ABSTRACT
BACKGROUND: The aim of the study was to develop an accurate, convenient, and easy microplate system for the identification of enterococcal species from clinical specimens. METHODS: The microplate identification method was composed of twelve biochemical tests and identification programs. The tests comprised in microplate were initially screened by a two-tube method, NaCl-esculin hydrolysis and pyrrolidonyl-beta-naphthylamide test; arginine dihydrolase, acid production from mannitol, sorbitol, sucrose, arabinose, raffinose, methyl-alpha-D-glucopyranoside, and ribose in the microplate; and pigment production and hemolytic pattern in blood agar plate. The performance of the microplate for identifying enterococci to the species level was evaluated in comparison with conventional reference tests and commercial kits. RESULTS: Among the 111 clinical isolates of Enterococcus species, the microplate system correctly identified 100% to genus level, and 91.0% to species level. All of E. casseliflavus, E. durans, and E. hirae were correctly identified by the microplate. The diagnostic sensitivity and specificity for identification of Enterococcus species were as follows: 100% and 96.7% in E. faecium, 93.5% and 100% in E. faecalis, 100% and 97.2% in E. raffinosus, and 33.3% and 98.1% in both E. avium and E. gallinarum. CONCLUSIONS: It is concluded that the microplate method offers a simple, cost-effective, rapid, and accurate identification system for the identification of most clinical isolates of Enterococcus species.
Subject(s)
Agar , Arabinose , Arginine , Enterococcus , Hydrolysis , Mannitol , Raffinose , Ribose , Sensitivity and Specificity , Sorbitol , SucroseABSTRACT
BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.
Subject(s)
Blotting, Southern , Clone Cells , DNA , Genomic Library , Glycoproteins , Herpes Simplex , Herpesvirus 2, Human , Phosphotransferases , Plasmids , SimplexvirusABSTRACT
BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.
Subject(s)
Blotting, Southern , Clone Cells , DNA , Genomic Library , Glycoproteins , Herpes Simplex , Herpesvirus 2, Human , Phosphotransferases , Plasmids , SimplexvirusABSTRACT
BACKGROUND: The accurate and rapid identification of enterococci can provide clinician's decision making of antimicrobial therapy because enterococci are usually multiresistant to commonly used antimicrobial agents and antimicrobial resistance patterns are different according to enterococcal species. Accuracy of identification system depends mainly on data base such as positive rate of biochemical reactions, relative frequency of occurrence of biotype, and isolation frequency of microorganisms. The purpose of this study was to analyze the isolation rate and biotype frequency of enterococci isolated from clinical specimens. METHODS: We used a simplified identification system for the identification of the enterococci from clinical specimens during the period of June 1998 to November 1998. Biochemical phenotypes of 500 isolates of enterococci were also analyzed by a simplified identification system consisting of eight conventional biochemical tests. RESULTS: Enterococci were isolated from urine (36.4%), wound (35.0%) and blood (7.2%) in order of decreasing frequency. Among the isolates, 67.8% were E. faecalis, 23.0% E. faecium, 2.2% E. hirae/durans, 2.0% E. casseliflavus, and 1.0% E. hirae. The simplified identification system of enterococci identified 93.6% of all isolates to species level. The system identified 98.5% of E. faecalis but only 89.6% of E. faecium. CONCLUSIONS: Our simplified identification system based on eight biochemical tests offer a simple, reliable and economic method for the identification of clinical isolates of enterococci, but further studies are needed for the improvement of accuracy and identification rate.
Subject(s)
Anti-Infective Agents , Decision Making , Enterococcus , Phenotype , Wounds and InjuriesABSTRACT
D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct infection and cytolysis of pancreatic beta cells. cDNA covering the major outer capsid protein (VP1) of EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J male mice, immunized with live recombinant BCG-VP1, became diabetic when challenged with highly diabetogenic EMC-D virus. But the control mice inoculated with normal BCG or rBCG transformed with vector alone developed diabetes in the same challenge. VP1-specific antibodies including neutralizing antibodies were markedly increased as time went on and reached to the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than following 4 weeks. Guinea pigs immunized with the live rBCG-VP1 showed strong delayed type hypersensitivity (DTH) to the VP1of EMC-D virus. It means that the live rBCG-VP1 elicit efficient humoral and cell-mediated imrnune responses against EMC-D virus, resulting in prevention of virus-induced diabetes in susceptible mice.
