BACKGROUND: Salmonella enterica serovars often have a broad host range and cause some gastrointestinal and systemic diseases. The diagnosis of typhoid fever or paratyphoid fever is made by ordinary culture methods and biochemical tests. However, a more rapid and alternative method of diagnosing these diseases is in need since the classical diagnostic method requires several days for a result. Some researchers have already reported serovar Typhi detection methods with PCR using the fliC-d gene and the Vi capsular antigen gene. METHODS: Thirty-six Salmonella strains isolated at Pusan National University Hospital from 1997 to 2004 were used for a rapid identification of S. enterica serovars Typhi and Paratyphi A with multiplex PCR that uses the O (rfbE, rfbS), H (fliC-d, fliC-a), and Vi (viaB) antigen genes. To further characterize these Salmonella strains, we used PCR to detect genes (invA and enterotoxin) for proposed virulence factors and performed antimicrobial susceptibility testing, serotyping and pulsed-field gel electrophoresis for epidemiological characteristics. RESULTS: Most strains were resistant to ampicillin. By PCR, tyv, prt, fliC-d and viaB genes were detected in serovar Typhi, whereas only fliC-a and prt genes were found in serovar Paratyphi A. In addition, invA and enterotoxin genes were detected in both strains. CONCLUSION: This method enabled us to identify and differentiate serovars Typhi and Paratyphi A by only a single PCR assay. That is, clinically important human pathogens were more rapidly and specifically detected and identified with multiplex PCR.
Ampicillin ; Busan ; Diagnosis ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins ; Genotype ; Host Specificity ; Humans ; Multiplex Polymerase Chain Reaction* ; Paratyphoid Fever ; Polymerase Chain Reaction ; Salmonella enterica* ; Salmonella* ; Serotyping ; Typhoid Fever ; Virulence Factors

Attaching and effacing Escherichia coli (AEEC) cause enteric infections in humans and animals. Attaching indicates the intimate attachment of bacteria to the enterocyte, and effacing relates to the localized effacement of brush border microvilli. Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) infections are characterized by the formation of attaching and effacing (AE) lesion on the intestinal epithelial cells. Therefore, they are often grouped together as AEEC. Development of multiplex PCR allowed us to type five of the most important genes implicated in the formation of the AE lesion. A total of 60 AEEC strains isolated from diarrheal patients were investigated by multiplex PCR for the presence of the insertion site of locus of enterocyte effacement (LEE) and LEE-related (eae, tir, espA, espB, and espD) genes. Associating the results of LEE genes typing in the AEEC strains, three different pathotypes are determined: eae(gamma)-tir(gamma)-espA(gamma)-espB(gamma)-espD(gamma) (O157:H7), eae(beta)-tir(beta)-espA(beta)-espB(beta)-espD(beta) (O26:H11), and eae(alpha)-tir(alpha)-espA(alpha)-espB(alpha)-espD(alpha) (O55:H6). These results indicate that AEEC are a heterogenous groups of organisms.
Animals ; Bacteria ; Enterocytes* ; Enterohemorrhagic Escherichia coli* ; Enteropathogenic Escherichia coli* ; Epithelial Cells ; Escherichia coli ; Humans ; Microvilli ; Multiplex Polymerase Chain Reaction

BACKGROUND: Most of the shigellosis outbreaks in Korea have been caused by Shigella sonnei since late 1990's. We analyzed 36 strains of S. sonnei isolated in South Korea from 1998 to 2001 by molecular epidemiologic tools to understand genetic relationship of the outbreaks. METHODS: The 36 strains of S. sonnei were tested for the presence of virulence genes (ial, ipaH, stx, set1A, set1B and sen) using polymerase chain reaction (PCR) method and for the production of Shiga-toxin using latex agglutination test. Seventeen representative strains were selected and their genetic relevance was analyzed by plasmid profile and pulsed-field gel electrophoresis(PFGE). RESULTS: By PCR, ipaH gene was detected in all 36 strains, set1B gene in 15 strains (41.7%), and sen gene in 16 strains (44.4%); all strains were negative for set1A gene. Although stx gene was positive in four strains by PCR method, the toxin was negative by latex agglutination test. The strains were differentiated into 11 groups by plasmid profile and 1 type with 3 subtypes (A-1, A-2and A-3) by PFGE. CONCLUSION: There was a wide range of diverse virulence genes present in the outbreak strains of S. sonnei. PFGE analysis indicated that all the strains tested were related with each other despite minor genotypic and phenotypic differences. A genetically identical clone of S. sonnei was estimated to be the cause of the outbreaks.
Clone Cells ; Disease Outbreaks ; Dysentery, Bacillary ; Genotype ; Korea* ; Latex Fixation Tests ; Molecular Epidemiology* ; Plasmids ; Polymerase Chain Reaction ; Shigella sonnei* ; Shigella* ; Virulence Factors* ; Virulence*

