforms on hands and on aprons were compared to the general microbial target value of <2.5 cfu.m-2 as suggested by literature. In this study, Matrix Laser Desorption Time of Flight Mass Spectrophotometer (MALDI-TOF MS) was found to be an accurate, rapid and cost effective method towards v identifying of foodborne pathogens and spoilage bacteria including yeast. Moreover, in recent years South Africa’s meat scandals have increased consumer awareness and the demand for food safety. Section 11 of the Meat Safety Act (Act no. 40 of 2000) stipulates that every abattoir must utilize an independent inspection service appointed by the department of agriculture to ensure that meat of high quality and wholesomeness is produced. However, once the meat and meat products leave the abattoir, they are under the jurisdiction of the local authorities who rely only on visual assessment as opposed to microbiological inspection in the maintenance of their hygiene and quality. Despite the high incidence of foodborne illnesses in both developed and developing countries; South African data on foodborne illness incidents is still insufficient. This could be attributed to the fact that in South Africa, legislation governing the acceptable standards of the levels of microbiota in the air and on food handlers’ hands is still inadequate. Additionally, lack of obligatory usage of Hazard Analysis Critical Control Point (HACCP) procedures in the meat premises poses a risk for economic productivity. In conclusion, the identification of airborne bacteria in the butcheries strongly suggests that in the planning of the existing establishments, the building layout, control of the traffic flow of personnel, the durability and imperviousness of floors, the ventilation system and the placement of the equipment were not carefully considered. This may play a role in the prevalence and proliferation of airborne microbes as the resulting establishments provide an environment conducive to the breeding of microbes. vi In regard to swabs, it was concluded that floors may present a high point of contamination possibly through aerosolization of microbial communities. Moreover, cleaning materials and hygiene practices need to be reviewed. The results of the administered questionnaire showed that food handlers should be sufficiently trained with regard to food quality management tools such as Good Manufacturing Practices (GMP), Hazard Analysis and Critical Control Point (HACCP) systems and food safety. The evaluation of meat contact surfaces for organic soils to determine their cleanliness using the rapid ATP bioluminescence testing can be convenient for everyone involved in the food chain since visual and touch inspection cannot be conclusive enough to meet regulatory requirements in terms of microbial counts.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The World Health Organisation (WHO) estimates that at least 75% of infants receive infant formula milk (IFM) either entirely or in conjunction with breast milk during the first four months after birth. The presence of the emerging pathogen Enterobacter sakazakii in IFM has been associated with rare but fatal cases of neonatal infections and deaths. There is thus a need for accurate methods for the rapid detection of E. sakazakii in foods. At present, the methods used to detect and identify this micro-organism are inadequate, controversial and contradictory. The aim of this study was to determine the most suitable method for E. sakazakii detection after evaluation of the currently available methods. A further aim was to optimise a polymerase chain reaction (PCR) method for the detection of only viable E. sakazakii cells utilising the DNA-intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA). The Food and Drug Administration (FDA) method for E. sakazakii detection was utilised to select 50 isolates from IFM and 14 from the environment, regardless of colony appearance. These isolates were identified by sequencing a 1.5 kilobase (kb) fragment of the 16S ribosomal DNA (rDNA) and by using the National Centre for Biotechnological Information (NCBI) database to confirm the closet known relatives. Seven of the 50 (14%) IFM isolates and six of the 14 (43%) environmental isolates were identified as E. sakazakii. The methods that were evaluated for accuracy in detecting and identifying these E. sakazakii isolates included yellow pigment production on tryptone soy agar (TSA), chromogenic Druggan-Forsythe-Iversen (DFI) and Enterobacter sakazakii (ES) agars and PCR using six different species-specific primer pairs described in the literature. The suitability of the FDA method was lowered by the low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively) of using yellow pigment production for E. sakazakii