Drug Resistance, Bacterial ; genetics ; Food Microbiology ; Gram-Negative Bacteria ; genetics ; isolation & purification ; Integrons ; Japan ; Seafood ; microbiology

Bacteriolysis ; physiology ; Bacteriophages ; genetics ; isolation & purification ; physiology ; England ; Gram-Negative Bacteria ; virology ; Humans ; International Cooperation

OBJECTIVETo establish a new method of multiplex semi-nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF).

METHODSAccording to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negative bacteria. All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time.

RESULTSBoth gram-positive and gram-negative bacteria amplified DNA fragment about 1,032 bp after first-step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram-positive and gram-negative bacteria respectively besides fragments of 1,032 bp; The detection limit for E. coli was 8 cfu/ml. The comparison of 62 CSF samples detected by both multiplex semi-PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8%, 95.7%, 88.2%, and 97.8% respectively for PCR.

CONCLUSIONThe result suggested that the multiplex semi-nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.

Cerebrospinal Fluid ; microbiology ; DNA Primers ; genetics ; DNA Probes ; genetics ; DNA, Bacterial ; cerebrospinal fluid ; isolation & purification ; Escherichia coli ; genetics ; isolation & purification ; Gram-Negative Bacteria ; genetics ; isolation & purification ; Gram-Positive Bacteria ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity ; Staphylococcus aureus ; genetics ; isolation & purification

BACKGROUND: Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. METHODS: Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. RESULTS: Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. CONCLUSIONS: The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.
Automation, Laboratory ; Candida albicans/genetics/*isolation & purification ; Disk Diffusion Antimicrobial Tests ; Gram-Negative Bacteria/genetics/*isolation & purification ; Gram-Positive Bacteria/genetics/*isolation & purification ; Humans ; RNA, Ribosomal, 16S/chemistry/genetics ; Retrospective Studies ; Sequence Analysis, RNA ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

No abstract available.
Aged ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Cefotaxime/analogs & derivatives/therapeutic use ; Cholangiopancreatography, Endoscopic Retrograde ; Gallstones/surgery ; Gram-Negative Anaerobic Bacteria/drug effects/genetics/*isolation & purification ; Gram-Negative Bacterial Infections/*diagnosis/drug therapy/microbiology ; Humans ; Male ; Metronidazole/therapeutic use ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Sequence Analysis, DNA ; Tomography, X-Ray Computed

No abstract available.
Anti-Bacterial Agents/*pharmacology ; Azabicyclo Compounds/*pharmacology ; Bacterial Proteins/genetics ; Ceftazidime/*pharmacology ; Cephalosporins/*pharmacology ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Bacterial/*drug effects ; Gram-Negative Bacteria/drug effects/*isolation & purification ; Humans ; Microbial Sensitivity Tests ; Penicillanic Acid/*analogs & derivatives/pharmacology ; Pseudomonas aeruginosa/drug effects/isolation & purification ; Real-Time Polymerase Chain Reaction