Subtyping of Listeria monocytogenes isolated from a South African avocado processing facility using PCR-RFLP and PFGE

The aim of this study was to subtype Listeria monocytogenes strains present in an avocado processing facility using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLPs) and pulsed-field gel electrophoresis (PFGE). The inlA gene of the L. monocytogenes isolates was PCR amplified and cut with the restriction endonuclease AluI or Tsp509I. Initial investigation of 64 L. monocytogenes isolates indicated that AluI was more discriminating and therefore an additional 76 isolates were subtyped with PCR-RFLPs using AluI. The PCR-RFLP with AluI resulted in three banding patterns. Subtyping of the isolates using PFGE was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The PFGE fingerprints generated by ApaI contained 5 clusters and resulted in better differentiation between the strains than digestion with AscI. The majority of strains were grouped in cluster I (n 1⁄4 22) and in cluster II (n 1⁄4 50). All of the clusters contained product, as well as environmental isolates and thus no correlations with specific sources of contamination could be established. The results of this study indicated that both molecular subtyping methods, PCR-RFLP and PFGE, were sensitive and specific enough to assess the diversity among the majority of the L. monocytogenes isolates. This is the first study in which L. monocytogenes isolates from a South African avocado processing facility were characterised with PCR-RFLP and PFGE. Results clearly point to the need for increased hygienic measures to prevent the dissemination of this pathogen during avocado processing. 2012 Elsevier Ltd. All rights reserved.