identification. DFI and ES agars were shown to be sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications was found to vary between 8% and 92%, with Esakf and Esakr being the most accurate of the primer pairs evaluated. The current FDA method for E. sakazakii detection requires revision in the light of the availability of more sensitive, specific and accurate detection methods. Based on the results obtained in this study, a new method is proposed for the detection of E. sakazakii in food and environmental samples. This proposed method replaces the culturing steps on violet red bile glucose agar (VRBGA) and TSA with culturing on chromogenic DFI or ES agar. For identification and confirmation of presumptive E. sakazakii isolates, the oxidase test, yellow pigment production and API biochemical profiling is replaced by DNA sequencing and/or species-specific PCR with the most accurate primer pair (Esakf and Esakr). The amendments to the current FDA method will reduce the time to detect E. sakazakii from approximately 7 days to 4 days and should prove to be more sensitive, specific and accurate for E. sakazakii detection. In this study, a novel PCR-based method was developed which was shown to be capable of discriminating between viable and dead E. sakazakii cells. This was achieved utilising the irreversible binding of bacterial DNA to photo-activated PMA or EMA in order to prevent PCR amplification from the dead cells. At concentrations of 50 and 100 μg.ml-1, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the PCR amplification from viable cells. EMA was equally effective in preventing PCR amplification from dead cells, however, it also inhibited PCR amplification from viable cells. PMA-PCR in particular, will be useful for assessing the efficacy of processing techniques, as well as for monitoring the resistance, survival strategies and stress responses of E. sakazakii. This will be an important step in the efforts to eliminate E. sakazakii from food and food production environments. / AFRIKAANSE OPSOMMING: Die Wêreld Gesondheidsorganisasie (WGO) beraam dat ten minste 75% van alle babas net baba formule melk (BFM) of BFM in kombinasie met moedersmelk in die eerste vier maande na geboorte kry. Die teenwoordigheid van die voortkomende patogeen Enterobacter sakazakii in BFM is al geassosieer met skaars maar noodlottige gevalle van neonatale infeksies en sterftes. Akkurate metodes word dus benodig vir die vinnige deteksie van E. sakazakii in voedsel. Die metodes wat huidiglik gebruik word vir die deteksie en identifikasie van hierdie mikroörganisme is onvoldoende, kontroversieël en teenstrydig. Die doel van hierdie studie was om die beste metode vir die deteksie van E. sakazakii te bepaal, na 'n evaluasie van die metodes wat huidiglik beskikbaar is. 'n Verdere doel was om 'n polimerase ketting reaksie (PKR) metode vir die deteksie van slegs lewensvatbare E. sakazakii selle te optimiseer deur gebruik te maak van die DNSbindende kleurstowwe, etidium mono-asied (EMA) en propidium mono-asied (PMA). Die Voedsel en Medisyne Administrasie (VMA) se metode vir E. sakazakii deteksie is gebruik om, ongeag van die kolonie kleur, 50 isolate vanuit BFM en 14 isolate vanuit die omgewing te kies. Hierdie isolate is geïdentifiseer deur die DNS volgorde van 'n 1.5 kilo-basis (kb) fragment van die 16S ribosomale DNS (rDNS) te bepaal en die Nationale Sentrum vir Biotegnologiese Informasie (NSBI) databasis te gebruik om die mees verwante spesie te bevestig. Sewe van die 50 (14%) BFM isolate en ses van die 14 (43%) omgewings isolate is geïdentifiseer as E. sakazakii. Die metodes wat geëvalueer is in terme van akkuraatheid vir deteksie en identifikasie van hierdie E. sakazakii isolate het PKR met ses verskillende spesie-spesifieke peiler pare soos beskryf in die literatuur, geel-pigment produksie op triptoon soja agar (TSA) en chromogeniese Druggan-Forsythe-Iversen (DFI) en Enterobacter sakazakii (ES) agars ingesluit. Die geskiktheid van die VMA metode is verlaag deur die lae sensitiwiteit, spesifisiteit en akkuraatheid (87%, 71% en 74% onderskeidelik) van geel pigment produksie vir E. sakazakii identifikasie. Chromogeniese DFI en ES agars was sensitief, spesifiek en akkuraat (100%, 98% en 98% onderskeidelik) vir die identifikasie van E. sakazakii. Die spesifisiteit van die PKR produkte het gewissel tussen 8% en 92%, en Esakf en Esakr is as die akkuraatste geëvalueerde peiler paar geidentifiseer. Die huidige VMA metode vir E. sakazakii deteksie vereis hersiening aangesien meer sensitiewe, spesifieke en akkurate deteksiemetodes voortdurend beskikbaar word. 