BACKGROUND/AIMS: Cronobacter sakazakii, an emergent pathogen is considered as a major concern to infants and neonates fed on reconstituted powdered infant milk formula. In conjunction with many other factors, biofilm forming capacity adds to its pathogenic potential. In view of the facts that infants are at highest risk to C. sakazakii infections, and emerging antibiotic resistance among pathogens, it is imperative to evaluate probiotic cultures for their efficacy against C. sakazakii. Therefore, pure probiotic strains were isolated from commercial probiotic products and tested for their antimicrobial and anti-biofilm activities against C. sakazakii. METHODS: A total of 6 probiotic strains were tested for their antibiotic susceptibility followed by antimicrobial activity using cell-free supernatant (CFS) against C. sakazakii. The inhibitory activity of CFS against biofilm formation by C. sakazakii was determined using standard crystal violet assay and microscopic observations. RESULTS: All the probiotic strains were sensitive to ampicillin, tetracycline, vancomycin and carbenicillin whereas most of the strains were resistant to erythromycin and novobiocin. Four of the 6 probiotic derived CFS possessed antimicrobial activity against C. sakazakii at a level of 40 μL. A higher biofilm inhibitory activity (>80%) was observed at initial stages of biofilm formation with weaker activity during longer incubation upto 48 hours (50%–60%). CONCLUSIONS: The study indicated the efficacy of isolated commercial probiotics strains as potential inhibitor of biofilm formation by C. sakazakii and could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii.
Ampicillin ; Biofilms ; Carbenicillin ; Cronobacter sakazakii ; Cronobacter ; Drug Resistance, Microbial ; Erythromycin ; Gentian Violet ; Humans ; Infant ; Infant, Newborn ; Milk ; Novobiocin ; Probiotics ; Tetracycline ; Vancomycin

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.
Agrobacterium tumefaciens ; genetics ; Bacterial Proteins ; genetics ; Carbenicillin ; pharmacology ; Cefotaxime ; pharmacology ; DNA, Bacterial ; genetics ; physiology ; Genetic Vectors ; genetics ; Hygromycin B ; pharmacology ; Magnaporthe ; drug effects ; genetics ; Mutagenesis, Insertional ; methods ; Mutation ; Oryza ; microbiology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transformation, Genetic ; genetics ; physiology

BACKGROUND: Kluyvera, a new genus in the family Enterobacteriaceae, has been rarely isolated from clinical specimens and regarded as an opportunistic pathogen. Although there were several case reports in Korea, most of them were reported at a genus level except a case of K. cyrocrescens. We isolated Kluyvera species from seven patients from July 1996 to January 1999. We identified them to species level and investigated their clinical significance. METHODS: The medical records of seven patients were reviewed for demographical findings, underlying diseases, diagnoses, the association of Kluyvera isolates with disease, antibiotic treatments, and clinical outcomes. Eight strains were identified and tested for the antimicrobial susceptibilities by MicroScan Neg Combo type 14 and 21 Panel(Dade Behring, USA). Five of the eight strains had been stored at -70degrees C and were tested for ascorbate fermentation, the ability to grow and ferment glucose at 5degrees C, and the zone of inhibition around carbenicillin and cephalothin. RESULTS: Kluyvera isolates were regarded as true pathogens in six of seven cases including Hickman-catheter associated sepsis(HCAS), empyema, peritonitis, necrotizing cholecystitis, sepsis, and liver abscess although the latter four cases yielded mixed cultures. While three of the six patients had underlying diseases, malignant lymphoma, hepatocellular carcinoma and stomach cancer, other three were previousely healthy. Most of them were improved with an empirical therapy, but Kluyvera species was repeatedly isolated from the HCAS case in spite of the antibiotic treatment; it was cured bacteriologically after the removal of the catheter. The five isolates were all confirmed to be K. ascorbata by positive ascorbate test, and failure to grow at 5degrees C. CONCLUSIONS: Six of the seven cases including three with no underlying diseases, isolates of Kluyvera species were found clinically significant, suggesting that Kluyvera species is potentially pathogenic in healthy individuals as well as compromized hosts. MicroScan system is capable of identifying Kluyvera species at the genus level, but not at the species level. The ascorbate test is simple and useful for differ entiation of K. ascorbata from K. cryocrescens.
Carbenicillin ; Carcinoma, Hepatocellular ; Catheters ; Cephalothin ; Cholecystitis ; Diagnosis ; Empyema ; Enterobacteriaceae ; Fermentation ; Glucose ; Humans ; Kluyvera* ; Korea ; Liver Abscess ; Lymphoma ; Medical Records ; Peritonitis ; Sepsis ; Stomach Neoplasms

