Impact of selected environmental factors on E.coli growth in river water and an investigation of carry-over to fresh produce

Van Blommestein, Anneri (2012-12)

Thesis (MSc)--Stellenbosch University, 2012.


ENGLISH ABSTRACT: The consumption of fresh produce has increased over the past few decades, but it has also resulted in an increase in foodborne outbreaks. Once fresh produce gets contaminated, microbes can survive or even multiply on the produce. There is, however, very little information available on what impact environmental conditions will have on the survival and growth of potential pathogens. Thus the purpose of the first phase of the study was to determine the impact of different environmental factors: carbon levels; temperature; incubation time; and initial microbial load on the growth of E.coli and other "indigenous" microbes present in the Plankenburg river water. Water from the Plankenburg River was incubated at 10° to 35°C and the E.coli and aerobic (ACC) loads determined at 0, 6, 12 and 24 h. The impact of different COD:N:P ratios on growth were also evaluated by altering the water values. It was found that in non-sterile river water the E.coli levels increased with increase in incubation temperature. Minimal growth took place between 10° and 20°C and the largest growth increases at 35°C. The data showed that E.coli dieoff was rapid when the nutrient levels were low. It was concluded that the carbon (COD) level is a major growth limiting factor in river water. The impact of adjusted carbon levels on the growth of E.coli present was then evaluated. The E.coli was found to die-off faster at the higher temperatures when the carbon levels were low (COD = <10 mg.L-1), possibly due to nutrient limitations. At higher carbon levels (COD = >100 mg.L-1), the growth profiles of E.coli showed major growth increases with no die-off during the 24 h period, probably because of the availability of sufficient nutrient levels. Even though standard methods can be used to identify if irrigation water is faecally contaminated it is important to confirm that organisms on fresh produce are from the faecally polluted irrigation water. The purpose of the second study was to determine the effect of daily irrigation on carry-over, the effect of “once-off” irrigation on the survival of E.coli on the produce, identifying types of E.coli in the irrigation water and those on irrigated fresh produce, and then linking the E.coli types. In the first trial, green beans planted in a tunnel and irrigated on a daily basis (14 days) with Plankenburg water and a “pure wild-type” E.coli at a concentration of 106 cfu.mL-1 were evaluated. The results showed that E.coli is carried over from irrigation water to the irrigated green beans, especially when the E.coli levels in the river water were high. After the enumeration steps, colonies from both the irrigation water and from the irrigated beans were selected (67 isolates from the green beans and 72 from the irrigation water). Unique phenotypic (API) profiles were generated for each isolate. The first indication of linking was confirmed based on the degree of phenotypic similarity using numerical clustering systems. The results showed that these isolates were related and originated from the same pollution source. The use of PCR further confirmed (uidA positive) that all the phenotypically identified strains were E.coli. With multiplex PCR, further linking confirmation was shown when strains tested positive for the mdh gene. The presence of EPEC strains in the irrigation water was also revealed. Thirteen E.coli isolates, which showed positive carry-over links between the irrigation water and the green beans, were further analysed using triplex PCR. However, no direct phylogenetic link to the three main phenotypic E.coli clusters was found. However the triplex PCR could be of value in identifying the source of faecal pollution. For further linking confirmation, DNA sequencing was done on selected phenotypically clustered strains. The oriC-locus sequencing was unsuccessful in distinguishing between the different E.coli strains while the dnaJ sequences showed clear differences and similarities between E.coli strains and some E.cloacae and K.pneumoniae strains respectively from the river water and from the beans. The probability of faecal coliforms being carried-over from irrigation water to fresh produce was shown. This showed the importance of a "multi-method" approach to confirm carryover.

