Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157:H7

Karimi, Farrokh,Mousavi, Amir,Salmanian, Ali Hatef,Alizadeh, Houshang,Rafati, Sima 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.4

Chloroplast genetic engineering offers an opportunity for high level expression and cost-effective recombinant protein production. Escherichia coli O157:H7 is one of the most important zoonotic pathogens causing hemorrhagic colitis (HC) and the life-threatening hemolytic-uremic syndrome in humans worldwide. The occurrence of zoonotic E. coli O157:H7 outbreaks in recent years has led to increased efforts in the development of safe and cost-effective immunogenic antigens against E. coli O157:H7. EspA and Tir/Intimin proteins are the important virulence factors which are encoded by the LEE locus of enterohemorrhagic E. coli. In this study, we hypothesized that the high level expression of the chimeric form of these effectors in chloroplasts and using tobacco transplastomic plants as an oral delivery system for the development of an edible-base vaccine would induce an immune response for the prevention of E. coli 0157:H7 attachment and colonization in animal model mice. The prokaryotic codonoptimized EIT protein was expressed in plastid genome via chloroplast transformation. Putative transplastomic plants were analyzed by PCR, and Southern blot analysis confirming chloroplast integration and homoplasmy in the T1 progeny. Immunoblotting and ELISA assays demonstrated that the EIT protein was expressed in chloroplasts and accumulated up to 1.4 % of total soluble protein in leaf tissue. In mice orally immunized with transplastomic tobacco plant leaves, high immunological responses (IgG and IgA specific antibodies) were detected in serum and feces. Finally, the challenging assay with E. coli O157:H7 in immunized mice showed reduced bacterial shedding.

Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157:H7

Farrokh Karimi,Amir Mousavi,Houshang Alizadeh,Ali Hatef Salmanian,Sima Rafati 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.4

Chloroplast genetic engineering offers an opportunity for high level expression and cost-effective recombinant protein production. Escherichia coli O157:H7 is one of the most important zoonotic pathogens causing hemorrhagic colitis (HC) and the life-threatening hemolytic- uremic syndrome in humans worldwide. The occurrence of zoonotic E. coli O157:H7 outbreaks in recent years has led to increased efforts in the development of safe and cost-effective immunogenic antigens against E. coli O157:H7. EspA and Tir/Intimin proteins are the important virulence factors which are encoded by the LEE locus of enterohemorrhagic E. coli. In this study, we hypothesized that the high level expression of the chimeric form of these effectors in chloroplasts and using tobacco transplastomic plants as an oral delivery system for the development of an edible-base vaccine would induce an immune response for the prevention of E. coli 0157:H7 attachment and colonization in animal model mice. The prokaryotic codonoptimizedEIT protein was expressed in plastid genome via chloroplast transformation. Putative transplastomic plants were analyzed by PCR, and Southern blot analysis confirmingchloroplast integration and homoplasmy in the T1 progeny. Immunoblotting and ELISA assays demonstrated that the EIT protein was expressed in chloroplasts and accumulated up to 1.4 % of total soluble protein in leaf tissue. In mice orally immunized with transplastomic tobacco plant leaves, high immunological responses (IgG and IgA specific antibodies) were detected in serum and feces. Finally, the challenging assay with E. coli O157:H7 in immunized mice showed reduced bacterial shedding.

Characterization of Diarrheagenic Escherichia coli Isolated from Fresh Beef, Pork, and Chicken Meat in Korean Markets

