One hundred and thirty trimethoprim-resistant R plasmids derived from of Escherichia coli isolated from clinical specimens and feces of healthy collegians were examined for incompatibility, EcoRI endonuclease restriction fragment pattern, and Southern hybridization with DHFR I, II, III, V, and VII probe. 1. Most trimethoprim-resistant R plasmids were resistant to ampicillin, tetracycline, chloramphenicol, gentamicin, and kanamycin, and showed multiple drug resistance and various antimicrobial resistance patterns. 2. Trimethoprim-resistant R plasmids ranged from 90 to 50 kilobase and 42.3% of R plasmids tested were classified to incompatibilty group Inc FI, Inc FII or Inc FIV, 3. Among 48 random selected R plasmids from various origin, 14 R plasmids (including 9 of 14 Inc FII plasmids and 3 of 14 Inc FI plasmids) hybridized with DHFR VII oligonucleotide probe but others did not respond to any of DHFR probes used. 4. Most R plasmids showed various EcoRI endonuclease fragments and different reaction sites by Southern hybridization. Six plasmids showed identical or nearly identical molecular weight, EcoRI endonuclease fragment patterns and different sites of Southern hybridization. But 2 Inc FII plasmids derived from urine and feces showed identical pattern. These findings, if confirmed by further studies, suggest that normal flora E. coli can act as reservoir of resistant genes and, consequently, as a factor in the dissemination of these genes among enteric pathogens and need to be examined further.
Ampicillin ; Chloramphenicol ; Deoxyribonuclease EcoRI ; Drug Resistance, Multiple ; Escherichia coli* ; Escherichia* ; Feces ; Gentamicins ; Immunodeficiency Virus, Feline ; Kanamycin ; Molecular Biology* ; Molecular Weight ; Plasmids ; R Factors ; Tetracycline ; Trimethoprim Resistance* ; Trimethoprim*

BACKGROUND: There have been few studies about the kinds of species causing surgical site infections and their resistance pattern in Korea. An increase of extended-spectrum beta-lactamase (ESBL) producing strains is a worldwide problem. However, there is not enough data on the prevalence of ESBL-producing strains in Korea and the true extent of this problem seems to be under-recognized. METHODS: Minimal inhibitory concentrations of gram-negative bacilli isolated from surgical site infections were tested using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards. To identify and characterize beta-lactamases, we performed conjugation test, isoelectric focusing, Southern hybridization, and polymerase chain reaction. RESULTS: A total of 54 strains of gram-negative enteric bacilli were identified:two strains of Acinetobacter spp., one of Citrobacter freundii, nine of Enterobacter cloacae, one of Enterobacter sakazakii, one of Escherichia coli, two of Klebsiella pneumoniae, one of Morganella morganii, one of Proteus vulgaris, 23 of Pseudomonas aeruginosa, four of Xanthomonas maltophila, and nine of Serratia marcescens. Three strains produced ESBL. CONCLUSION: Various species of gram-negative organisms isolated from surgical site infections showed complex antibiograms to various beta-lactams, even to the new generation of antibiotics. A large proportion of these strains showed conjugally transferable, plasmid-mediated, beta-lactam resistance. Some strains were ESBL-producing. This evidence suggests that there has been a molecular evolution of beta-lactamase genes to a great extent in Korea, possibly due to indiscriminate use of antibiotics.
Acinetobacter ; Agar ; Anti-Bacterial Agents ; beta-Lactam Resistance* ; beta-Lactamases ; beta-Lactams ; Citrobacter freundii ; Cronobacter sakazakii ; Drug Resistance ; Enterobacter cloacae ; Escherichia coli ; Evolution, Molecular ; Isoelectric Focusing ; Klebsiella pneumoniae ; Korea ; Microbial Sensitivity Tests ; Morganella morganii ; Polymerase Chain Reaction ; Prevalence ; Proteus vulgaris ; Pseudomonas aeruginosa ; Serratia marcescens ; Xanthomonas