Subject(s)
Animals , Humans , Male , Mice , Antibodies , Antibodies, Neutralizing , Bacillus , Capsid Proteins , Clone Cells , DNA, Complementary , Guinea Pigs , Hypersensitivity , Immunization , Insulin-Secreting Cells , Mycobacterium bovisABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Herpes Simplex , Simplexvirus , Skin , VaginaABSTRACT
Bovine growth hormone (bGH) gene was expressed in an insect spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into s. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication patters of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern lot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml (106 cells) by radioimmunoassay.
Subject(s)
Baculoviridae , Cell Line , Clone Cells , DNA , DNA, Complementary , Growth Hormone , Insecta , Nucleopolyhedroviruses , Plasmids , Polymerase Chain Reaction , Radioimmunoassay , SpodopteraABSTRACT
The peneicillin binding protein gene(mecA gene) is present in the methicillin-resistant Staphylococcus strains but not in the susceptible ones. The goal of the present study was to establish experimental evidences which might use polymerase chain reaction(PCR) for culture confirmation and eventually clinical diagnosis of methicillin resistant Staphylococcui. Two primers (5'-AAAATCGATGGTAAAGGTTGGC-3', 5'-AGTTCTGCAGTACCGGATTTGC-3') based on the known DNA sequence of the mecA gene from methicillin-resistant Staphylococcus aureus were used in PCRs to screen for the presence of this gene in Staphylococcal isolates from various clinical settings. When the primers were used to copy the DNA of the mecA gene, only 533 base-pair DNA fragment was appeared. The product indicates a positive PCR result for methicillin-resistant Staphylococcal isolates. In contrast, from the DNA of the methicillin-sensitive Staphylococcal isolates the 533bp was not amplified. Results obtained with PCR were generally consistent with those of standard microbiological assays. The mecA gene in methicillin-high resistant Staphylococci was located on the approximately 5.56kb Hind III restriction fragment. The 533bp probe was hybridized to the 5.56kb Hind III restriction fragment of mecA-positive S. aureus. No hybridization was occured in the mecA-negative strain. The mecA gene was cloned, named pHL-1201 and verified by colony hyhridization. The 533bp probe was hybridized to the approximately 5.56kb Hind III restriction fragment of the DNA obtained from pHL-1201. PCRs with the primers successfully distinguished methicillin-resistants from methicillin-susceptible strains of S. aureus and S. epidermidis.
Subject(s)
Base Sequence , Carrier Proteins , Clone Cells , Cloning, Organism , Diagnosis , DNA , Methicillin Resistance , Methicillin-Resistant Staphylococcus aureus , Polymerase Chain Reaction , Staphylococcus aureus , StaphylococcusABSTRACT
Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the peal-pol clone (Lee et al 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind 75 phage promoter and the 6x His region of the pQE-30 expression vector, and it was called pQEVP1. Again, the 6xHis-tagged VP1 DNA fragment in the pOEVPl was cleaved with EcoRl and transferred into the VP1 site of the pBacVPl, resulting pBacHis-VPl recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (Lacz-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay, The recombinant virus was named VP1-HcNPV-1. The 6xHis-tagged VP1 protein produced by the pQEVPl was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was 2.0x10(5) pfu/ml at 7 days postinfection.
Subject(s)
Bacteriophages , Baculoviridae , Blotting, Western , Chromatography , Clone Cells , DNA , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Genome , Inclusion Bodies , Infectious pancreatic necrosis virus , Molecular Weight , Nucleopolyhedroviruses , Recombination, Genetic , RNAABSTRACT
No abstract available.
Subject(s)
DNA , Genome , Nucleopolyhedroviruses , Promoter Regions, GeneticABSTRACT
No abstract available.