Sixty-eight clinical isolates of Stenotrophomonas maltophilia from inpatients of 2 university hospitals in Taegu were epidemiologically analyzed by using the minimum inhibitory concentrations of 25 antimicrobial drugs, biochemical reaction, pulsed-field gel elctropgoresis (PFGE), and PCR with enterobacterial repetitive intergenic consensus sequences as primer (ERIC-PCR). 1. All the strains were susceptible to minocycline. More than 57% were susceptible to sulfisomidine (Su), ciprofloxacin (Ci), Ofloploxacin (Of), nalidixic acid (Na), and chloramphenicol (Cm), and 19apprx35% to ceftazidime (Cd), trimethoprim (Tp), Ticacillin-clavulanic acid, and cefoperazone-sulbactam. Most isolates were resistant to beta-lactam antibiotics such as ampicillin (Ap), carbenicillin (Cb), cefotaxim (Ct), cefoxitin (Cx), and aminoglycosides including gentamicin (Gm), tobramycin (Tb), amikacin (Ak). 2. All the isolates were multiply resistant of 5 to 17 drugs and showed 40 different resistance pattern types. 3. All the strains showed very similar biochemical reactions except beta-galactosidase and nitrate reduction test. Fourteen strains selected randomly were classified 10 different pattern type by PFGE and ERIC-PCR. These two methods showed identical result. Four strains isolated from wound in 1994 showed similar MIC pattern and identical API 20NE profile, PFGE, and ERIC-PCR pattern indicating episodes of cross-infection among patients. These results indicate that PFGE or ERIC-PCR profile has comparable discriminatory power for epidemiological typing of S. maltophilia.
Amikacin ; Aminoglycosides ; Ampicillin ; Anti-Bacterial Agents ; beta-Galactosidase ; Carbenicillin ; Cefotaxime ; Cefoxitin ; Ceftazidime ; Chloramphenicol ; Ciprofloxacin ; Consensus Sequence ; Daegu ; Gentamicins ; Hospitals, University ; Humans ; Inpatients ; Microbial Sensitivity Tests ; Minocycline ; Nalidixic Acid ; Polymerase Chain Reaction ; Stenotrophomonas maltophilia* ; Stenotrophomonas* ; Sulfisomidine ; Tobramycin ; Trimethoprim ; Wounds and Injuries

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

BACKGROUND: The number of reported cases of contact lens related ulcerative keratitis has markedly increased. Pseudomonas aeruginosa is one of the most virulent pathogen in contact lens wearers with corneal ulcer. It causes a rapidly spreading ulcer with copious mucopurulent discharge and occasionally corneal perforation. We analyzed the clinical and microbiological findings of 20 cases of culture-proven pseudomonas keratitis in contact lens wearers with a brief review of literature. METHODS: Twenty cases of Pseudomonas keratitis in contact lens wearers were reviewed retrospectively. All patients were seen at the department of ophthalmology, Konyang Hospital during the period from April 1994 to September 1997. RESULTS: The male to female ratio was 9 to 11. The age distribution ranged from 16 to 64 years: 10 patients in their 20s, 7 in their 30s, and each in their 10s, 50s, and 60s, respectively. The duration of lens wear before developing keratitis was variable from 5 weeks to 12 years. Seven cases(35%) of them had a history of over night wearing of contact lens. The size of corneal ulcer varied from 1 to 8 mm in the longest diameter. Hypopyon was seen in 80% of patients. Almost all P. aeruginosa were resistant to ampicillin, cephalothin, chloramphenicol, and carbenicillin, but susceptible to imipenem, ciprofloxacin, and piperacillin. CONCLUSION: Pseudomonas keratitis is a rapidly spreading ulcer with various complications. It has markedly increased in contact lens wearers. To prevent further increase, education, early diagnosis, and treatment are important.
Age Distribution ; Ampicillin ; Carbenicillin ; Cephalothin ; Chloramphenicol ; Ciprofloxacin ; Corneal Perforation ; Corneal Ulcer ; Early Diagnosis ; Education ; Female ; Humans ; Imipenem ; Keratitis ; Male ; Ophthalmology ; Piperacillin ; Pseudomonas aeruginosa ; Pseudomonas* ; Retrospective Studies ; Ulcer*

