Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a variety
of food sources. It is the cause of the food-borne disease, listeriosis that shows
symptoms such as meningitis, encephalitis and abortion. Different strains of L.
monocytogenes exist and not all are thought to be pathogenic to humans. The aim of
this study was to evaluate and compare conventional methods, culturing on selective
(Oxford agar) and chromogenic (RAPID’L.mono agar) media, as well as speciesspecific
and multiplex polymerase chain reaction (PCR) methods for the detection and
identification of 94 L. monocytogenes isolates from various areas in an avocado
processing facility, as well as the final product. To achieve a better understanding of
the genetic diversity of the confirmed L. monocytogenes strains isolated from the
avocado facility, two subtyping techniques, PCR-restriction fragment length
polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), were
employed.
All of the isolates were identified as Listeria species on both Oxford and
RAPID’L.mono agar. On the RAPID’L.mono agar, 76 of the 94 isolates produced
colonies typical of L. monocytogenes, with the remaining 18 showing colonies typical of
L. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfully
amplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same
80 isolates the multiplex PCR successfully amplified 800, 517 and 238 base pair (bp)
fragments of the inlA, inlC and inlJ genes, respectively. The remaining 14 isolates
included the 13 isolates identified as L. innocua, as well as an isolate identified as L.
monocytogenes on RAPID’L.mono. The results obtained on the Oxford agar showed a
100 % positive correlation when compared to the PCR results in identifying Listeria
species, while the RAPID’L.mono had a 4 % false negative result in identifying L.
monocytogenes compared to the PCR results.
Sixty-four of the confirmed L. monocytogenes isolates were subtyped using PCRRFLP
and PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene was
successfully amplified for all of the isolates, followed by digestion with the restriction
enzymes, AluI and Tsp509I. AluI produced three different banding patterns and
Tsp509I produced two different banding patterns. Subtyping of the isolates using PFGE
was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The
restriction fragments were resolved by PFGE and the fingerprints were classified into
four clusters. In the combined analyses, cluster I contained forty-eight isolates (n=48), cluster II 1 isolate (n=1), cluster III fifteen isolates (n=15) and cluster IV 1 isolate (n=1).
The PCR-RFLP results had a 98 % correlation with the PFGE results.
The results of this study indicated inconsistencies between the results obtained
by conventional and molecular detection methods for the identification of L.
monocytogenes. Species-specific and multiplex PCR, however, proved useful to
accurately detect and identify L. monocytogenes in a shorter period of time and could
replace the use of conventional agar during identification. Both PCR-RFLP and PFGE
proved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP being
less expensive and results obtainable in a shorter period of time. / AFRIKAANSE OPSOMMING: Listeria monocytogenes is ‘n patogeen afkomstig van voedsel wat uit ‘n verskeidenheid
voedselbronne geisoleer kan word. Dit is die oorsaak van die voedsel afkomstigde
siekte, listeriosis met simptome soos harsingvliesontsteking, ensefalitis en aborsie. ‘n
Verskeidenheid L. monocytogenes stamme bestaan, maar nie almal word as
patogenies beskou nie. Die doel van hierdie studie was om konvensionele metodes,
naamlik mikrobiologiese kweking op selektiewe (Oxford agar) en chromatografiese
(RAPID’L.mono agar) media, sowel as spesies-spesifieke en multipleks polimerase
ketting reaksie (PKR) metodes te evalueer en vergelyk vir die deteksie en identifikasie
van 94 L. monocytogenes isolate geisoleer vanuit verskeie areas in ‘n avokado
prosesseringsfasiliteit sowel as die finale produk. Om ‘n beter begrip van die genetiese
diversiteit van die isolate wat as L. monocytogenes bevestig is te verkry, is twee
subtiperingstegnieke, PKR-restriksiefragmentlengte polimorfisme (PKR-RFLP) en
pulsveld jel-elektroforese (PVJE) toegepas.
Beide Oxford en RAPID’L.mono agar het al die isolate as Listeria spesies
geidentifiseer. Op die RAPID’L.mono agar het 76 van die 94 isolate kolonies tipies van
L. monocytogenes gevorm, 13 kolonies was tipies van L. innocua (n=13) en vyf kolonies
tipies van L. ivanovii (n=5). Die spesies-spesifieke PKR het ‘n 730 basis paar (bp)
streek van die hly geen suksesvol geamplifiseer vir 80 van die 94 isolate. Die
multipleks PKR het 800, 517 en 238 bp fragmente van die inlA, inlC and inlJ gene
onderskeidelik, vir dieselfde 80 isolate suksesvol geamplifiseer. Die oorblywende 14
isolate het die 13 isolate wat as L. innocua geïdentifiseer is en die een isolaat wat as L.
monocytogenes op RAPID’L.mono geïdentifiseer is ingesluit. Resultate verkry met die
Oxford agar het 100 % ooreengestem met die PKR resultate vir die identifikasie van
Listeria spesies. Die RAPID’L.mono het ‘n 4 % vals negatiewe resultaat gelewer in
vergelyking met die PKR resultate.
Vier-en-sestig van die bevestigde L. monocytogenes isolate is gesubtipeer deur
PKR-RFLP en PVJE. Tydens die PKR-RFLP analise is ‘n 733 bp fragment van die inlA
geen suksesvol geamplifiseer, gevolg deur vertering met die restriksie-ensieme, AluI
and Tsp509I. AluI het drie verskillende bandpatrone opgelewer en Tsp509I twee
verskillende bandpatrone. Subtipering deur PVJE is uitgevoer deur makro-restriksie
van die genomiese DNA met ApaI en AscI. Die restriksie fragmente is geskei deur
PVJE en die vingerafdrukke is in vier groepe geklassifiseer. Groep I het 48 isolate
(n=48), groep II 1 isolaat (n=1), groep III 15 isolate (n=15) en groep IV 1 isolaat (n=1) gehad tydens die gekombineerde analise. Die PKR-RFLP resultate het 98 %
ooreengestem met die van die PVJE.
Die resultate van hierdie studie het teenstrydighede tussen die resultate van
konvensionele en molekulêre deteksie metodes opgelewer vir die identifikasie van L.
monocytogenes. Die spesies-spesifieke en multipleks PKR het egter beide goed te pas
gekom vir die akkurate deteksie en identifikasie van L. monocytogenes en kan heel
moontlik die gebruik van konvensionele agar tydens identifikasie vervang. Beide PKRRFLP
en PVJE was nuttig vir die subtipering van L. monocytogenes isolate. PKR-RFLP
is egter ‘n goedkoper tegniek en die resultate is in ‘n korter tydsperiode beskikbaar.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17912 |
| Date | 12 1900 |
| Creators | Bester, Ingrid Muriel |
| Contributors | Witthuhn, R. C., Cameron, M., Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 91 p. : ill. |
| Rights | Stellenbosch University |
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