| Listeria monocytogenes is a bacterial foodborne pathogen responsible for human and animal listeriosis, an illness characterized by meningitis, encephalitis, septicaemia.More and more studies showed that the prevalence of L.monocytogenes in pork is higher than in other food. Pulse-field gel electrophoresis(PFGE) is the recently developed method to type of L.monocytogenes,a method characterized by high reproducibility, discriminatory power , typability and has been the best standanrd for subtype of bacteria. For many times, it has been used to identify specific pathogenic organisms, trace the foodborne illness and search the relationship between different foodborne illness break out in different regions/time. The objective of this study is to (i)investigate the prevalence of L.monocytogenes in pig slaughter factory and healthy sheep in jiangsu province; (ii) establish a method for subtype L.monocytogenes by pulse-field gel electrophoresis; (iii) subtype different L.monocytogenes isolates by PFGE, draw phylogenetic trees and gain similar matrices.1. Prevalence of L.monocytogenes in a pig slaughter factory and healthy sheep in Jiangsu provinceIn this study, we investigated the prevalence of L.monocytogenes in 680 meat and environment samples of slaughter-processing-sale flow with the PCR method based on hly gene developed previously. The results showed that there were not any L.monocytogenes in the whole slaughter line, howere, L.monocytogenes were present in samples of hogpen environment,ice-room meat and market pork with the prevalence of 1%,3% and 6.9% respectively. These datas suggested the importence of disinfection of environment regularly. The prevalence of pork in market was the highest in all kinds of samples, which suggested we should pay more attention to reduce contamination from external factors. It was also investigated the prevalence of L.monocytogenes in 422 samples of healthy sheep and the results showed that the the highest prevalence was 12.8%—from samples of environment, then 2.1%—from samples of ewe during lactation. There were not any L.monocytogenes in pregnant ewe,lamb during lactation,ram lambs. The results suggested that strict disinfection according to operating regulations was very important, besides, improvement of quality of hay, decreasement of herd density, strengthen exercise of sheep were effective measures to keep sheep healthy.2. Establish a method for subtyping L.monocytogenes by pulse-field gel electrophoresisThe key steps of PFGE were: mixing bacterial suspension with agarose to make plugs; to release chromosome of the fixed bacteria; washing the plugs; restriction digestion of L.monocytogenes DNA in agarose plugs; plused field electrophoresis; analysis of the restriction endonuclease digestion profiles (REDPs). We optimized the method from following aspects: the best density of bacteria; appropriate treatment before digestion of DNA; the process of washes; digestion time and enzyme; parameters for PFGE; analysis method. Through exploring the method, the good REDPs of the reference strains were got, and finally the optimum conditions for PFGE were determined. (1) adjust the optical density of bacterial cell suspensions to 1.3(range of 1.25 to 1.35) at 610 nm; (2) a 10 min lysozyme treatment step before mixture with agarose, choose the agarose with low melting point to substitute Seakem Gold Agarose(SKG), mix cell suspensions and agarose with the same volume; (3) the process of washes: wash plugs twice with preheated water for 45 minutes, and then wash plugs twice with preheated TE buffer for 45 minutes. Add TE containing PMSF for 30 minutes, then wash plugs with water and TE buffer; (4) restriction digestion: choose AscI to digest L.monocytogenes DNA; a ice-bath treatment for 30 minutes before digestion; the volume of enzyme 2ul/slice(40U/ul); incubate for at least 3 hr; (5) parameters for PFGE: agradient,6.0V/cm; angle,120°; temperature,12℃; switch time,4.0s-40.0s; run time,22h; (6) use Quantity One to analyse the results, draw phylogenetic trees and gain similar matrices. The methods developed in this study had the features of high reproducibility, discriminatory power and typability, besides, it had the advantages of shorter time from culturing bacteria to gaining results and lower costs of experiments. Also, the method has reference value to other Gram-positive bacteria, and provides a sound technical platform for tracing L.monocytogenes. 3. Antimicrobial resistance of Listeria monocytogenes isolates and their subtyping by pulsed-field gel electrophoresisIn this study, the antibiotic sensitivity of 50 L.monocytogenes strains isolated from food,environment and clinic was tested. The test methods and results interpretation were according to NCCLs manual"Anti-microbial drug sensitivity test performance standards". Results showed that there were 3 strains sensitive to all 15 kinds antibiotics tested and 47 strains produced 11 kinds of phenotypes spectrum. At the same times, their PFGE subtypes were performed. Results showed that the highest similarity coefficient was 94.0% and the corresponding strains could be found in the RTE food products of the same market. L. monocytogenes found in the same kind of food in the same market had high degree of similarity, and similar results were got from L. monocytogenes isolates of the fresh food products and sewage in the same market. It was noteworthy that one clinical isolate was similar to the strain isolated from RTE food product. The epidemiological values of subtyping by antibiotic resistance and PFGE, by RAPD and PFGE were compared. These results showed that with a high resolution, excellent repeatability characteristics, PFGE was the ideal subtyping method to study the molecular epidemiology of L.monocytogenes.
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