초록 ▼

Ⅳ. 연구개발결과
식중독세균은 작물과 어떠한 반응도 없으며 식물병원세균의 TTSS(type 3 secretion system)를 식중독균에 발현시키는 경우에도 역할을 하지 못한다. 그러나 작물 바깥 또는 내부에서 생존이 가능하다. 식중독세균 중 황색포도상구균과 근연종인 포도알균 60균주를 분리하여 동정법을 확립하고 독소, 항생제 내성을 분석하고 병원성과 바이오필름 생성능력의 관계를 평가하였다. 또한 바이오필름을 대량생산하는 포도알균과 황색포도상구균바이오필름 관련유전자(bap)의 발현 비교분석 및 바이오필름 형성이 황색포도상구균

Ⅳ. 연구개발결과
식중독세균은 작물과 어떠한 반응도 없으며 식물병원세균의 TTSS(type 3 secretion system)를 식중독균에 발현시키는 경우에도 역할을 하지 못한다. 그러나 작물 바깥 또는 내부에서 생존이 가능하다. 식중독세균 중 황색포도상구균과 근연종인 포도알균 60균주를 분리하여 동정법을 확립하고 독소, 항생제 내성을 분석하고 병원성과 바이오필름 생성능력의 관계를 평가하였다. 또한 바이오필름을 대량생산하는 포도알균과 황색포도상구균바이오필름 관련유전자(bap)의 발현 비교분석 및 바이오필름 형성이 황색포도상구균이의 초기 부착능에 미치는 영향 확인하였다.
독소형 황색포도상구균을 대상으로 돌연변이 라이브러리를 제작하고 이를 이용하여 Entrotoxin B, D의 발현분석하고 이들 유전자를 포함하는 유전체의 서열 분석을 마쳤다.

Abstract ▼

1. Genetics and physiologies of food-borne pathogens on agricultural produces
Recently, research into the interaction of human bacterial pathogens and plant has risen with the number of clinical cases of food-borne illness caused by contaminated fresh produce. Certain human bacterial pathogens ar

1. Genetics and physiologies of food-borne pathogens on agricultural produces
Recently, research into the interaction of human bacterial pathogens and plant has risen with the number of clinical cases of food-borne illness caused by contaminated fresh produce. Certain human bacterial pathogens are not proven to be plant pathogens yet. To identify whether enteric bacteria can cause similar phenomenon when it is internalized into plants, we performed hypersensitive response assay. All tested bacteria belong to enterobacteriaceae family did not show HR, and disease symptom also was not observed. To monitor survival and multiplication of enteric bacteria, we inoculated bacteria with 106 CFU/ml concentration into leaf of various plants including tomato, pepper, and tobacco (those are Solanaceae family), and Chinese cabbage (Brrasicae family). Yersinia enterocolitica was disappeared within 4 days, however E. coli O157:H7 and Salmonella enterica were survived over 2 weeks. To find out reaction of plant immune response after enteric bacterial infection, plant immune response related gene expression were analyzed using RT-PCR analysis. Both salicylic acid and jasmonic acid pathways were turned-on, simultaneously. When we introduced type three secretion system and plant cell wall degrading enzyme into enteric bacteria, it couldn’t macerate Chinese cabbage. These results demonstrate that enteric pathogens can use plants as vectors and leads to the risk of human illness.
Staphylococci including Staphylococcus aureus have recognized as an important opportunistic pathogen and food-borne pathogen. The consumption of leaf vegetable has been increased in Korea. We isolated coagulase negative Staphylococcus and Staphylococcus aureus from leaf vegetables. For identification of Staphylococcus, API Staph ID system, analysis of 16S rRNA sequence and confirmation of species-specific genes were used. There is no well-organized database for CNS distribution in agricultural products, and few studies have used species-specific PCR method for CNS. In this study, we developed new method using sodA gene encoding the manganese-dependent superoxide dismutase in Staphylococci to evaluate the identification of CNS. All Staphylococci were identified by PCR of sodA gene and confirmed by 16S rRNA sequencing. The identification rates with species-specific PCR of sodA gene were 100 %. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely staphylococcal species. To study genetic diversity among the isolates, genes associated with toxins, antibiotic susceptibility, genetic relationships were analyzed using PCR, the disk diffusion method, and pulsed-field gel electrophoresis (PFGE). At least one enterotoxin encoded genes (sea, seb, sec, sed, and see) were detected in 20.75% of isolates from leaf vegetables. In antibiotic resistance test, 96.23% of S. aureus isolates from leaf vegetables were resistant against penicillin. We generated a dendrogram displaying the composite S. aureus isolates from various origins based on a combination of the toxin genes, antibiotic susceptibility and patterns of PFGE. The distinct composite-type among the isolates was clustered into 8 major groups. 5 kinds of pathogenicity test (Lipase, Lecithinase, Hemolysis, Protease, DNase) were performed. Almost CNS has lipase, lecithinase, hemolysis, protease activities. It was confirmed high DNase activity from some kinds of CNS strains. Biofilm formation is known as a major factor determining pathogenicity of staphylococci in such device-associated infections. S. haemolyticus, S. xylosus species were showed strong biofilm activities. Moreover these two species were helped S. aureus biofilm initial attachment. This study can be used an important data for study controlling of staphylococci as opportunistic human pathogen of agricultural products.
2. Virulence related gene expression of Staphylococcus aureus on agricultural products
Staphylococci including Staphylococcus aureus have recognized as an important opportunistic pathogen and food-borne pathogen. The consumption of leaf vegetable has been increased in Korea. We isolated Staphylococcus aureus from leaf vegetables. To study pathogenicity including regulation of enterotoxins, genes associated with toxins, antibiotic susceptibility and genetic relationships were analyzed using PCR, the disk diffusion method, and pulsed-field gel electrophoresis (PFGE). S. aureus D-1 had 3 different enterotoxin genes (seb, sed, and sej) and low antibiotic resistance. S. aureus D-1 strain was constructed mutant library. Enterotoxin B gene was encoded on Staphylococcal pathogenicity island (SaPI) of chromosomal DNA and enterotoxin D and J genes on native plasmid. The native plasmid, pD-1, was sequenced and analysed. It was 29,963-bp. For genetic research, integrase vectors were constructed. Integrative vectors based on the lysogenic bacteriophage site-specific recombination system were constructed using Gibson assembly method. Gibson assembly is a high throughput, single tube and isothermal reaction method for the gathering of multiple DNA fragments. Integrative vectors can replicate in Escherichia coli but cannot replicate in Staphylococcus. Insert DNA in these vectors can only be maintained by integrating into the chromosome in Staphylococcus. To improve vector system, pS4194 and pK4623 were constructed by assembling various genetic elements. p15A replication origin was used to make both vectors. MCS (multiple cloning sites) of pDL278 was used to increase restriction enzyme sites. Antibiotic resistant gene of pS4194 was used with spectinomycin adenyltransferase gene derived from pDL276. Antibiotic resistant gene pK4623 was used with aminoglycoside tranferase gene derived from pDL278. Size of vector pS4194 and pK4623 are under 5kb. These characteristics of new integrative plasmid vector system are more effective to genetic modification of Staphylococcus than previous vector systems.