AFRIKAANSE OPSOMMING: Tydens die afgelope paar dekades het die verbruik van vars groente en vrugte vermeerder, maar dit het ook gepaard gegaan met ‘n toename in voedselverwante uitbrekings. Indien vars groente of vrugte gekontamineer word, kan die mikrobes daarop oorleef en ook vermeerder. Daar is min informasie beskikbaar oor die impak van omgewingskondisies op die oorlewing en groei van potensiële patogene. Dus was die doel van die eerste fase van die studie om die impak van verskillende omgewingsfaktore te bepaal: koolstofvlakke; temperatuur; inkubasietyd; en aanvanklike mikrobiese lading op die groei van E.coli en ander “inheemse” mikrobes wat teenwoordig is in die Plankenburg Rivier water. Water van die Plankenburg Rivier was geïnkubeer by 10° tot 35°C en die E.coli en aërobe kolonie tellings (AKT) was bepaal by 0, 6, 12 en 24 h. Die impak van verskeie CSB:S:F verhoudings op groei, was ook geëvalueer deur die waarders van die rivierwater te verander. Dit was gevind dat in die nie-steriele rivierwater, die E.coli vlakke vermeerder het soos die inkubasie temperatuur vermeerder het. Minimale groei het plaasgevind by 10° en 20°C en die meeste groei by 35°C. Die data het gewys dat die E.coli vinnig afgesterwe het as die nutriënt vlakke laag was. Die gevolgtrekking was dat die koolstofvlakke (CSB) die hoof faktor is wat die groei beperk in rivierwater. Die impak van die aangepasde koolstofvlakke op die groei van E.coli teenwoordig was ook geevalueer. Daar was gevind dat die E.coli vinniger afsterf by die hoër temperature as die koolstofvlakke laag is (CSB = <10 mg.L-1), omdat die nutriente moontlik beperk is. Tydens die hoë koolstofvlakke (CSB = >100 mg.L-1), het die E.coli groeiprofiele baie groei getoon met geen afsterwe tydens die 24 h periode nie, omrede dat daar moontlik genoeg nutriente beskikbaar was. Al kan standaard metodes gebruik word om fekale kontaminasie in besproeiingswater te identifiseer, is dit belangrik om te kan bevestig of die organismes op vars groente of vrugte van fekale gekontamineerde besproeiingswater is. Die doel van die tweede fase van die studie was om die effek van daaglikse besproeiing op oordrag te bepaal, effek van eenkeer se besproeiing op die oorleweing van E.coli op groenboontjies, identifisering van E.coli tipes in die besproeiingswater en op die groenboontjies, asook die koppeling van E.coli tipes. In die eerste proef was groenboontjies geplant in ‘n tonnel en daagliks besproei (14 dae) met Plankenburg water en ‘n “suiwer wilde-tipe” E.coli met ‘n konsentrasie van 106 cfu.mL-1. Die resultate het getoon dat E.coli oorgedra was van die besproeiingswater na die besproeide groenboontjies, veral toe die E.coli vlakke in die rivierwater hoog was. Na al die isoleringsstappe, is kolonies van die besproeiingswater en van die besproeide groenboontjies geselekteer (67 isolate van die groenboontjies en 72 van die besproeiingswater). Unieke fenotipiese (API) profiele was gegenereer vir elke isolaat. Die eerste indikasie van koppeling was bewys deur die graad van soortgelyke fenotipiese profiele deur numeriese groepering. Die resultate het gewys die isolate was verwant en oorspronklik van dieselfde bron van kontaminasie. Die gebruik van PKR het verder bewys (uidA positief) dat al die fenotipiese geidentifiseerde isolate E.coli was. Verdere koppeling was geidentifiseer met die multiplex PKR deurdat die isolate positief getoets het vir die mdh geen. Die teenwoordigheid van die EPEC isolate in die besproeiingswater was ook gevind. Dertien E.coli isolate, wat positiewe oordrag verbinding getoon het tussen die besproeiingswater en die groenboontjies, was verder geanaliseer deur triplex PKR. Geen direkte fenotipiese verbinding met die drie hoof fenotipiese E.coli groepe was egter gevind nie. Die triplex PKR kan wel van waarde wees om die bron van die fekale besoedeling te identifiseer. Vir verdere koppeling bevestiging, was DNA volgorde gedoen op isolate van geselekteerde fenotipiese groepe. Die oriC-locus volgorde was onsuksesvol om te onderskei tussen die verskillende E.coli tipes, terwyl die dnaJ volgordes duidelike verskille en ooreenstemmings getoon het tussen die E.coli asook party E.cloacae en K.pneumoniae tipes, onderskeidelik van die rivierwater en boontjies. Die waarskynlikheid van oordrag van fekale kolvorme vanaf besproeiingswater na vars goedere was getoon. Dit wys die belangrikheid van ‘n “multi-metode” benadering om oordrag te bewys.

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