To use in the identification of Legionella pneumophila isolates, one Legionella genus-specific, an two L. pneumophila species-specific monoclonal antibodies (MAbs) were produced. Reactivities of the MAbs with Legionella species and non-Legionella strains were tested by immunoblot. MAb 7D8-F1, MAb 7D8-A9, and MAb 1G12-C11 reacted only with protein. MAb 7D8-F1 recognized a genus-specific epitope in the protein ranged from 28 to 34 kDa in the tested 40 species with 61 serogroups. MAb 7D8-A9 and MAb 1G12-C11 reacted strongly with 49 kDa and 70 kDa protein from 18 L. pneumophila serogroups but did not react with 39 non-L. pneumophila species, respectively. In addtion, 17 non-Legionella strains, including Coxiella burnetii, Klebsiella pneumoniae, Mycoplasma pneumoniae, Pseudomonas areoginosa, Francisella tularensis, and Staphylococcus pneumoniae did not show cross-reacivities with the three MAbs. The MAbs reacted with 28 environmemtal isolates and three clinical isolates previously identified as L. pneumophila. When the immunoblot patterns were divided into four types (type I~IV) by using MAb 7D8-F1, all the 31 isolates belonged to the type II. These results indicate that the MAbs were highly specific to Legionella and can be used for the identification of L. pneumophila isolates on the genus and species levels.
Antibodies, Monoclonal* ; Coxiella burnetii ; Francisella tularensis ; Klebsiella pneumoniae ; Legionella pneumophila ; Legionella* ; Mycoplasma pneumoniae ; Pneumonia ; Pneumonia, Mycoplasma ; Pseudomonas ; Staphylococcus

AIMTo explore new agents of quinolone derivatives with high activity against Gram-positive organisms.

METHODSdl-7-(4,4-Dimethyl-3- aminomethylpyrrolidinyl)-quinolones were designed and synthesized, and their activity against Gram-positive organisms was tested in vitro.

RESULTSTen target compounds were obtained. The structures of these compounds were confirmed by 1H NMR, MS. The target compounds with dl-4,4-dimethyl-3-( methyl) aminomethylpyrrolidine side chains had high activity against Gram-positive organisms. Especially the MIC values of compound 22 for 4 strains of Gram-positive resistant bacteria (two strains of MRSA and two of MRSE) were 0.015 -0.5 mg x L(-), which exhibited more potent activities than gatifloxacin (4 - 128 times). Its MIC value for Pseudomonas aeruginosa 03-5 (0.008 mg x L(-1)) was 4 times as that of gatifloxacin (0.03 mg x L(-1)).

CONCLUSIONThe compound 22 showed high activity against Gram-positive organisms in vitro and it is worth of more investigation.

Anti-Bacterial Agents ; chemical synthesis ; pharmacology ; Gram-Negative Aerobic Bacteria ; drug effects ; Gram-Positive Bacteria ; drug effects ; Microbial Sensitivity Tests ; Molecular Structure ; Pseudomonas aeruginosa ; drug effects ; Quinolones ; chemical synthesis ; pharmacology ; Staphylococcus epidermidis ; drug effects

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.
Bordetella ; Bordetella bronchiseptica ; Bordetella parapertussis ; Bordetella pertussis ; Discrimination (Psychology) ; DNA ; DNA, Ribosomal ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Whooping Cough

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.
Bordetella ; Bordetella bronchiseptica ; Bordetella parapertussis ; Bordetella pertussis ; Discrimination (Psychology) ; DNA ; DNA, Ribosomal ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Whooping Cough

Animals ; Animals, Domestic ; China ; epidemiology ; Coinfection ; Coxiella burnetii ; Francisella tularensis ; Tick-Borne Diseases ; epidemiology ; transmission ; veterinary

No abstract available.
Achromobacter denitrificans/isolation & purification ; Alcaligenes/isolation & purification ; Bordetella Infections/*microbiology ; Bordetella bronchiseptica/isolation & purification ; Crush Injuries/*microbiology ; Fractures, Bone/*microbiology ; Humans ; Male ; Middle Aged ; Surgical Wound Infection/*microbiology ; Tibial Fractures/microbiology

BACKGROUND: Recently a novel plasmid-mediated resistant mechanism that conferred high-level resistance to aminoglycoside via methylation of 16S rRNA was reported. The aims of this study were to determine the prevalence of the 16S rRNA methylase genes and to characterize the coresistance to other antibiotics in Gram-negative bacilli. METHODS: Consecutive non-duplicate Gram-negative bacilli were isolated from clinical specimens at a Korean secondary- and tertiary-care hospital from July 2006 to June 2007. The antimicrobial susceptibility was tested by the CLSI agar dilution method,and PCR was performed to detect the 16S rRNA methylase genes in the arbekacin-resistant isolates. RESULTS: In Gram-negative bacilli, the proportions of 16S rRNA methylase gene-positive isolates were 5% (75/1,471) in the secondary-carehospital and 4% (48/1,251) in the tertiary-care hospital, and the positive rates by species were 1% Escherichiae coli 16% (10/1,062), Klebsiella pneumoniae 16% (75/460), K. oxytoca 2% (1/44), Citrobacter spp. 9% (7/82), Enterobacter spp. 2% (4/181), Serratia marcescens 6% (6/100), Proteus miriabilis 4% (2/57), Achromobacter xylosoxidans 20% (1/5), Pseudomonas aeruginosa < 1% (1/505), Acinetobacter spp. 10% (11/112), and Stenotrophomonas maltophilia 2% (1/66), respectively. Among 16S rRNA methylase-positive isolates from secondary- and tertiary-care hospitals, 93% (70/75) and 90% (43/48), respectively, were armA positive, and others, except one rmtA positive isolate, were positive for the rmtB gene, according to PCR results. The rates of ESBL-positive and cefoxitin-resistant K. pneumoniae were 59% and 92%,s respectively. In addition, 91% of 16S rRNA methylase-producing K. pneumoniae were positive for qnrB. There were no MBL producers among 16S rRNA methylase-producing Pseudomonas and Acinetobacter species. CONCLUSION: The novel aminoglycoside-resistant mechanisms involving16S rRNA methylase were prevalent and widely distributed among Gram-negative bacilli in Korea, and other resistance mechanisms were commonly associated with 16S rRNA methylase-mediated resistance in Korea.
Achromobacter denitrificans ; Acinetobacter ; Agar ; Anti-Bacterial Agents ; Citrobacter ; Enterobacter ; Escherichia ; Klebsiella pneumoniae ; Korea ; Methylation ; Methyltransferases ; Pneumonia ; Polymerase Chain Reaction ; Prevalence ; Proteus ; Pseudomonas ; Pseudomonas aeruginosa ; Serratia marcescens ; Stenotrophomonas maltophilia