A total of 35 strains of Shigella sonnei, 21 strains isolated in South Korea from 2000 to 2001 and 14 strains isolated in Japan from 2001 and 2002 were analyzed for antimicrobial susceptibility, plasmid profile and molecular epidemiology. Using polymerase chain reaction (PCR) method, the strains were tested for the presence of virulence genes. And then reversed passive latex agglutination(RPLA) test was used to determine if the strain was Shiga-toxin producing. Their random amplified polymorphic DNA (RAPD) patterns were examined. Pulsed-field gel electrophoresis (PFGE) patterns were also analyzed. Most strains showed multiple resistance to more than four antimicrobial agents, i.e., tetracycline, erythromycin, streptomycin and vancomycin. All South Korea strains were susceptible to chloramphenicol and gentamicin, while all Japan strains were susceptible to kanamycin and cefoperazone. The antibiogram could be classified into 6 groups. By PCR, ipaH gene was detected from all strains, but set1A and set1B genes were not. Sen and ial genes were detected from 19 strains (54.3 %). Especially, stx gene was positive in 11 of the 35 strains by PCR method but not confirmed by RPLA method. The strains were differentiated into 12 groups by plasmid profile and 6 arbitrary groups (a to f) by RAPD analysis. The isolates could be grouped into 5 (A to E) PFGE patterns including 3 subgroups A-1, A-2 and A-3. Type A was the major type (82.9 %).
Anti-Infective Agents ; Cefoperazone ; Chloramphenicol ; DNA ; Drug Resistance, Microbial* ; Electrophoresis, Gel, Pulsed-Field ; Erythromycin ; Gentamicins ; Japan* ; Kanamycin ; Korea* ; Latex ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Plasmids ; Polymerase Chain Reaction ; Shigella sonnei* ; Shigella* ; Streptomycin ; Tetracycline ; Vancomycin ; Virulence

Enteropathogenic Escherichia coli (EPEC) strains possess genes for attaching and effacing (eae) and EPEC adherence factor (EAF) plasmid. It is necessary to develop molecular techniques for the evaluation of EPEC isolates. A total of 183 E. coli isolates from neonates admitted to Pusan National University Hospital were investigated by polymerase chain reaction (PCR) and DNA colony hybridization. Of the 183 isolates tested, 10 (5.5%) were positive for eae by PCR and DNA colony hybridization and confirmed to be EPEC. Ten EPEC isolates showed 3 different adherence patterns: seven strains had diffuse adherence, two localized adherence-like adherence, and one aggregative adherence. They were also examined by antimicrobial susceptibility tests, serotyping, and molecular epidemiological typing such as pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. The EPEC isolates could be divided into 9 different antimicrobial resistance patterns, 6 serotypes, 4 PFGE patterns, and 5 RAPD patterns. This result indicates that the EPEC isolates from neonates were originated from different sources.
Busan ; DNA ; Electrophoresis, Gel, Pulsed-Field ; Enteropathogenic Escherichia coli* ; HeLa Cells* ; Humans ; Infant, Newborn* ; Plasmids ; Polymerase Chain Reaction ; Serotyping

