Phylogeny and molecular identification of Cronobacter strains isolated from south African food products

Strydom, Amy (2011-03)

Thesis (MSc Food Sc)--University of Stellenbosch, 2011.

Thesis

ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter.

AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak. Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle virulensie. Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee afsonderlike groepe gedeel met 'n 93% vertrouens vlak. Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr. muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr. malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met malonaatbenutting-analises. 'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme (BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon. DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig. Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf spesies van Cronobacter kan verseker.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/6506
This item appears in the following collections: