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

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/ cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (P<0.01) when macrophages were incubated with V. vulnificus (100 bacteria/ cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may play a role in the pathogenesis of V. vulnificus septicemia.
Animals ; Cell Survival ; Cholesterol ; Erythrocytes ; Humans ; Macrophages ; Macrophages, Peritoneal* ; Membranes ; Mice* ; Perforin ; Sepsis ; Shock ; Trypan Blue ; Vibrio vulnificus* ; Vibrio* ; Virulence

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

Vibrio vulnificus, a halophilic vibrio is an estuarine gram-negative bacteria that is associated with severe and frequently fatal wound infections and life-threatening septicemia. Bacteriocins are defined as antibacterial substance produced by various species of bacteria which are usually active against closely related organisms. Bacteriocins have found widespread application in epidemiological studies as specific markers of bacteria. It was proposed by Ha et al. (1990. J. Korean. Soc. Microbiol. 25: 586.) to give the bacteriocins produced by V. vulnificus the name "vulnificins". In the present study, a total of 72 strains of V. vulnificus isolated from patients and oysters were subjected to screen potential producers and indicators of vulnificin, applying ultraviolet induction method. Sensitivity of several strains of Serratia marcesans, Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhi and Yersinia enterocolitica to vulnificins were also examined out. All the tested strains of V. vulnificus produced vulnificins active against indicator strains with various different inhibitory patterns. The spectrum of vulnificin activity and sensitive spectrum of indicator strains were considerably broad. Interestingly, almost all strains of S. marcescens, P. aeruginosa, Salmonella sp., Shigella sp. and Y. enterocolitica tested were sensitive to 1-7 vulnificin(s). Taken together, the present study demonstrated that all of the isolates of V. vulnificus produced vulnificins and that 8 good vulnificin producers and 10 good indicators were detected. These strains can be employed efficiently for establishing vulnificin typing scheme of V. vulnificus and for the detection of bacteriocinogeny and sensitivity in V. vulnificus. Biological role of vulnificin remains to be further elucidated.
Bacteria ; Bacteriocins ; Gram-Negative Bacteria ; Humans ; Ostreidae ; Pseudomonas aeruginosa ; Salmonella ; Salmonella typhi ; Sepsis ; Serratia ; Shigella ; Shigella flexneri ; Vibrio vulnificus* ; Vibrio* ; Wound Infection ; Yersinia enterocolitica

Staphylococcus aureus infections are often life-threatening. Relatively little is known about the host response to these infections, in particular, the implication of apoptosis induced by this microorganism. In this study, we have shown that S. aureus was cytotoxic to J774A.1 cell, a murine macrophage cell line. The cell death mediated by S. aureus occurred through apoptosis, as shown by increase in the proportion of fragmented host cell DNA. Although phagocytosis and NO production had important role in the induction of apoptosis, the contact between bacteria and host cells was not essential for this pathway. A certain bacterial product could also induce typical caspase-dependent apoptosis of J774A.1 cell. It is expected that new interpretation may be possible to host-parasite relationship based on these results.
Animals ; Apoptosis* ; Bacteria ; Cell Death ; Cell Line* ; DNA ; Host-Parasite Interactions ; Macrophages* ; Mice* ; Phagocytosis ; Staphylococcus aureus* ; Staphylococcus*

Streptococcus mutans is the most important causative bacteria of dental caries among the oral bacteria. Lactococcus lactis 1370 was isolated from the oral cavity of child. The effect of Lactococcus lactis 1370 on the formation of artificial plaque by Streptococcus mutans was studied. 1. The insoluble substances and bacteria were much more attached on the wall of disposable cuvette in the culture of Streptococcus mutans than in the combined culture of Streptococcus mutans and Lactococcus lactis 1370. 2. The mean weight of produced artificial plaque on the wires in the beaker was 131.7 mg in the culture of Streptococcus mutans only, whereas being reduced to 6.4 mg in the combined culture of Streptococcus mutans and Lactococcus lactis 1370 (p<0.05). The viable cell didn't show the significant difference between them after culturing. 3. When Streptococcus mutans was cultured in the media containing culture supernatant of Lactococcus lactis 1370 cultured in M17 broth containing 0.5% yeast extract and 5% sucrose, the mean weight of produced artificial plaque was 8.0 mg on the wires, whereas being 125.4 mg in the media without culture supernatant of Lactococcus lactis 1370 (p<0.05). The viable cell didn't show the significant difference between them after culturing. 4. When Streptococcus mutans was cultured in the media containing soluble polymer produced by Lactococcus lactis 1370, the mean weight of produced artificial plaque was significantly reduced compared with being cultured in the media without soluble polymer (p<0.05). The viable cell didn't show the significant difference between them after culturing. 5. The soluble polymer produced by Lactococcus lactis 1370 was glucan. 6. The glucan produced by Lactococcus lactis 1370 was water-soluble glucan containing alpha-1,6-glucose linkage as the main linkage. These results suggest that the artificial plaque formed by Streptococcus mutans is inhibited by water-soluble glucan produced by Lactococcus lactis 1370.
Bacteria ; Child ; Dental Caries ; Humans ; Lactococcus lactis* ; Lactococcus* ; Mouth ; Polymers ; Streptococcus mutans ; Sucrose ; Yeasts