One hundred and twenty-two strains of E. coli isolated from urinary tract infection were examined for antibiogram, transferability of trimethoprim (Tp) resistance, incompatibility with F group plasmid and southem hybridization with DHFR I, II, and III probe of Tp-resistant R plasmids. 1. Among 172 Gram negative bacilli isolated from urinary tract infection, 122 (70.9%) were E. coli and 75 strains of them were resistant to trimethoprim (Tp). Most of Tp-resistant isolates were also resistant to penicillins (ampicillin, carbenicillin, and ticarcillin), aminoglycosides (kanamycin and gentamicin), and sulfisoxazole but almost all strains were susceptible to cephalosporins. 2. Most of Tp-resistant strains and E. coli transconjugant derived from them showed multiple drug resistance and various antimicrobial resistance patterns. 3. Thirty-three Tp-resistant strains (45.2%) transferred 35 Tp-resistant plasmids to E. coli recipients but among them 6 transconjugants did not show retransfer of resistance and plasmid DNA were not detected in 2 transconjugants after resistance transfer. 4. Tp-resistant R plasmids ranged from 157 to 67 kb and 8 R plasmids were classified to incompatibilty group IncFI or IncFII ranging from 120 to 83 kb. Three and two R plasmids belonged to IncFII showed similar molecular weight, resistance pattern, and reaction site by southern hybridization with DHFR I probe. Twenty-five plasmids specifically responded on various EcoRI endonuclease fragments to DHFR I probe but not to DHFR II or DHFR III probe. These findings suggest that most of Tp- resistant R plasmids from urine isolates of E. coli were derived from various sources but some plasmids including IncFII R plasmids were probably originated from same or similar sources.
Aminoglycosides ; Carbenicillin ; Cephalosporins ; Deoxyribonuclease EcoRI ; DNA ; Drug Resistance, Multiple ; Escherichia coli* ; Escherichia* ; Microbial Sensitivity Tests ; Molecular Weight ; Penicillins ; Plasmids ; R Factors ; Sulfisoxazole ; Trimethoprim Resistance* ; Trimethoprim* ; Urinary Tract Infections

One hundred of clinical isolates of Klebsiella spp. from three hospitals were analyzed by phenotypic and genotypic characteristics for epidemiologic investigation. Almost all isolates of Klebsiella spp. showed highly resistance to ampicillin, and carbenicillin and 4.5-7.9% of K. pneumoniae isolates were also resistant to cefotaxime and ceftazidime, and 10-15% to aminoglycoside antibiotics except amikacin. However, all strains were highly susceptible to imipenem, cefotetan, amikacin and ciprofloxacin. All Koxytoca strains were susceptible to antimicrobials tested except Ap, Am and Cb. Twelve strains of K. pneumoniae hybridized with TEM or SHV probe and extended spectrum B-lactamases from 7 strains were TEM type. Eleven conjugative R plasmids and their parental strains were analyzed. Among them, three couples of plasmids showed identical or nearly identical resistance phenotypes of B-lactams and aminoglycosides, molecular weights, and pI values by isoelectric focusing, and hybridized fragment patterns with TEM probe by Southern hybridization, EcoR1 restriction endonuclease fragment patterns. Their parental strains were isolated from sputum, tissue, and ascites of patients and had similar characteristics. These results indicate that the epidemic strains or epidemic plasmids were present in this hospital and antimicrobial resistance anlysis can be used to discriminate clinical isolates of multi-resistant K. pneumoniae.
Amikacin ; Aminoglycosides ; Ampicillin ; Anti-Bacterial Agents ; Ascites ; Carbenicillin ; Cefotaxime ; Cefotetan ; Ceftazidime ; Ciprofloxacin ; DNA Restriction Enzymes ; Epidemiology* ; Family Characteristics ; Humans ; Imipenem ; Isoelectric Focusing ; Klebsiella* ; Molecular Weight ; Parents ; Phenotype ; Plasmids ; Pneumonia ; R Factors ; Sputum

