Proteins in Outer Membrane Vesicles Produced by Burkholderia cepacia are Responsible for Pro-inflammatory Responses in Epithelial Cells

Gram-negative bacterial pathogens produce outer membrane vesicles (OMVs) and this secreted cargo plays a role in host-pathogen interactions. OMVs isolated from Burkholderia cepacia induce the cytotoxicity and pro-inflammatory responses both in vitro and in vivo, but OMV components associated with host pathology have not been characterized. This study analyzed the proteomes of OMVs produced by B. cepacia ATCC 25416 and investigated whether proteins in B. cepacia OMVs were responsible for host pathology in vitro. Proteomic analysis revealed that a total of 265 proteins were identified in B. cepacia OMVs. Of the 265 OMV proteins, 179 (67.5%), 32 (12.1%), 27 (10.2%), 17 (6.4%), and 10 (3.8%) were predicted to be located in the cytoplasm, inner membrane, periplasmic space, outer membrane, and extracellular compartment, respectively. Several putative virulence factors were also identified in B. cepacia OMVs. B. cepacia OMVs slightly induced the cytotoxicity in lung epithelial A549 cells, but there was no difference in cytotoxic activity between intact OMVs and proteinase K-treated OMVs. B. cepacia OMVs stimulated the expression of pro-inflammatory cytokine and chemokine genes in A549 cells, but the expression of these cytokine genes was significantly inhibited in A549 cells incubated with proteinase K-treated OMVs. In conclusion, our results suggest that proteins in B. cepaciaOMVs are directly responsible for pro-inflammatory responses in lung epithelial cells.

Proteins in Outer Membrane Vesicles Produced by Burkholderia cepacia are Responsible for Pro-inflammatory Responses in Epithelial Cells

Gram-negative bacterial pathogens produce outer membrane vesicles (OMVs) and this secreted cargo plays a role in host-pathogen interactions. OMVs isolated from Burkholderia cepacia induce the cytotoxicity and pro-inflammatory responses both in vitro and in vivo, but OMV components associated with host pathology have not been characterized. This study analyzed the proteomes of OMVs produced by B. cepacia ATCC 25416 and investigated whether proteins in B. cepacia OMVs were responsible for host pathology in vitro. Proteomic analysis revealed that a total of 265 proteins were identified in B. cepacia OMVs. Of the 265 OMV proteins, 179 (67.5%), 32 (12.1%), 27 (10.2%), 17 (6.4%), and 10 (3.8%) were predicted to be located in the cytoplasm, inner membrane, periplasmic space, outer membrane, and extracellular compartment, respectively. Several putative virulence factors were also identified in B. cepacia OMVs. B. cepacia OMVs slightly induced the cytotoxicity in lung epithelial A549 cells, but there was no difference in cytotoxic activity between intact OMVs and proteinase K-treated OMVs. B. cepacia OMVs stimulated the expression of pro-inflammatory cytokine and chemokine genes in A549 cells, but the expression of these cytokine genes was significantly inhibited in A549 cells incubated with proteinase K-treated OMVs. In conclusion, our results suggest that proteins in B. cepaciaOMVs are directly responsible for pro-inflammatory responses in lung epithelial cells.

Isolation and Genetic Characterization of Orientia tsutsugamushi from Scrub Typhus Patients in Gyeongsangnam-do, Korea

Orientia tsutsugamushi (O. tsutsugamushi), which is endemic to an Asia-Pacific region, has increased its incidence and caused annually around 10 thousand patients infected with scrub typhus in Korea in the past several years. In the present study, we isolated 44 O. tsutsugamushi from the patients with febrile illness accompanied with or without an eschar in Gyeongsangnam-do, Korea. These isolates were characterized by genetic analysis of the major outer membrane protein, the 56-kDa type-specific antigen (tsa56), which is unique to O. tsutsugamushi. Two types of sequences of tsa56, designated by JJ1 and JJ2, were determined from 37 and 7 isolates of the 44 isolates, respectively. JJ1 and JJ2 showed 74.7~90.8% identity in nucleotide sequence and 66.1~90.5% identity in amino acid sequence with 33 reference strains except for Boryong and Kuroki. JJ1 and JJ2 had 100 and 99.9% nucleotide identity to Boryong strain, and 99.9 and 99.8% to Kuroki, which has been known to be similar to Boryong, respectively. In addition, they showed 77.9~ 81.4% nucleotide identity with the cluster of Gilliam-related genotypes, whereas they showed higher nucleotide identity (89.6~90.8%) with the cluster of Karp-related genotypes. To our knowledge, this is the first report to isolate O. tsutsugamushi and characterize their genotype as the Boryong in Jinju and West Gyeongsangnam-do, Korea, even though it has been reported that the Boryong was the predominant genotype in isolates from chiggers, domestic rodents, and patients in the southern part of Korea. Furthermore, our isolates could be useful source to study on the pathophysiology and epidemiology of scrub typhus in Korea.

Investigation of the Requirements of 'Good Teaching' to Improve Teaching Professionalism in Medical Education / 한국의학교육

PURPOSE: The purpose of this study is to survey the awareness of faculty (F) and students (S) on 'good teaching' and to analyze an example of good lesson, finally to identify the requirements of 'good teaching' in medical education. METHODS: Quantitative and qualitative methods were applied simultaneously. First, survey using a questionnaire was analyzed by frequency analysis and verified using chi-square-test, Mann-Whitney test. Second, the case of good teaching and qualitative data were analyzed by 'priori codes coding' and 'open coding'. RESULTS: The results of survey are as follows: Both faculty and students regarded lessons that taught important content easy to understand (F: 50%; S: 69.1%); Lessons that allow students make sure important information (F: 48.6%; S: 51.4%); Lessons that prepare and plan considering the student's level and interest (F: m=3.78; S: m=3.76) were good lessons. Faculty wanted lessons that improve student's academic achievement (35.7%), while students choose lessons that deliver curriculum effectively using appropriate teaching method (47.1%). According to the results of case analysis, it turned out that characteristics of good teaching were as follows: Thorough plan and preparation of content, various teaching methods and materials, encouragement of intellectual inquiry and curiosity, active interaction between faculty and students, clear feedback and reasonable evaluation. CONCLUSION: Requirements of good teaching are open to faculty at school of medicine and are to be utilized as guidelines to monitor and improve their instruction.

Development of Computer-Based Test (CBT) and Student Recognition Survey on CBT / 한국의학교육

PURPOSE: The purpose of this study is to understand student recognition of CBT as well as its strengths and weaknesses, and to explore the improvement methodologies for the effective development and implementation of CBT. METHODS: A questionnaire survey was conducted twice (before and after implementation of CBT) with a total of 17 multiple-choice and 2 essay-type questions. The multiple-choice questions were analyzed by frequency analysis and the essay-type questions were coded by content analysis. RESULTS: The results are as follows. First, the overall satisfaction with CBT was shown to be high. Second, students listed the merits of CBT as follows: simple correction of answers (before: 89.4%; after: 80.8%), presentation of realistic materials (before: 72.9%; after: 84.7%), prompt feedback on grades (before: 60.3%; after: 71.1%), shortened exam time and effective time scheduling (before: 86.5%; after: 66.4%), accurate estimation of abilities (before: 70.2%; after: 36.6%), and assistance in academic improvement (before: 70.9%; after: 22.1%). Drawbacks of CBT were: inconvenience of review (before: 70.9%; after: 22.1%), inconvenient screen organizations (before: 0%; after: 48.1%), possibility of cheating (before: 73.9%; after: 31.8%), and equality issue of test (before: 47.3%; after: 17.3%). CONCLUSION: Assessment paradigms are currently shifting from summative evaluation to formative evaluation, from one-off assessment to continuous assessment, and from output assessment to process assessment. Therefore, CBT must be expanded to move from result-oriented summative evaluation to formative evaluation continuously monitoring the student learning process.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from Clinical Specimens

A total of 58 vancomycin-resistant E. faecium (VREF) was isolated from 3 hospitals located in Daegu, Korea. The VREF isolates were evaluated for the antimicrobial susceptibility pattern and resistance determinants against vancomcin, aminoglycosides, and macrolides. The multilocus sequence types (MLST) were determined to characterize the clonal diversity of the VREF isolates. The VREF isolates were highly resistance to teicoplanin, erythromycin, ciprofloxacin, gentamicin, and streptomycin, whereas quinupristin-dalfopristin and linezolid were the most susceptible drugs. All isolates carried the vanA gene. The aac6'-aph2" (n=53) and aadE (n=27) genes were detected in the high-level aminoglycoside resistant (HLAR) isolates. The aac6'-aph2" gene was located in the conjugally transferable plasmids. The ermB and ermA genes were detected in the 54 and 3 VREF isolates, respectively. The VREF isolates showed 11 different sequence types (ST). The VREF isolates belonging to ST192 was the most prevalent (n=19), but detected in one hospital, whereas the isolates belonging to ST203 (n=11) were detected in 3 hospitals. These results suggest that the VREF isolates resistant to aminoglycosides and erythromycin are originated from different clones and specific VREF clones are spread in the study hospitals.

Genetic Diversity of Stenotrophomonas maltophilia Isolated from Clinical Specimens

Stenotrophomonas maltophilia is a multi-drug resistant pathogen that has been isolated with increasing frequency from the hospitalized patients. A total of 202 S. maltophilia was isolated from three university hospitals and analysed by molecular typing for an epidemiologic investigation. All isolates were tested by antimicrobial susceptibility, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). The RAPD and PFGE patterns were recorded and analysed by the unweighted-pair group method with arithmetic average method. Two or more isolates were considered to be clonally related if their PFGE pattern exhibited > or =80% similarity. Trimethoprim/ sulfamethoxazole and ciprofloxacin were the most active antimicrobial agents tested. The majority of the isolates found to be genetically unrelated by PFGE. The genetically related isolates were recovered from the same patient. The result demonstrates a high genetic diversity of S. maltophilia isolates from clinical specimens. The clonal diversity of S. maltophilia from the hospitalized patients is partly due to the strains originated from the hospital environments, but not horizontal transfer between the patients

Antimicrobial Resistance and Integrons Found in Commensal Escherichia coli Isolates from Healthy Humans

The emergence and spread of antimicrobial resistance among the pathogenic and commensal Enterobacteriaceae are of great concern worldwide. We characterized the antimicrobial resistance and integrons found in commensal Escherichia coli from healthy humans in the community. Class 1 integrase (intl1) and class 2 integrase (intl2) genes were identified in 22 (13.3%) and 2 (1.2%) of 165 E. coli isolates, respectively. dfrA17-aadA5 and dfrA1-aadA2 were the most common class 1 integrons. The prevalence of each type of class 1 integron among commensal E. coli isolates during 2001~2003 was similar to that of clinical E. coli isolates from hospital-acquired infections during 1994~1999. The resistant rates of commensal E. coli isolates carrying intl1 to ampicillin, streptomycin, gentamicin, sulfamethoxazole, trimethoprim, chloramphenicol, and tetracycline were significantly higher than those of intl1-negative E. coli isolates (p<0.05). Integrons were directly associated with multidrug resistance in commensal E. coli isolates. It is hypothesized that multidrug-resistant Enterobacteriaceae from hospital-acquired infections are a potential reservoir for integrons associated with resistance genes found in commensal E. coli isolates in the community

Learning Styles in Medical School / 한국의학교육

PURPOSE: The purpose of this study is to investigate the learning styles of students at Kyungpook National University(KNU) School of Medicine and to assess its implications on teaching-learning methods in medical school. METHODS: 571 students in all years of medical school were invited to complete a questionnaire via online. For this study, Felder's Index of Learning Styles(ILS) was used, which included 49 items. Felder's ILS measures the learner's relative preference for each of the five dichotomous learning style dimensions. RESULTS: 242(42.3%) students completed the questionnaire. More than 80% preferred sensitive information and more than 60% preferentially took in visual information. Both males and females preferred sensing and visual learning materials. On average, The students at KNU school of Medicine were reflective, sensing, visual, global and inductive. CONCLUSION: From these results, we suggested some effective methods of teaching-learning based on the students' preferences and some perspectives for future works.