BACKGROUND: There have been few studies about the kinds of species causing surgical site infections and their resistance pattern in Korea. An increase of extended-spectrum beta-lactamase (ESBL) producing strains is a worldwide problem. However, there is not enough data on the prevalence of ESBL-producing strains in Korea and the true extent of this problem seems to be under-recognized. METHODS: Minimal inhibitory concentrations of gram-negative bacilli isolated from surgical site infections were tested using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards. To identify and characterize beta-lactamases, we performed conjugation test, isoelectric focusing, Southern hybridization, and polymerase chain reaction. RESULTS: A total of 54 strains of gram-negative enteric bacilli were identified:two strains of Acinetobacter spp., one of Citrobacter freundii, nine of Enterobacter cloacae, one of Enterobacter sakazakii, one of Escherichia coli, two of Klebsiella pneumoniae, one of Morganella morganii, one of Proteus vulgaris, 23 of Pseudomonas aeruginosa, four of Xanthomonas maltophila, and nine of Serratia marcescens. Three strains produced ESBL. CONCLUSION: Various species of gram-negative organisms isolated from surgical site infections showed complex antibiograms to various beta-lactams, even to the new generation of antibiotics. A large proportion of these strains showed conjugally transferable, plasmid-mediated, beta-lactam resistance. Some strains were ESBL-producing. This evidence suggests that there has been a molecular evolution of beta-lactamase genes to a great extent in Korea, possibly due to indiscriminate use of antibiotics.
Acinetobacter ; Agar ; Anti-Bacterial Agents ; beta-Lactam Resistance* ; beta-Lactamases ; beta-Lactams ; Citrobacter freundii ; Cronobacter sakazakii ; Drug Resistance ; Enterobacter cloacae ; Escherichia coli ; Evolution, Molecular ; Isoelectric Focusing ; Klebsiella pneumoniae ; Korea ; Microbial Sensitivity Tests ; Morganella morganii ; Polymerase Chain Reaction ; Prevalence ; Proteus vulgaris ; Pseudomonas aeruginosa ; Serratia marcescens ; Xanthomonas

Eighty-nine isolates of Enterobacter spp. from two university hospitals were analyzed by phenotypic and genotypic characteristics for epidemiologic investigation. Most strains were isolated from sputum, urine, wound, pus and catheter tip. Most isolates of Enterobacter spp. were resistant to ampicillin, cefazolin and cefoxitin and 39% of E. cloacae isolates were also resistant to other cephalosporins and aminoglycoside antibiotics except amikacin but all strains were highly susceptible to imipenem and ciprofloxacin. Twenty-six antimicrobial resistance patterns were obtained from E. clacae, but E. aerogenes showed only 4 patterns. Fourty-two plasmid profiles were identified, but plasmid was not detected from 28.4% of E. cloacae and 58% of E. aerogenes. Six biotypes from E. cloacae and three biotypes from E. aerogenes were obtained by carbohydrate metabolism. Fourteen strains of E. cloacae carried conjugative R plasmids and these plasmids were further analyzed. Among them, ten plasmids showed identical antibiogram, molecular weight, and pI value by isoelectric focusing and nearly identical restriction endonuclease fragment pattern. Their parental strains had identical antibiogram, biotype, plasmid profile, and were isolated from 4 different specimens including 6 catheter tips of different patients. But most clinical isolates showed various types of combination and seemed to be different strains. These results indicate that the epidemic strain were present in this hospital and the combination of antibiogram and plasmid analysis can be used to discriminate the epidemic strains of multi-resistant E. cloacae.
Amikacin ; Ampicillin ; Anti-Bacterial Agents ; Carbohydrate Metabolism ; Catheters ; Cefazolin ; Cefoxitin ; Cephalosporins ; Ciprofloxacin ; Cloaca ; DNA Restriction Enzymes ; Enterobacter* ; Hospitals, University ; Humans ; Imipenem ; Isoelectric Focusing ; Microbial Sensitivity Tests ; Molecular Weight ; Parents ; Plasmids ; R Factors ; Sputum ; Suppuration ; Wounds and Injuries

A clinical isolate of Klebsiella pneumoniae K7746 produced the extended-spectrum beta-lactamase (ESBL) SHV-12. A 6.6 kb BamHI fragment containing the blaSHV-12 gene of K7746 strain was cloned into pCRScriptCAM vector resulting in the recombinant plasmid p7746-C1. The restriction map of 3.6 kb inserted DNA and sequences immediately surrounding blaSHV-12 of p7746-C1 were homologous to plasmid pMPA2a carrying blaSHV-2a. In addition, both blaSHV-12 and blaSHV-2a were expressed from a common hybrid promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the blaSHV promoter itself. The results indicate that blaSHV-12 and blaSHV-2a may have evolved from a common ancestor in the sequential order of blaSHV-2a first, followed by blaSHV-12. Furthermore, by the PCR mapping method using primers corresponding to the IS26 and blaSHV, the association between IS26 and blaSHV was studied in 12 clinical isolates carrying blaSHV-2a, 27 clinical isolates carrying blaSHV-12, and 5 reference strains carrying blaSHV-1 to blaSHV-5. All 39 strains carrying blaSHV-2a or blaSHV-12 were positive by the PCR, providing confirmative evidence that IS26 has been involved in the evolution and dissemination of blaSHV-2a and blaSHV-12. But 5 reference strains carrying blaSHV-1 to blaSHV-5 were negative by the PCR. Therefore, we concluded that the molecular evolutionary pathway of blaSHV-2a and blaSHV-12 may be different from that of other blaSHV-ESBL, e.g., blaSHV-2, blaSHV-3, blaSHV-4, and blaSHV-5.
beta-Lactamases ; Clone Cells ; DNA ; Klebsiella pneumoniae ; Plasmids ; Polymerase Chain Reaction