조용선,구민선,장혜진 한국미생물·생명공학회 2020 한국미생물·생명공학회지 Vol.48 No.2

Diarrhea is a major public health concern associated with pathogenic Escherichia coli infections. Foodborne pathogenic E. coli can lead to large diarrheal outbreaks and hence, there is a need to estimate the frequency of pathogenic E. coli load in the various types of meat available in markets. In the present study, we classified and characterized diarrheagenic E. coli isolates collected from 399 raw meat samples from retail sources in Korea. Shiga toxin-producing E. coli (STEC) were detected in 11 (9.7%) samples, including nine strains (8.0%) in beef and two strains (1.8%) in chicken. The frequency of the detected virulence markers were as follows: astA, 28.3%; escV,18.6%; eaeA,17.7%; ent, 7.0%; EHEC-hly, 4.4%; stx1, 3.5%; and stx2, 3.5%. We did not observe any typical EPEC, EIEC, or ETEC virulence determinants in any of the samples. The STEC serotype O26 was detected in one sample, but no other serogroups (O91, O103, O128, O157, O145, O111, and O121) were found. Further research is needed to better understand the virulence mechanism of STEC serotypes, their ecology, and prevalence in animals, food, and the environment. These results will help improve risk assessment and predict the sources of food poisoning outbreaks.

All blood, No stool: enterohemorrhagic Escherichia coli O157:H7 infection

윤장원,Carolyn J. Hovde 대한수의학회 2008 Journal of Veterinary Science Vol.9 No.3

Enterohemorrhagic Escherichia coli serotype O157:H7 is a pathotype of diarrheagenic E. coli that produces one or more Shiga toxins, forms a characteristic histopathology described as attaching and effacing lesions, and possesses the large virulence plasmid pO157. The bacterium is recognized worldwide, especially in developed countries, as an emerging food-borne bacterial pathogen, which causes disease in humans and in some animals. Healthy cattle are the principal and natural reservoir of E. coli O157:H7, and most disease outbreaks are, therefore, due to consumption of fecally contaminated bovine foods or dairy products. In this review, we provide a general overview of E. coli O157:H7 infection, especially focusing on the bacterial characteristics rather than on the host responses during infection.

Infectious diseases ; Comparative study on the epidemiological aspects of enterohemorrhagic Escherichia coli infections between Korea and Japan, 2006 to 2010

( Won Chang Lee ),( Young Hwan Kwon ) 대한내과학회 2016 The Korean Journal of Internal Medicine Vol.31 No.3

Background/Aims: To compare the epidemiological aspects of enterohemorrhagic Escherichia coli (EHEC) between Korea and Japan by analyzing the current state of EHEC infection outbreaks and related risk factors. Methods: We investigated the epidemiological aspects of EHEC infection cases between Korea and Japan from 2006 to 2010. The following factors were analyzed: national prevalence rate (PR), regional prevalence rate, epidemic aspects (i.e., Cases related to gender), male to female morbidity ratio, age, and seasonal distribution. Results: In total, there were 254 cases of EHEC with an average PR of 0.11 per 100,000 populations in Korea from 2006 to 2010. During the same period in Japan, there were 20,883 cases of EHEC with an average PR of 3.26 per 100,000 populations. The PR in Japan was significantly higher than that in Korea (p < 0.01). In both countries, more females than males had EHEC infections, with the highest incidence of infections (> 50%) observed for individuals younger than 9 years. EHEC is an emerging zoonosis and may be caused by consumption of raw or undercooked meat products from ruminants. Conclusions: This study provides a quantitative analysis of the epidemiological aspects and risk factors of EHEC infections in Korea and Japan and will provide insight on effective future strategies to reduce these infections.

Characterization of Diarrheagenic Escherichia coli Isolated from Fresh Beef, Pork, and Chicken Meat in Korean Markets

Cho, Yong Sun,Koo, Min Seon,Jang, Hye Jin The Korean Society for Microbiology and Biotechnol 2020 한국미생물·생명공학회지 Vol.48 No.2

Diarrhea is a major public health concern associated with pathogenic Escherichia coli infections. Food-borne pathogenic E. coli can lead to large diarrheal outbreaks and hence, there is a need to estimate the frequency of pathogenic E. coli load in the various types of meat available in markets. In the present study, we classified and characterized diarrheagenic E. coli isolates collected from 399 raw meat samples from retail sources in Korea. Shiga toxin-producing E. coli (STEC) were detected in 11 (9.7%) samples, including nine strains (8.0%) in beef and two strains (1.8%) in chicken. The frequency of the detected virulence markers were as follows: astA, 28.3%; escV,18.6%; eaeA,17.7%; ent, 7.0%; EHEC-hly, 4.4%; stx1, 3.5%; and stx2, 3.5%. We did not observe any typical EPEC, EIEC, or ETEC virulence determinants in any of the samples. The STEC serotype O26 was detected in one sample, but no other serogroups (O91, O103, O128, O157, O145, O111, and O121) were found. Further research is needed to better understand the virulence mechanism of STEC serotypes, their ecology, and prevalence in animals, food, and the environment. These results will help improve risk assessment and predict the sources of food poisoning outbreaks.