Epidemiology of Shigellosis in Korea

Shigellosis is an acute diarrheal disease caused by bacteria of the genus Shigella. Following the occurrence of a large outbreak of shigellosis as well as sporadic cases since 1998, shigellosis has been a major health problem in Korea. There have been major changes in epidemiology during the last five decades concerning shigellosis in terms of total incidence of shigellosis, prevalence of certain serogroups, selection of specific clones, and introduction of new Shigella clones. S. dysenteriae was the most prevalent species until the early twentieth century, S. flexneri was the most prevalent until the late 1980s, and S. sonnei has been the most prevalent since 1990. Diverse serotypes of S. dysenteriae (4 serotypes), S. flexneri (8 serotypes), and S. boydii (4 serotypes) were found during the Korean War and many of these Korean endemic Shigella strains circulated in the community until the late 1970s. However, the endemic strains of S. dysenteriae, S. boydii, and S. sonnei disappeared in the late 1980s. A new clone of S. sonnei that was introduced between the late 1980s and the early 1990s was responsible for a large proportion of shigellosis in recent years. S. flexneri serotype 4a was the most frequently found during the Korean War and then the incidence of S. flexneri 2a gradually increased with time. S. flexneri isolates detected from 1991 to 1997 were all serotype 2a. However, the diverse clones of S. flexneri reemerged in Korea since 1999. It has not been determined whether the S. flexneri strains from the 2000s were the descendants of the Korean endemic strains or imported new strains, but the PFGE patterns were different between S. flexneri strains from the 1980s and 2000s. The widespread of new S. sonnei strains and the persistence of S. flexneri strains are responsible for the endemicity of shigellosis in Korea.

Effects of Chronic Alcohol Exposure on Dopaminergic and Serotonergic Neurotransmission System in Rat / 대한정신약물학회지

OBJECTIVE: This study investigates the effects of chronic alcohol exposure on rat brain THmRNA expression, TH (tyrosine hydroxylase) acitivity, and TPH (tryptophan hydroxylase) activity which are important in synthesis of dopamine and serotonin and other components of both the dopaminergic and serotonergic systems of the rat brain. METHODS: Rats were fed a liquid diet containing alcohol for 4 weeks. We investigated effects of chronic alcohol exposure on dopaminergic systems as follows. We evaluated expression of THmRNA in LC, VTA and substantia nigra by using in-situ hybridization and measured activity of TH by using immunoassay. We used HPLC for simultaneous measurement of dopamine, DOPAC and HVA in the cerebral cortex, striatum, hypothalamus, hippocampus, mid brain, hind brain, and cerebellum. Also we investigated serotonergic systems as follows. We evaluated expression of TH mRNA in the dorsal raphe nucleus by using radioprobe and measured the activity of TPH by using enzyme immunoassay. We used HPLC for simultaneous measurement of 5-HT and 5-HIAA in the cerebral cortex, striatum, hypothalamus, hippocampus, mid brain, hind brain, and cerebellum. RESULTS: Alcohol exposure for 4 weeks increased the expression of TH mRNA in the ventral tegmental area and the locus ceruleus but not in the substantia nigra. The 4 weeks of alcohol exposure did not cause significant changes in levels of dopamine and metabolites in the different areas of the brain, nor was it associated with changes in the maximal binding and affinity (Kd) of anterior striatal dopamine D2 receptor. Alcohol exposure for 4 weeks had no effect on the expression of TPH mRNA or on the activity of TPH in the dorsal raphe nucleus and the hypothalamus. CONCLUSION: We reported at first that chronic alcohol exposure could increase TH mRNA in the locus ceruleus. In a previous study of acute alcohol treatment, there is increase of dopamine metabolism but in this study, we did not observe any changes in dopamine metabolism in the different areas of the brain. Also we did not see any significant changes in the synthesis and metabolism of serotonin after 4 weeks of chronic alcohol exposure compared with control. Therefore, synthesis and metabolism of serotonin was affected in the acute phase. And, as previous reports have suggested, any changes caused by alcohol returned to previous levels via adaptation and regulatory mechanisms.