Acinetobacter species encounters frequently with clinical specimens and now accounts for a substantial proportion of endemic nosocomial infections in Korea. Recent trends indicate that the antimicrobial resistant strains of Acinetobacter species are increasing. Sixty-one strains were isolated from specimens of patients suspected of nosocomial infections during 1991 to 1996. At present, phenotypic identification of Acinetobacter using biochemical test may not be reliable and resulted in the difficulty to clarify the source of infections and epidemiological study of hospital-acquired infections. Aware of the importance of rational taxonomic proposal for these isolates, correct species identification of these organisms by molecular typing method was carried out. A total of fifty-four strains of A. calcoaceticus-A. baumannii complex species which were identified to genospecies 2 and 13 by biochemical characteristics was subjected to identify by ribotyping using restriction endonuclease EcoRI, ClaI, and SalI. Of fifty-four strains, twenty-five strains were identified as A. baumannii (genospecies 2) and twenty-one strains as genospecies 13, and six strains changed to genospecies 3, and the rest two strains were confirmed as A. haemolyticus (genospecies 4). This result suggests that the ribotyping may be of value for identification of genospecies and epidemiological information of Acinetobacter strains.
Acinetobacter baumannii* ; Acinetobacter calcoaceticus* ; Acinetobacter* ; Cross Infection ; DNA Restriction Enzymes ; Humans ; Korea ; Molecular Typing ; Ribotyping*

BACKGROUND: Detection of extended-spectrum beta-lactamase (ESBL) expression is difficult in ordinary clinical laboratories. The Etest has been introduced into clinical settings for the rapid identification of ESBL. The principle behind the Etest is to compare the minimal inhibitory concentration (MIC) of ceftazidime alone with the MIC of ceftazidime with clavulanic acid. The aim of this study was to evaluate the efficiency of the Etest for the detection of ESBL in Korea, where antimicrobial resistance rates are high. METHODS: The double disk synergy test and the Etest were performed simultaneously. The results of the clinical isolates were compared to those of strains producing TEM-1, TEM-2, and SHV-1 as negative controls. The results of the double disk synergy test and the E-test were confirmed by isoelectric focusing of beta-lactamase extracted from suspicious ESBL-producing strains. RESULTS: MIC determination using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards revealed that a total of 48 strains were resistant or intermediate against one or more antibiotics of the third generation cephalosporins. These strains included five strains of E. coli, 14 of S. marcescens, seven of K. pneumoniae, 18 of Enterobacter spp., and four of Citrobacter spp. Sixteen (33%) of the strains, including five strains of E. coli, three of S. marcescens, five of K. pneumoniae, and three of Enterobacter spp. were ESBL- producing strains that were confirmed by double disk synergy test. Thirteen (81%) of the strains of ESBL- producing organisms were detected by Etest, but the remaining three strains (19%) were undetectable by Etest alone. CONCLUSION: The accuracy of Etest for the detection of ESBL was not high, but the efficiency of Etest as the primary screening method of a large number of clinical isolates was appreciable regarding efficiency and rapidity.
Agar ; Anti-Bacterial Agents ; beta-Lactamases* ; Ceftazidime ; Cephalosporins ; Citrobacter ; Clavulanic Acid ; Enterobacter ; Gram-Negative Bacteria ; Isoelectric Focusing ; Korea ; Mass Screening ; Pneumonia