One hundred and eighteen strains of Escherichia coli isolated from clinical specimens were epidemiologically analyzed for antimicrobial resistance, EcoRI restriction endonuclease analysis, southern hybridization with TEM and SHV probe of conjugative R plasmids. 1. Sixty-two to 73% of E. coli isolates were resistant to ampicillin, carbenicillin, sulfisomidine, and tetracycline, and 20-27% to kanamycin, gentamicin, tobramycin, and nalidixic acid. However more than 93% were susceptible to cephalosporins and all strains were highly susceptible to cefotetan, imipenem, aztreonam, and amikacin. 2. Twelve strains were susceptible to all drugs tested and the multiple resistant strains showed 65 resistance pattern types. 3. Thirty-six resistant strains(34%) transferred R plasmids to E. coli RG488 or RG176 by mixed culture. Fifty-six plasmids with 31 different resistant phenotype were obtained from them. 4. Some of 15 plasmids derived from 10 strains showed identical or similar EcoRI restriction endonuclease digestion patterns, hybridized fragment patterns with TEM probe by southern hybridization, and resistance levels of j3-lactams and aminoglycosides. These results indicate that the epidemic strains or plasmids were present in this hospital and molecular genetic analysis of R plasmids can be used to discriminate clinical isolates of multi- resistant E. coli.
Amikacin ; Aminoglycosides ; Ampicillin ; Aztreonam ; Carbenicillin ; Cefotetan ; Cephalosporins ; Digestion ; DNA Restriction Enzymes ; Escherichia coli* ; Escherichia* ; Gentamicins ; Imipenem ; Kanamycin ; Molecular Biology ; Nalidixic Acid ; Phenotype ; Plasmids ; R Factors ; Sulfisomidine ; Tetracycline ; Tobramycin

BACKGROUND: During the period from April to June 1996, an outbreak of typhoid fever occurred in SuYoung-Ku, Pusan city. We performed the molecular epidemiological analysis and drug susceptibility test for 48 isolates of Salmonella typhi from the outbreak. METHODS: Chromosomal DNA of S. typhi was digested with the restriction endonuclease Spe I and the resulting restriction polymorphism was observed on pulsed field gel electrophoresis (PFGE). Phage typing was performed using Vi-phages, and antimicrobial susceptibility test was done by Disc diffusion method. RESULTS: On phage typing, 2 isolates were M1 phage type, 2 E1 phage type and 44 untypable. The PFGE analysis on 14 to 16 chromosomal DNA restriction fragments ranged from 50 Kb to 1000kb showed 2 different patterns of restriction fragments, divided into group A and B. Forty-seven isolates belonged to group A that were allocated into A1 (42 strains), A2 (1 strain), A3 (3 strains), A4 (1 strain), and one strain was grouped into group B. In the F-value of genetic similarity of the epidemic S. typhi strains, there was low similarity (F value:0.2-0.258) between group A strains and group B strains. However, the subgroup strains (A1-A4) showed high similarity (F value:0.8-0.897) each other. The drug susceptibility test showed susceptibility to ampicillin, carbenicillin, cephalothin, chloramphenicol, tobramycin, gentamicin, kanamycin, nalidixic acid, neomycin, polymyxin B, streptomycin, tetracycline, trimethoprim-sulfamethoxazole, ciprofloxacin and norfloxacin. CONCLUSION: This study showed that phage typing and PFGE were very useful as a tool to investigate molecular analyses of epidemic S. typhi strains this study.
Ampicillin ; Bacteriophage Typing ; Bacteriophages* ; Busan* ; Carbenicillin ; Cephalothin ; Chloramphenicol ; Ciprofloxacin ; Diffusion ; DNA ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field* ; Epidemics ; Gentamicins ; Kanamycin ; Nalidixic Acid ; Neomycin ; Norfloxacin ; Polymyxin B ; Salmonella typhi* ; Salmonella* ; Streptomycin ; Tetracycline ; Tobramycin ; Trimethoprim, Sulfamethoxazole Drug Combination ; Typhoid Fever