Prevalence of Specific Clone of Salmonella enterica Serovar Typhimurium Clones in the Swine Population of Kyungpook Province During 1998 to 2000

A total of 40 Salmolella enterica serovar Typhimurium (S. typhimurium) strains were isolated from clinical specimens of swine at 10 farms in Kyungpook province from 1998 to 2000. We investigated the clonal relationship of S. typhimurium isolates by antimicrobial susceptibility, plasmid profile, and Southern hybridization analysis with tetA, and pulsed-field gel electrophoresis (PFGE). All S. typhimurium isolates showed identical biochemical characteristics and were resistant to tetracycline, streptomycin, and sulfamehtoxazole. They were classified into 5 groups by antimicrobial resistance patterns. S. typhimurium isolates carried 3 to 5 plasmids and were classified into 5 groups by plasmid profiles. Southern hybridization showed that tetA gene was located in 21 kb of plasmid. S. typhimurium isolates from 9 different farms showed identical or similar PFGE patterns, which indicates clonal origin of the strains. All S. typhimurium isolates, except one isolate from 1998, seemed to belong to be one clone by the combination of three epidemiological typing methods. These data demonstrated that a specific clone of Salmolella enterica serovar Typhimurium was widely spread in swine farms in Kyungpook province.

Degradation of the Retinoblastoma Tumor Suppressor Protein by the Human Papillomavirus-16 E7 Variants

Human papillomavirus type 16 (HPV-16) plays an etiological role in benign and malignant epithelial tumors. A critical event in HPV transformation of human cells is the inactivation of retinoblastoma protein (pRB) by the E7 protein. The metabolic half-life of pRB is decreased in cells that express high-risk HPV E7 proteins. The present study investigated the frequency of HPV-16 E7 variants in Korean women and compared the pRB degradation activity of E7 variant proteins. Of the 40 HPV-positive specimens from a total of 91 tissue specimens, 21 HPV-16 positive specimens were studied by sequencing analysis to determine the variation of E7 gene. The most frequent E7 variant was N29S (57%). The HPV-16 E7 variant was more prevalent in invasive cervical cancer tissue specimens than in those from low grade clinical stage. The degradation of pRB in HaCaT cells by HPV-16 E7 variant proteins was investigated by western blot analysis. There was no significant difference in pRB degradation activity between the HPV-16 E7 prototype protein and E7 variant proteins. The pRB degradation activity did not differ among HPV-16 E7 variants. These results suggest that the E7-induced degradation of pRB is important in cervical tumorigenesis; however, there was no relation between the pRB degradation activity and the variations in HPV-16 E7 protein among Korean women.

Molecular Epidemiology of Nalidixic Acid Resistance in Shigella sonnei Isolates

Twenty-six nalidixic acid-resistant Shigella sonnei strains isolated from 1982 to 2001 and 56 nalidixic acid-resistant mutants induced by quinolone drugs from susceptible wild strains were analyzed by sequencing the gyrA gene. All the 22 nalidixic acid-resistant isolates from 1998 to 2001 showed identical amino acid substitution of Ser to Leu (TCG --> TTG) at codon 83 while 7 different mutation types were detected in artificially induced nalidixic acid-resistant mutants. Asp87 (GGC) type was observed most commonly among mutants induced by nalidixic acid while Ser83 (TTG) type was common among mutants induced by ciprofloxacin or norfloxacin. All the isolates collected between 1998 and 2001 showed identical or nearly identical pulsed-field gel electrophoresis pattern. These results suggest that the explosive increase of S. sonnei infection after 1998 was mainly due to the spread of restricted number of clones resistant to nalidixic acid.