Staphylococcus aureus continues to be the main cause of surgical site infections. Recently, methicillin-resistant S. aureus (MRSA) has been known to be resistant to many kinds of antibiotics and causes the problem of neonatal nosocomial infection. Antibiotic sensitivity tests which have been routinely used to detect MRSA in the laboratory depend on the culture conditions. Therefore it is necessary to develop a new method based on a molecular biological technique in order to overcome these problems. We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to staphylococcal-specific regions of the 16S rRNA genes), ii) provide an indication of the likelihood that the Staphylococci present in the specimen are resistant to oxacillin (based on the amplication of the mecA gene, encoding penicillin-binding protein 2'(PBP-2'), which is known to confer resistance to the bacteriostatic action of methicillin). In this study, 67 S. aureus strains were isolated from the neonatal intensive care unit and general neonatal nursery at Pusan National University Hospital, Busan, Korea, between January and July 2003. Methicillin resistance was tested by the oxacillin disk diffusion method and the MIC method. We performed the multiplex PCR to amplify the mecA gene, encoding PBP-2'. We tested it by multiplex PCR and compared the results with the antimicrobial susceptibilities. Different results were obtained from 2 MRSA (4.65%), suggesting that the PCR method should be performed at the same time for a more accurate clinical test of MRSA
Anti-Bacterial Agents ; Busan ; Cross Infection ; Diagnosis ; Diffusion ; Infant, Newborn ; Intensive Care, Neonatal ; Korea ; Methicillin Resistance* ; Methicillin-Resistant Staphylococcus aureus* ; Multiplex Polymerase Chain Reaction* ; Nurseries ; Oxacillin ; Penicillin-Binding Proteins ; Polymerase Chain Reaction ; Staphylococcal Infections ; Staphylococcus aureus ; Thiram

PURPOSE: Nosocomial infection with Staphylococcus aureus, especially methicillin resistant S. aureus, has become a serious concern in the neonatal intensive care unit. The aim of this study is to investigate the virulence factors, and the relationship between the antibiotic resistance and the associated genes of Staphylococcus aureus isolated from nasal cavity of neonates. METHODS: Fifty one isolates of S. aureus were obtained from nasal swab taken in 28 neonates in the NICU and nursery of Pusan National University Hospital between February and May, 2001. They were tested in regard to antibiotic susceptibility, coagulase test and typing, plasmid DNA profile, as well as reactivity to enterotoxin A-E(sea, seb, sec, sed, see) genes and toxic shock syndrome toxin-1(tst) gene by polymerase chain reaction(PCR). Associated genes such as mecA, mecR1, mecI, and femA were also determined by PCR. The origin of MRSA strains was assessed using DNA fingerprinting by arbitrarily-primed polymerase chain reaction(AP-PCR). RESULTS: Twenty three(45.1%) and six(11.8%) isolates were resistant to oxacillin and vancomycin respectively. Multidrug resistance to three or more of the antibiotics tested was observed in 51.0% of the isolates. Forty two isolates were coagulase positive and twenty two isolates had mecA gene. Sixteen isolates had both mecA and femA genes and had type I-III plasmids. 64.7% of isolates carried sec gene, and 80.4% carried tst gene. DNA fingerprinting by AP-PCR for 12 MRSA strains showed 10 distinct patterns, suggesting different origins. CONCLUSION: We confirmed that the prevalence of nasal carriage of S. aureus and the incidence of antimicrobial-resistant S. aureus, especially vancomycin resistance, is very high in neonates who were admitted in NICU and nursery. It is possible that these pathogens are responsible for serious nosocomial infections in neonates. The need for improved surveillance and continuous control of pathogens is emphasized.
Infant, Newborn ; Humans ; Incidence

A total of 136 strains of Escherichia coli, isolated from rectal swabs of neonates at the neonatal intensive care unit of Pusan National University Hospital during the period from February to April of 2001 and March to April of 2002 were serotyped. The presence of eaeA, aggA, bfpA, astA, LT, and ST genes was test by PCR. Four enteroaggregative E. coli (EAggEC) strains were isolated in 2001 and eight in 2002. In 2001, three strains of enterotoxigenic E. coli (ETEC) were isolated and these strains were tested for antimicrobial susceptibility. ETEC isolates were detected by a reverse passive latex agglutination (RPLA) test for the production of heat-labile enterotoxin. The strains were analyzed by using plasmid profiling, random amplified polymorphic DNA (RAPD), and pulsed field gel electrophoresis (PFGE) methods. The O serotypes could be assigned to 29 isolates (21.3%): O166, 6; O167, 5; O86a, O6 and O127a, 4 each; O8, 2; and O28ac, O44, O158, O20, 1 each. There were 107 untypable isolates. EAggEC isolates were typed as O86a in 4, O127a in 4, and untypable in 4 strains. Three isolates of ETEC were typed as O6. The PCR detected aggA gene in 12 strains (8.8%), but bfpA, eaeA, EAST-1, and ST genes were not detected. LT gene was detected in 3 strains (2.2%) by PCR and DNA probe hybridization, LT production was confirmed in those strains by a latex bead aggregation method. Out of the 15 EAggEC and ETEC strains, eleven (80.0%) were multi-drug resistant to more than 3 antibiotics. Thirteen groups were classed by antibiogram and most strains were susceptible to gentamicin, kanamycin, and cefoperazone, but resistant to ampicillin (100%), cephalothin (66.7%), cefuroxime (46.6%), and tetracycline (46.7%). All isolates possessed a 60 kbp plasmid and 15 strains possessed smaller plasmids. Twelve EAggEC and 3 ETEC strains were differentiated into 5 and 3 groups by the plasmid profiling. RAPD and PFGE grouped the EAggEC isolates into 7 and 6 groups, respectively. The RAPD and PFGE patterns matched in 11 isolates (91.7%) among the 12 EAggEC strains, but the plasmid profile analysis and antibiogram showed no correlation. Three ETEC strains showed different plasmid profiles, and RAPD and PFGE patterns.
Agglutination ; Ampicillin ; Anti-Bacterial Agents ; Busan ; Cefoperazone ; Cefuroxime ; Cephalothin ; DNA ; Electrophoresis, Gel, Pulsed-Field ; Enteropathogenic Escherichia coli* ; Enterotoxigenic Escherichia coli ; Enterotoxins ; Epidemiologic Studies* ; Escherichia coli ; Gentamicins ; Humans ; Infant, Newborn* ; Intensive Care, Neonatal ; Kanamycin ; Latex ; Microbial Sensitivity Tests ; Microspheres ; Plasmids ; Polymerase Chain Reaction ; Tetracycline ; Virulence*

BACKGROUND: Nineteen strains of Shigella sonnei isolated from the patients were examined regarding their biochemical characterization, serotype, and antibiotics resistance, and then analyzed for plasmid DNA profile. METHODS: Strains were tested for possession of set1A, set1B, sen, ipaH, ial, stx and invE genes using the polymerase chain reaction (PCR) method and were analyzed using the pulsed-field gel electrophoresis (PFGE) pattern against 7 outbreak isolates (10 strains). RESULTS: These strains had the typical biochemical characterization of S. sonnei with positive ornithine decarboxylase and -galactosidase activity, but were negative in mannitol fermentation. Serotype were identified as the I phase in 13 strains (68.0%) and the II phase in 6 strains (32.0%). All strains were resistant to erythromycin, vancomycin, tetracycline, and penicillin. The antibiogram type showed 4 groups from I to IV. The strains showed 8 types of plasmid profiles and were designated as P1 to P8. By the PCR, the ipaH gene and the set1B gene were detected from all of the 16 strains. The invE was detected from 9 strains (56.3%), and the sen gene was detected from 5 strains. All strains were negative for the Stx and the set1A gene. High-molecular-weight genomic DNA was prepared from 7 outbreak isolates (10 strains) and digested with the restriction endonuclease XbaI. Restriction fragment patterns of chromosomal DNA were demonstrated by PFGE. XbaI produced about 23 fragments in all strains with the their size ranged from 40 to 680 kb. Ten strains could be differentiated to 3 patterns by chromosomal DNA fingerprint. CONCLUSIONS: All of the Shigella sonnei strains that were isolated from Busan Province showed similar chromosomal DNA fragment patterns, while the Japanese differed in chromosomal DNA fingerprint pattern. PFGE is useful for the epidemiological study of Shigella sonnei associated endemic diarrhea.
Anti-Bacterial Agents ; Asian Continental Ancestry Group ; Busan ; Diarrhea ; DNA ; DNA Fingerprinting ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field ; Erythromycin ; Fermentation ; Genotype ; Humans ; Mannitol ; Microbial Sensitivity Tests ; Ornithine Decarboxylase ; Penicillins ; Plasmids ; Polymerase Chain Reaction ; Shigella sonnei* ; Shigella* ; Tetracycline ; Vancomycin ; Virulence Factors* ; Virulence*

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.
Anti-Bacterial Agents ; Chloramphenicol ; DNA ; Fermentation ; Humans ; Hydrolysis ; India ; Japan ; Korea ; Microbial Sensitivity Tests ; Oxacillin ; Penicillins ; Polymerase Chain Reaction ; Seawater* ; Tetracycline ; Urease ; Vancomycin ; Vibrio parahaemolyticus* ; Vibrio* ; Virulence Factors* ; Virulence*