OBJECTIVETo study the types of subspecies of Francisella tularensis from China and to investigate the genetic relationships between F. tularensis strains from China and from other countries.

METHODSTen strains of F. tularensis isolated from China were amplified by using typing primers C1/C4 and RD1. On the basis of the lengths of the polymerase chain reaction (PCR) products, it was concluded that these strains of F. tularensis belonged to the same subspecies. At the same time, the fopA, tul4, and 16S rRNA genes of the 10 strains were amplified, and a three-gene based phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis software version 4.0.

RESULTSThe 10 strains of F. tularensis from China were all identified as belonging to subspecies holarctica (type B). We found no direct relationship between the genotypes of F. tularensis subsp. holarctica and the geographical area from where they were isolated.

CONCLUSIONThe F. tularensis strains isolated from North China mainly belong to subspecies holarctica (type B). The strains of F. tularensis subsp. holarctica from China may have evolved earlier than those from Europe and North America.

China ; Francisella tularensis ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

Aims: This study aimed to isolate and evaluate the indigenous fluorescent Pseudomonas spp. with bio-control potential against Rhizoctonia solani and promoting growth in chilli seedlings. Methodology: A total of 120 fluorescent bacterial were isolated from the healthy chilli rhizosphere soil from the seven major chilli cultivation localities in Terengganu, Malaysia. Only 115 Gram negative fluorescent isolates were further invitro screened for antagonistic activities against R. solani and plant growth-promoting properties. The 50 most effective fluorescent Pseudomonads antagonist against R. solani with minimum percentage inhibition of radial growth (PIRG) of 65% were selected. Hierarchical cluster analysis was further conducted with two dendrograms derived from SPSS Statistic 20 to facilitate the comparison between these 50 isolates for antagonistic and growth-promoting properties. A total of 40 fluorescent isolates within the most potential cluster were further selected and identified using 16S rRNA sequencing. Thirty four fluorescent isolates were identified as Pseudomonas spp. and six isolates as Burkholderia spp. The top 13 ranked fluorescent Pseudomonas spp. from the scoring index were evaluated for seed germination and vigor index in chilli seedlings. There was no significant difference in germination rate between fluorescent Pseudomonas inoculated with control. However, vigor index of chilli seeds pre-inoculated with fluorescent P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) were significantly increased with 4684.9, 4657.3 and 4401.0 over control (P ≤ 0.05). Conclusion, significance and impact of study These selected fluorescent isolates: P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) have the potential to be developed as biofungicide against R. solani and as growthpromoter in chilli production system.
Pseudomonas fluorescens ; Rhizoctonia ; Seedlings

PURPOSE: Study on the pathogen and the pathogen-related disease require the information at both cellular and organism level. However, lack of appropriate high-quality antibodies and the difference between the experimental animal models make it difficult to analyze in vivo mechanism of pathogen-related diseases. For more reliable research on the infection and immune-response of pathogen-related diseases, accurate analysis is essential to provide spatiotemporal information of pathogens and immune activity to avoid false-positive or mis-interpretations. In this regards, we have developed a method for tracking Francisella tularensis in the animal model without using the specific antibodies for the F. tularensis. MATERIALS AND METHODS: A dual reporter plasmid using GFP-Lux with putative bacterioferritin promoter (pBfr) was constructed and transformed to F. tularensis live vaccine strain to generate F. tularensis LVS (FtLVS)-GFP-Lux for both fluorescence and bioluminescence imaging. For vaccination to F. tularensis infection, FtLVS and lipopolysaccharide (LPS) from FtLVS were used. RESULTS: We visualized the bacterial replication of F. tularensis in the cells using fluorescence and bioluminescence imaging, and traced the spatio-temporal process of F. tularensis pathogenesis in mice. Vaccination with LPS purified from FtLVS greatly reduced the bacterial replication of FtLVS in animal model, and the effect of vaccination was also successfully monitored with in vivo imaging. CONCLUSION: We successfully established dual reporter labeled F. tularensis for cellular and whole body imaging. Our simple and integrated imaging analysis system would provide useful information for in vivo analysis of F. tularensis infection as well as in vitro experiments, which have not been fully explained yet with various technical problems.
Animals ; Antibodies ; Fluorescence ; Francisella tularensis* ; Francisella* ; Immunodeficiency Virus, Feline ; In Vitro Techniques ; Methods ; Mice ; Models, Animal ; Plasmids ; Vaccination ; Whole Body Imaging