Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2005. / It is estimated by the World Health Organisation (WHO) that thousands of millions of cases of foodborne diseases occur world–wide every year. Enterobacter sakazakii is a member of the family Enterobacteriaceae and has been identified as an occasional contaminant of powdered infant formula milk (IFM). Enterobacter sakazakii is an opportunistic emerging pathogen and has the ability to cause a severe form of neonatal meningitis. This organism was referred to as “yellow pigmented Enterobacter cloacae” until 1980 after which it was renamed as E. sakazakii. The current method for the detection of E. sakazakii is very time consuming and includes pre–enrichment, enrichment in Enterobacteriaceae enrichment broth, subsequent plating on violet red bile glucose agar and subculturing on tryptone soy agar. In this study a polymerase chain reaction (PCR) method was developed for the identification of the presence of E. sakazakii in infant food products. A part of the 16S ribosomal RNA (rRNA) gene from E. sakazakii was amplified using the primer pair Esak2 and Esak3. An internal amplification control (IAC) was constructed as part of the PCR detection method. The 850 base pair (bp) E. sakazakii PCR product was digested with AluI and the two fragments containing the primer binding sites were ligated, resulting in a 240 bp IAC. During this study a positive band for both the target DNA (850 bp) and the IAC (240 bp) was simultaneously observed when the IAC was added to the PCR mixture at a concentration of 0.72 pg.ml-1. Four of 22 South African food products tested positive for the presence of E. sakazakii, using both the PCR and recommended culturing methods. The PCR method was used successfully for the detection of E. sakazakii within three days and thus provides a possible alternative and improvement on the recommended current culturing methods. Other microorganisms present in the products tested included Escherichia coli, Klebsiella pneumoniae, Raoultella terrigena (“Klebsiella terrigena”) and Chryseomonas luteola. Since E. sakazakii is usually present in low numbers in food products, it is possible that these few cells are unevenly distributed in the products, making it important to take multiple samples when evaluating IFM and thereby ensuring that even low numbers of this pathogen are detected.

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.