Comparative Analysis of Detection Methods for Food-borne Pathogens in Fresh-cut Agricultural Materials

Hye-Jeong Jang(장혜정),Hye-Jeong Kim(김혜정),Ji-in Park(박지인),Sun-Nyoung Yu(유선녕),Bo-Bae Park(박보배),Gang-Ja Ha(하강자),Soon-Cheol Ahn(안순철),Dong-Seob Kim(김동섭) 한국생명과학회 2021 생명과학회지 Vol.31 No.1

건강에 대한 관심 증대와 1인 가구 증가라는 사회구조적인 변화로 이용하기 편리한 농산물에 대한 소비가 증가하고 있다. 대부분의 신선 농산물은 가열하지 않고 섭취하는 경우가 많기 때문에 식품 매개 병원체에 쉽게 노출될 수 있어 세계적으로 과채류가 원인인 식중독 사고의 보고가 증가하고 있다. 이에 본 연구에서는 신선 농산물의 미생물학적 품질을 평가하고 식중독균 검출 방법을 비교 분석하고자 하였다. 신선 농산물 중 채소류 129건을 구입하여 배양기반 방법으로 식중독균을 분석한 결과, non-pathogenic Escherichia coli (3.9%), Bacillus cereus (31.8%), Clostridium perfringens (5.4%), Yersinia enterocolitica (0.8%), enterohemorrhagic E. coli (0.8%)가 검출되었다. 이러한 식중독균의 분석에는 증균 배양과정이 중요하게 작용을 하며 균주의 순수 분리 및 확인 동정에까지 상대적으로 많은 시간과 노력이 요구된다. 따라서 증균 배양의 과정 없이 식중독균을 신속하게 검출 할 수 있는 PCRDGGE를 수행하여 배양 기반의 분석법과 비교하였다. 비병원성 대장균은 배양 기반 방법에서 검출되지 않았음에도 PCR-DGGE에서는 검출된 경우가 2건이 있었다. 본 연구에서 사용한 대장균 정량 분석방법은 시료를 10배 희석한 후 배양하는 과정에서 시료의 손실 가능성과 검출 한계가 높은 단점으로 PCR-DGGE가 균종의 확인에 더욱 용이할 것으로 보였다. 저위해성 식중독균은 배양 기반 방법보다 PCR-DGGE에서 검출 한계가 높은 것으로 보였다. 고위해성 식중독균은 배양 기반 방법보다 PCR-DGGE (10 CFU/g)에서 검출 한계가 낮아 균종 확인과 검출에 용이하다고 판단되었고 이를 통해 채소류에서 고위해성 식중독균의 잠재적 위험성을 확인하였다. 본 연구의 결과는 신선 농산물의 미생물 위해 평가와 기준 설정을 위한 기초 자료로 활용될 수 있으며 신선 농산물 관련 식중독균 검출 방법의 개선과 식중독 발생 예방에 기여할 것으로 기대한다. The consumption of fresh-cut agricultural materials is increasing due to increased public interest in health and the increase of single-person households. Most fresh-cut agricultural materials can be eaten without heating, thus easily exposing the consumer to food-borne pathogens. As a result, food-borne diseases are increasing worldwide. In the analysis of food-borne pathogens, it is important to detect the strains, but this is time consuming and laborious. Alternative detection methods that have been introduced, include polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), which is performed without prior culturing. Samples of fresh-cut agricultural materials, such as vegetables, were analyzed by the culture-based method. In 129 samples, non-pathogenic Escherichia coli (3.9%), Bacillus cereus (31.8%), Clostridium perfringens (5.4%), Yersinia enterocolitica (0.8%), and enterohemorrhagic E. coli (0.8%) were detected. Eight samples contaminated with bacteria were randomly selected, further analyzed by PCR-DGGE, and compared with the culture-based method. Two cases detected non-pathogenic E. coli by PCR-DGGE only, despite a lack of detection by the culture method. It was supposed there was possibility of sample loss during its 10-fold dilution for appropriate cultivation. In the detection of high-risk food-borne pathogens, it was found that the detection limit was lower in PCR-DGGE than in the culture-based method (10 CFU/g). This suggests that PCR-DGGE can be alternatively used to detect strains. On the other hand, low-risk food-borne pathogens seem to have higher detection limits in PCR-DGGE. Consequentially, this study contributes to the improvement of food-borne pathogen detection and the prevention of its related-diseases in fresh-cut agricultural materials.