Clonal Types and Antimicrobial Susceptibilitiy Patterns in Methicillin-Resistant Staphylococcus aureus Isolates from a Korean Hospital

A total of 54 non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical specimens and 3 MRSA isolates from healthy medical staffs were obtained from Kyungpook National University Hospital. They were analyzed for clonal types by multilocus sequence typing, protein A gene (spaA) typing, the staphylococcal cassette chromosome mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). The MRSA isolates were examined for antimicrobial susceptibility. Clinical MRSA isolates were classified into 4 clonal complexes, 4 sequence types (STs), 5 spaA types, 4 PFGE patterns, and 3 SCCmec types with variants. On the basis of ST, ST239 (n=25) and ST5 (n=24) were the most frequently encountered. MRSA isolates belonging to ST239 were genotypically homogenous, while those belonging to ST5 showed variations in spaA and SCCmec types. Of the 3 MRSA isolates from healthy medical staffs, one was genotypically identical to MRSA isolates belonging to ST5 and the other two ST239. All MRSA isolates were susceptible to vancomycin and teicoplain. Only 4% of isolates were resistant to rifampin, while 91% of isolates were resistant to ciprofloxacin. The resistance rate of MRSA isolates belonging to ST239 against trimethoprim-sulfamethoxazole (SXT) was significantly higher than that of the isolates belonging to ST5 (76% vs 0%, p<0.001). In summary, ST239 and ST5 were responsible for most MRSA infections and healthy medical staffs also carried these MRSA strains. The susceptibility of the ST239 clone against SXT, which was commonly used for oral therapy to treat MRSA infection, was significantly different from the ST5 clone.

Epidemiologic analysis of Ampicillin Resistance in Shigella sonnei Isolates by blaTEM Hybridization

Forty-seven ampicillin-resistant R plasmids derived from 218 Shigella sonnei isolates from Daegu and Gwangju areas from 1980 to 2000 were epidemiologically compared by fragments of restriction endonuclease patterns by EcoRI and SmaI, and by Southern hybridization with a blaTEM-1 probe. All the ampicillin-resistant strains isolated in the 1980S carried a conjugative R plasmid responsible for multiple resistance other than ampicillin, and an ampicillin-resistance plasmid. Ampicillin-resistant strains isolated in the 1990S harbored single conjugative R plasmid encoding ampicillin resistance along with variable antimicrobial resistances. The restriction endonuclease digestion patterns and Southern hybridiztion analysis of conjugative R plasmids showing identical resistance pattern and a same size showed different fragment and Western blotting patterns according to different isolation years and areas, while identical patterns were observed among the plasmids derived from a same isolation year and area. These findings suggest that ampicillin resistance among S. sonnei isolates was due to introduction of ampicillin-resistant R plasmids originated from different sources.

The Achievement of Medical Students Admitted after Baccalaureate Level / 한국의학교육

PURPOSE: Kyungpook National University School of Medicine initiated a pilot project to admit small number of qualified students who received BS degree or above in the field of sciences in 1996. This study was conducted to evaluate effectiveness of the pilot project. METHODS: Students' academic grades, outcome of their opinion surveys on adaptability to the school life, difficulties and problems from the classes of 1996 to 2001 were analyzed. RESULTS: A total of 57 students were admitted to this program over the past 7-year period, 54.8% of them from natural science major and 36.8% from engineering. Students admitted with BS degree were performing above average in the class by % rank. There was no significant difference in academic performance between the natural science and the engineering major. Of 20 graduated, only one took career in basic medical science (physiology). The students thought undergraduate education was helpful in maturing personal characters than in knowledge or skill attainment. CONCLUSION: The achievement of medical students admitted after baccalaureate level was comparable to that of students with two years of premedical education.