'n Nuwe metode, gebaseer op die resultate van hierdie studie, word voorgestel vir die deteksie van E. sakazakii in voedsel- en omgewingsmonsters. Die voorgestelde metode vervang die kwekingsstap op violet rooi gal glukose agar (VRGGA) en TSA deur kweking op chromogeniese DFI of ES agars. Verder word die oksidase toets, geel pigment produksie en API biochemiese profiele van vermoeidelike E. sakazakii isolate vervang deur DNS volgorde bepaling en/of spesie-spesifieke PKR met die mees spesifieke peiler paar (Esakf and Esakf) vir die identifikasie en bevestiging van E. sakazakii. Die voorgestelde wysigings van die VMA metode sal die tydsduur van E. sakazakii identifikasie van 7 dae na 4 dae verminder, en behoort ook meer sensitief, spesifiek en akkuraat te wees vir die deteksie van E. sakazakii. 'n Nuwe PKR-gebaseerde metode wat tussen lewensvatbare en dooie E. sakazakii selle kan onderskei is in hierdie studie ontwikkel. Dit is bereik deur die onomkeerbare binding van bakteriële DNS aan lig-geaktiveerde EMA of PMA om die PKR amplifisering van dooie selle te voorkom. Konsentrasies van 50 en 100 μg.ml-1 PMA het PKR amplifikasie heeltemal geïnhibeer, terwyl geen inhibisie van lewensvatbare selle bespeur kon word nie. EMA was ook suksesvol in die voorkoming van die PKR amplifikasie van dooie selle, alhoewel daar ook 'n mate van DNS inhibisie was tydens die amplifikasie van lewensvatbare selle. PMA-PKR kan ook van nut wees vir die assessering van die doeltreffendheid van prosesseringstegnieke, en ook vir die waarneming van die weerstandigheid, oorlewingsstrategieë en stresresponse van E. sakazakii. Dit sal 'n belangrike stap wees in pogings om E. sakazakii van voedsel en voedsel produksieomgewings te elimineer.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2010 / South Africa currently faces one of the highest HIV prevalence rates in the world. As this prevalence rises, the strain placed on its hospitals is likely to increase due to the shortage of beds. The devastating effects of HIV/AIDS initiated the establishment of a hospice which is a non-governmental organisation whose goal is the provision of care for terminally ill patients, either in their homes, in hospitals or in a hospice’s own in-patients wards. Part of the hospice’s mission is to offer palliative care without charge to anyone who requires it. The basic elements of hospice care include pain and symptom management, provision of support to the bereaving family and promoting a peaceful and dignified death. This also includes the provision of cooked foods to the patients using the kitchen facilities of the hospices for this activity. It is well known that the kitchen is particularly important in the spread of infectious disease in the domestic environment due to many activities that occur in this particular setting. Food and water safety is especially important to the persons infected with the human immunodeficiency virus (HIV) or with immunodeficiency syndrome (AIDS).It is estimated that food-borne pathogens (disease–causing agents) are responsible for 76 million illnesses, some resulting in death, in the United States alone every year. In one study of patients with AIDS, two-thirds had diarrhoeal disease and in two-thirds of these, the following enteric pathogens were identified: Salmonella, Shigella, Listeria, Yersnia, Cryptosporidium, Entamoeba histolylica and Campylobacter sp. In an epidemiological study of patients with HIV infection a close association was found between consumption of raw or partially cooked fish and antimicrobial-resistant Mycobacterium avium complex. Antibiotic resistance in food-borne pathogens has become a reality and this poses a serious threat to the medical fraternity since it diminishes the effectiveness of treatment. This study was undertaken to determine the prevalence of foodborne pathogens including bio aerosols isolated from the kitchen surfaces and food handler’s before and after cooking. The antibiotic resistance of the isolated pathogens was further determined to assess their impact on treatment. The following microbiota were isolated: Total viable counts (TVC), Coliforms, Escherichia coli, Staphylococcus aureus, Pseudomonas and presumptive Salmonella. The hospices had high counts of E.coli and S.aureus on the cutting boards for the breakfast session compared to the traditional home based kitchens. It was speculated that this could have originated from crosscontamination via the foodhandler’s hands and the food served. It is evident from the results that hospices lack a management system regarding the prevalence of E. coli as it was present on the cutting boards throughout the food preparation sessions. Gram negative organisms (coliform and P. aeruginosa) were in particular both resistant to oxacillin and this pose a great challenge in this particular setting. This can be addressed by putting emphasis on hygiene as a strategy per se for reducing antibiotic resistance.