Members of the genus Acinetobacter are recognized as newer pathogens of the nosocomial infection with an increasing frequency in recent years. Strains that belonged to A. calcoaceticus-A. baumannii complex (genomic species 1, 2, 3, and 13TU) were major groups associated with nosocomial infection. Phenotypic identification was unreliable and laborious method to classify Acinetobacter strains into 19 genomic species. Rapid and reliable identification of clinical isolates is essential to diagnosis and epidemiology of Acinetobacter. We investigated the suitability of amplified ribosomal DNA restriction analysis (ARDRA) to identify genomic species of 131 Acinetobacter isolates. The 16S rRNA genes (ribosomal DNA) were enzymatically amplified and the amplified PCR products were restricted independently with the enzymes, AluI, CfoI, and MboI. Genomic species of Acinetobacter was classified by the combinations of restriction patterns. The analysis was showed that restriction profiles were characteristic for each genomic species. One hundred fourteen isolates were identified as A. baumannii, twelve were identified as genomic species 13TU, and one was identified as genomic species 3. Four isolates were found to be unknown organisms. All of the isolates which were identified to A. baumannii by phenotypic tests were completely discriminated into A. baumannii and genomic species 13TU by ARDRA. This study demonstrates that ARDRA is a rapid and simple techniques for the identification of Acinetobacter species according to the genomic species.
Acinetobacter baumannii* ; Acinetobacter calcoaceticus* ; Acinetobacter* ; Cross Infection ; Diagnosis ; DNA, Ribosomal* ; Epidemiology ; Genes, rRNA ; Polymerase Chain Reaction

Among wild chipmunks, Tamias sibiricus, captured in Kyunggi and Kangwon province in Korea, 1997, seropositivity for Orientia tsutsugamushi was determined. Serological test for Orientia tsutsugamushi infection was performed using indirect immunofluorescent antibody technique (IFA). Of 243 wild chipmunks, 61 against Gilliam strain and 64 against Karp strain of Orientia tsutsugamushi were IFA positive. Seropositivity against Gilliam strain was shown 33.3% in Kyunggi and 23.5% in Kangwon province, and against Karp strain was shown 33.3% and 25.4%, respectively.
Gangwon-do ; Gyeonggi-do ; Korea* ; Orientia tsutsugamushi* ; Sciuridae* ; Serologic Tests

ITSI-5.8S-ITSII rDNA region was amplified from the reference strains and clinical isolates with ITS1 and ITS4 primers. These primers amplified DNA fragments of 550 bp in Microsporum audouinii and Trichophyton violaceum, 700 bp in Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton tonsurans, and 750 bp in Microsporum ferreugineum and Microsporum canis. The restriction enzyme patterns of PCR products digested with 13 restriction enzyme including PstI were distint among the genera, whereas identical in the same species. Examination of the ITS (Internal Transcribed Spacers)1 nucleotide sequence revealed that there was the genetic difference in each genera and species. Phylogenetic relationship among each species showed that the Trichophyton mentagrophytes was more closely related Trichophyton tonsurans than Trichophyton rubrum, and Microsporum gypseum was less related than Microsporum spp.
Arthrodermataceae* ; Base Sequence* ; DNA* ; DNA, Ribosomal ; Microsporum ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length* ; Ribosomes* ; Trichophyton

Bacteroides fragilis and Escherichia coli, normal colonic inhabitants, are the most frequently isolated bacteria in infected tissues, particularly in intraabdominal abscesses. This study was designed to determine whether enteric bacteria may alter the B. fragilis-induced expression of proinflammatory cytokines in mouse peritoneal tissue (MPT). After C57BL/6 mice were inoculated with abscess-forming mixture containing B. fragilis in the presence or absence of E. coli, RNA was extracted from MPT. Expression of interleukin (IL)-1alpha and tumor necrosis factor (TNF)alpha mRNA was assessed using RT-PCR and standard RNA. Each cytokine protein was also measured by ELISA. The co-inoculation of E. coli into mouse peritoneal cavity advanced the onset of abscess development by B. fragilis infection. When mouse was co-infected with E. coli and B. fragilis intraperitoneally, there was a synergistic increase in the expression of IL-1alpha and TNFalpha mRNA in MPT and this was paralleled by increased cytokine protein secretion. Mixed inoculation of heat-killed E. coli and B. fragilis did not cause a synergistic increase in those cytokine mRNA expression. These results suggest that enteric bacteria may significantly affect proinflammatory cytokine signal produced by host peritoneal cavity in response to B. fragilis infection.
Abscess ; Animals ; Bacteria ; Bacteroides fragilis* ; Bacteroides* ; Coinfection* ; Colon ; Cytokines ; Enterobacteriaceae ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli* ; Escherichia* ; Gene Expression* ; Interleukins ; Mice ; Peritoneal Cavity ; RNA ; RNA, Messenger ; Tumor Necrosis Factor-alpha