Molecular Epidemiology of Shigella sonnei Isolated from 1994 to 1999 in Daegu area

A total of 78 Shigella sonnei strains isolated from 1994 to 1999 in Daegu were screened by biochemical test, antimicrobial susceptibility test, plasmid profiling, and epidemiologic analysis. Among them, 26 representative strains with different properties were selected and their molecular epidemiological characteristics were studied. Among the 78 strains, two strains did not ferment raffinose and another two strains did not produce colicin. The 26 selected strains were differentiated into 15 plasmid profiles, 11 antimicrobial, 14 enterobacterial repetitive intergenic consensus sequencebased PCR (ERIC-PCR) pattern, 10 pulsed-field gel electrophoresis (PEGE) pattern types. When all of the typing methods were combined, the strains could be differentiated into 15 types. However, significant correlation among different testing methods was not observed. PEGE analysis indicated that all the strains tested were related despite minor genotypic and phenotypic differences. Our data could not address whether these genetic diversity of strains is due to different circulating strains originated from a number of clone or due to minor genetic variation.

Epidemiologic analysis of Shigella sonnei Isolates by using Antimicrobial Resistance Gene Probes

Thirty-four Shigella sonnei isolates from 6 outbreaks and sporadic cases from May 1999 until January 2000 in Daegu and 9 regions of Gyeongsangbuk-Do were epidemiologically analyzed by plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization with 2 antimicrobial resistance gene probes, tetA and dfrA1. In outbreak cases, resistance pattern in all of the strains was identical: they were resistant to tetracycline (Tc), streptomycin (Sm), sulfisomidine (Su), trimethoprim (Tp), and nalidixic acid (Na). In sporadic cases, Tc, Sm, Su, Na, ampicillin (Ap), and kanamycin (Km) pattern and TcSmSuTpApNa pattern were additionally observed. Isolates from the same outbreak showed identical plasmid profile and PFGE pattern. Most of different outbreak strains and sporadic strains showed different plasmid profiles, and identical or different PFGE patterns, while all of the isolates shared common tetA gene on a non-conjugative 18.3 kbp R plasmid carrying resistance to tetracycline, streptomycin, and sulfisomidine, and dfrA1 gene on the chromosome. Non-conjugative R plasmids derived from all of the isolates were confirmed to be identical by the Southern hybridization analysis of restriction endonuclease treated or non-treated plasmid profiles using the tetA probe. The same strains also reacted with dfrA1 probe at the same-sized DNA fragment (60 kbp) on pulsed-field gel electrophoresis of total genomic DNA. Our findings suggested that epidemic strains of Shigella sonnei prevalent in the Daegu and Gyeongsangbuk-Do area during the test period should have originated from an identical or closely related strain source although most of strains did not show the same plasmid profile and PFGE pattern.

Genomic Analysis of Human Papillomavirus Type 16 in Cervical Cancer

To study the correlation of human papillomavirus (HPV) infection with clinical stage in cervical abnormalities, 17 cases of normal cervical tissue and 69 cases of abnormal cervical tissue (cervical intraepithelial neoplasia and invasive cervical cancer) was examined by PCR with HPV-specific consensus primers. One case (5.9%) of normal cervical tissue and 42 cases (60.9%) of abnormal cervical tissues harbored HPV. To investigate the integration of HPV genome in 24 cases of HPV 16 positive cervical cancer, E2 gene of HPV 16 was amplified. Integration of HPV 16 was found in 7 cases (29.2%) with E2 disruption. All samples with E2 disruption were from invasive cervical cancer. A multiplex PCR for the mapping of integrated HPV 16 genome with an anchor primer and indicator primers showed that 11 cases (45.8%) were disrupted somewhere in HPV genome but E6, E7, and LCR regions were conserved in all cases. Seven types of integrated HPV genome from long- (7,062 bp) to short-conserved type (3,204 bp) with various deletions were detected by the multiplex PCR. These results show that integration can be detected more accurately by multiplex PCR than by E2 PCR, and E2 disruption is not a critical event of integration