BACKGROUND: Detection of extended-spectrum beta-lactamase (ESBL) expression is difficult in ordinary clinical laboratories. The Etest has been introduced into clinical settings for the rapid identification of ESBL. The principle behind the Etest is to compare the minimal inhibitory concentration (MIC) of ceftazidime alone with the MIC of ceftazidime with clavulanic acid. The aim of this study was to evaluate the efficiency of the Etest for the detection of ESBL in Korea, where antimicrobial resistance rates are high. METHODS: The double disk synergy test and the Etest were performed simultaneously. The results of the clinical isolates were compared to those of strains producing TEM-1, TEM-2, and SHV-1 as negative controls. The results of the double disk synergy test and the E-test were confirmed by isoelectric focusing of beta-lactamase extracted from suspicious ESBL-producing strains. RESULTS: MIC determination using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards revealed that a total of 48 strains were resistant or intermediate against one or more antibiotics of the third generation cephalosporins. These strains included five strains of E. coli, 14 of S. marcescens, seven of K. pneumoniae, 18 of Enterobacter spp., and four of Citrobacter spp. Sixteen (33%) of the strains, including five strains of E. coli, three of S. marcescens, five of K. pneumoniae, and three of Enterobacter spp. were ESBL- producing strains that were confirmed by double disk synergy test. Thirteen (81%) of the strains of ESBL- producing organisms were detected by Etest, but the remaining three strains (19%) were undetectable by Etest alone. CONCLUSION: The accuracy of Etest for the detection of ESBL was not high, but the efficiency of Etest as the primary screening method of a large number of clinical isolates was appreciable regarding efficiency and rapidity.
Agar ; Anti-Bacterial Agents ; beta-Lactamases* ; Ceftazidime ; Cephalosporins ; Citrobacter ; Clavulanic Acid ; Enterobacter ; Gram-Negative Bacteria ; Isoelectric Focusing ; Korea ; Mass Screening ; Pneumonia

Conjugative R plasmids derived from 74 clinical isolates of Serratia marcescens were epidemiologically analyzed for antimicrobial resistance, EcoRI restriction endonuclease analysis and Southern hybridization with DHFR, TEM and SHV probe. 1. Resistance frequency of isolates against various B-lactam antibiotics was changed by year. 2. Twenty (27%) resistant strains transferred 32 R plasmids to E. coli or Klebsiella by mixed culture. Most strains isolated from 1994 to 1996 transferred only trimethoprim resistance but most strains isolated from 1997 did resistances against gentamicin (Gm) and B-lactams including ampicillin (Ap), carbenicillin (Cb), cefazolin (Cz), cefaloridine (Cl), cefamandole (Cn). 3. Ten plasmids of GmApCbCzC1Cn or GmApCbCzC1 pattern and 3 plasmids of TcSuGmTbApCbCzC1 pattern respectively showed identical EcoRI restriction endonuclease digestion patterns and hybridized fragment patterns with TEM-1 probe by Southern hybridization. These results indicate that the epidemic plasmids carrying blamM gene were present in this hospital in 1997 and molecular genetic analysis of R plasmids can be used to discriminate S. marcescens isolates for epidemiologic studies.
Ampicillin ; Anti-Bacterial Agents ; Carbenicillin ; Cefamandole ; Cefazolin ; Cephaloridine ; Digestion ; DNA Restriction Enzymes ; Epidemiologic Studies ; Epidemiology* ; Gentamicins ; Klebsiella ; Molecular Biology ; Plasmids ; R Factors ; Serratia marcescens* ; Serratia* ; Trimethoprim Resistance

Ninety-two strains of Serratia marcescens isolated from 5 hospitals were analyzed for plasmid profile, antimicrobial drug resistance pattern, biotyping, and production of pigment. Ninety-three percents of strains were resistant to chloramphenicol (Cm), tetracycline (Tc), sulfisoxazole (Su), cefazolin (Cz), ampicillin (Ap), and rifampin (Rf). A majority of strains were susceptible to amikacin (Ak), ciprofloxacin (Ci), and cefotaxim (Ct). Fifty-four resistance patterns were found in 94 strains and the most prevalent resistance pattern was CmTcSuApCzRf. Seventeen (17.4%) isolates could transfer their partial resistance to E. coli or Klebsiella pneumoniae by conjugation. Twenty-seven plasmid profiles in 54 strains (58.7%) were detected, however no predominant patterns were seen in isolates from each hospital. Eleven biotypes were detected. The common types were A3b (29.4%) and A8b (27.1%), predominant types were found in each hospital. Twenty strains from 4 of 5 hospitals showed consistence of 3 types. These results indicate that plasmid profile analysis, Grimont biotyping, and resistance pattern type of strains in combination are useful as an epidemiological tool for S. marcescens isolates and some of isolates were confirmed as nosocomial strains.
Amikacin ; Ampicillin ; Cefazolin ; Cefotaxime ; Chloramphenicol ; Ciprofloxacin ; Drug Resistance, Microbial ; Epidemiologic Studies* ; Klebsiella pneumoniae ; Plasmids ; Rifampin ; Serratia marcescens* ; Serratia* ; Sulfisoxazole ; Tetracycline