Original Articles : Whole genome-scale transcriptome analysis of the amyristolyated enterohemorrhagic Escherichia coli O157:H7

( Jang W Yoon ),( Ka Hee Kwon ),( Yong Ho Park ) 한국예방수의학회 2013 예방수의학회지 Vol.37 No.4

Previously, we demonstrated the presence of a second copy of LPS myristoyl transferase in enterohemorrhagicEscherichia coli O157:H7, an important zoonotic diarrheagenic food-borne pathogen; the pO157-encoded ecf (an eae-conservedfragment) and the chromosomally-encoded lpxM (also referred to as msbM) genes. Although both genes share the samefunction as an LPS myristoyl transferase, the pO157-encoded ecf is thermoregulated via an intrinsically curved DNA whilethe chromosomal lpxM is regulated by the PhoP/Q two component regulatory system. However, it is unclear why E. coli O157:H7 carries two copies of LPS myristoyl transferase that are differentially regulated. In this study, a whole genome-scaletranscriptome specific to E. coli O157:H7 was carried out for identification of the genes differentially expressed in theamyristoylated E. coli O157:H7. The results identified a total of 110 EHEC genes that were up- or down-regulated in theamyristoylated E. coli O157:H7 strain, including genes associated with virulence (26.36%), metabolism (20.91%), transport(10.91%), signal transduction (4.55%), genetic information processing (3.64%), stress response (2.73%), regulatory function(2.73%), motility/adherence (3.64%), cell envelope (2.73%), cell division (1.82%) and ORFs of unknown function (17.27%). Of particular interest, the expression of LEE pathogenicity island genes was significantly influenced by LPS structural defects.

Antibacterial Effect of Crude Extracts of Kaempferia parviflora (Krachaidam) against Cronobacter spp. and Enterohemorrhagic Escherichia coli (EHEC) in Various Dairy Foods: A Preliminary Study

( Dana Jeong ),( Dong-hyeon Kim ),( Jung-whan Chon ),( Hyunsook Kim ),( Soo-kyung Lee ),( Hong-seok Kim ),( Jin-hyuk Yim ),( Kwang-young Song ),( Il-byung Kang ),( Young-ji Kim ),( Jin-hyeong Park ),( 한국유가공기술과학회 2016 Journal of Dairy Science and Biotechnology (JMSB) Vol.34 No.2

Rhizomes of Kaempferia parviflora (Zingiberaceae) have been used in traditional Thai medicine for health promotion. In this study, the antibacterial activity of ethanol extract of K. parviflora against Cronobacter spp. and enterohemorrhagic Escherichia coli (EHEC) was investigated using paper disc dilution method. The results revealed that the ethanol extract exhibited antibacterial activity against Cronobacter spp. and EHEC. With an increasing concentration of K. parviflora ethanol extract, larger zones of inhibition of Cronobacter spp. and EHEC strains tested were observed. Therefore, its antibacterial activity suggested that K. parviflora could be used as a natural additive to ascertain food safety of various dairy products.

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Adriana Hamond Regua-Mangia,Alice Gonçalves M. Gonzalez,Aloysio M. F. Cerqueira,João Ramos C.Andrade 대한수의학회 2012 Journal of Veterinary Science Vol.13 No.2

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.