Detection and molecular subtyping of Listeria Monocytogenes isolated from a South African avocado processing facility

[摘要] ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a varietyof food sources. It is the cause of the food-borne disease, listeriosis that showssymptoms such as meningitis, encephalitis and abortion. Different strains of L.monocytogenes exist and not all are thought to be pathogenic to humans. The aim ofthis study was to evaluate and compare conventional methods, culturing on selective(Oxford agar) and chromogenic (RAPID'L.mono agar) media, as well as speciesspecificand multiplex polymerase chain reaction (PCR) methods for the detection andidentification of 94 L. monocytogenes isolates from various areas in an avocadoprocessing facility, as well as the final product. To achieve a better understanding ofthe genetic diversity of the confirmed L. monocytogenes strains isolated from theavocado facility, two subtyping techniques, PCR-restriction fragment lengthpolymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), wereemployed.All of the isolates were identified as Listeria species on both Oxford andRAPID'L.mono agar. On the RAPID'L.mono agar, 76 of the 94 isolates producedcolonies typical of L. monocytogenes, with the remaining 18 showing colonies typical ofL. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfullyamplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same80 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 isolatesincluded 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 a100 % positive correlation when compared to the PCR results in identifying Listeriaspecies, 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 PCRRFLPand PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene wassuccessfully amplified for all of the isolates, followed by digestion with the restrictionenzymes, AluI and Tsp509I. AluI produced three different banding patterns andTsp509I produced two different banding patterns. Subtyping of the isolates using PFGEwas carried out by macrorestriction of the genomic DNA with ApaI and AscI. Therestriction fragments were resolved by PFGE and the fingerprints were classified intofour 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 obtainedby conventional and molecular detection methods for the identification of L.monocytogenes. Species-specific and multiplex PCR, however, proved useful toaccurately detect and identify L. monocytogenes in a shorter period of time and couldreplace the use of conventional agar during identification. Both PCR-RFLP and PFGEproved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP beingless expensive and results obtainable in a shorter period of time.

[发布机构] Stellenbosch University