축산물유래 Listeria monocytogenes의 분자역학적 연구
Molecular Epidemiological Study of Listeria Monocytogenes Isolates from Animal Products
축산물 Listeria monocytogenes 분자역학 수의학;
- 원문 URL
This study investigated the epidemiology of Listeria monocytogenes, a food-borne pathogen. The epidemiology of food-borne pathogens is of great importance for clarifying bacterial origin and preventing bacterial contamination and infection. This work examined 68 L. monocytogenes strains, including 11 reference strains and 57 isolates from imported US beef, domestic meats (beef, pork, chicken meat), raw milk, and milk plants. 1. The first objective was to evaluate the production of virulence proteins, such as hemolysin (LLO) and lecithinase (LCP), the adsorption of Congo red (CRA), and to detect virulence genes using the polymerase chain reaction (PCR). In the study of virulence protein production, 68 (100%), 62 (91.2%), and 54 (79.4%) of the 68 L. monocytogenesstrains were positive for LLO production, the LCP test, and the CRA test, respectively, while strains of other species, such as L. innocua, L. gray, L. murrayi, and L. welshimeri, were not. There were no significant differences between L. monocytogenes serotypes and the ability to produce LLO or LCP. L. monocytogenesstrains had very high hemolytic titers (2 to 16 fold), while the other Listeria species, other than L. ivanovii and L. seeligeri, did not. The hemolysin activities of L. monocytogenes, L. ivanovii, and L. seeligeri usually exceeded 1.0 HU/mg, while those of other Listeriaspp. were less than 0.04 HU/mg. In the PCR assay, all of the L. monocytogenes strains contained the hlyA, plcA, plcB, inlA, and inlB virulence genes and produced a product of the expected size. In the PCR of the actA gene, the expected 385-bp product was seen in 39 (57.4%) L. monocytogenesstrains, while an unexpected 268-bp product was seen in 29 (42.6%) strains. Most L. monocytogenes strains isolated from Hanwoo beef produced the 385-bp actA gene product, while strains of imported US beef usually produced the 286-bp actA gene product. By contrast, no virulence gene products were amplified in the other Listeria spp. 2. A random amplified polymorphic DNA (RAPD) technique and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) were optimized and combined to develop a standard molecular epidemiological analysis of L. monocytogenes. There was great genetic variability among the isolates, which produced 24, 24, 30, and 34 RAPD patterns with primers HLWL85, HLWL74, D87, and MMT1, respectively, and 26 ERIC-PCR patterns. The discriminatory power of the RAPD method was highest using primer D87 (DI=0.958. S=80%) and that of RAPD combined with ERIC-PCR analysis was superior (DI=0.978, S=70%). A few genotypes were specific for L. monocytogenesstrains belonging to a particular serotype. The isolates could be classified into 38 genotypes using composite profiles combining the RAPD analyses with four different primers and the ERIC-PCR analysis. Of these, 34 genotypes were specific to origin. Using the combined method, the strains isolated from imported beef (US) and the reference strains could be distinguished completely from the strains isolated in Korea. Four isolates of strains from imported beef (US) shared very high genetic similarity (S=83%). 3. Protein profiles were investigated to compare virulent strains of L. monocytogenes Scott A and avirulent RI strains using 2-dimensional gel electrophoresis (2-DE) maps and 2-DE immunoblot profiles. In the 2-DE analysis, RI and Scott A gave rise to approximately 340 and 390 protein spots on the gels, respectively. There were few differences in spot location betweenthe two strains, but most of the spots were placed at the same pI and MW on the gels. Of these, 17 proteins involved in either metabolic pathways or protein synthesis were identified in the MALDI-TOF MS analysis, including Phosphoglycerate mutase, 2,3-bisphosphoglycerate-independent, ATP synthase F1, alpha subunit, 6-phosphogluconate dehydrogenase, DNA polymerase Ⅲ, beta chain, Enolase, Glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase (alpha subunit), Elongation factor Ts, Oxidoreductase, aldo/keto reductase family, Pyridoxine biosynthesis protein, PTS system mannose-specific, factor IIAB, Fructose-1,6-bisphosphate aldolase, Malonyl CoA-acyl carrier protein transacylase, CodY protein (Bacillus subtilis), Triose phosphate isomerase, ATP binding cassette (ABC) transporter, and Ribosomal protein L10. From the spots, the ATP binding cassette (ABC) transporter was specifically identified from the Scott A strain in a peptide mass fingerprinting (PMF) analysis, and the protein participating in regulating active energy metabolism might be present in virulent strains of L. monocytogenes. In the 2-DE immunoblot profiles, spots specific to Scott A were present in the range 66.8 kDa/pI 4.86-5.28, while spots specific to RI were placed in the range 38.8 kDa/pI 4.85-5.05 and 34.8 kDa/pI 4.34-5.33. In addition, the protein band at 60 kDa was thought to be an immunodominant band specific for Scott A in the SDS-PAGE immunoblot analysis. These antigenic protein bands and spots might serve as markers for distinguishing virulent and non-virulent strains.