| Staphylococcus aureus is clinically important pathogenic bacteria, is the hospital infection is one of the main pathogenic bacteria community infection common pathogenic bacteria. The pathogenic can cause skin and soft tissue pyogenic infection, also can cause pneumonia, arthritis, osteomyelitis, endocarditis, bacteremia and severe infection such as meningitis.For a long time, the identification of microorganisms pathogenic and resistance analysis relay on the culture of phenotypic survey method. Traditional culture method operation trival and take a long time. And for those who raise request demanding or unable to culture of bacteria, the culture method is disadvantage. The disadvantage of the culture method, not only delay the diagnosis of the disease, but also led to experience administration and rational use of antibiotics. According to shortages of the cultivation, this study aims to establish molecular biology fast detection clinical infectious staphylococcus aureus and analysis of the resistance gene.Part1Fluorescence in situ hybridization rapidly detects Staphylococcus aureusBackgroud and Objective:Establish a fluorescence in situ hybridization method of rapid detection Staphylococcus aureus, and evaluation of this method in Staphylococcus aureus infections caused with different specimens.Methods:The probe specific for Staphylococcus aureus were designed based on the conserved16SRNA sequences. All strains were test by FISH test and the traditional culturing techniques.Results:By using fluorescence in situ hybridization (FISH) with rRNA-targeted, fluorescently labeled oligonucleotide probes, Staphylococcus aureus pathogens present in clinical samples were identified within3-4h. Compared to conventional methods of bacterial identification, the FISH method produced positive results for>95.0%of the samples tested. Conclusions:FISH method has the potential to become an extremely useful diagnostic tool for Staphylococcus aureus infections because it has a quick turnaround time and high accuracy. Part2Rapid Identification of Staphylococcus aureus:Comparison between FISH and PCR methodsBackgroud and Objective:Staphylococcus aureus (S. aureus) is the most important pathogen in the genus Staphylococcus. The ability to rapidly and accurately distinguish between S. aureus and non-S. aureus bacteria (coagulase-negative staphylococci [CoNS]) is essential for the appropriate therapeutic use of antibiotics and the timely intervention for infection control. Several methods have been reported as effective in the rapid identification of S. aureus, but molecular biology methods have the most potential.Methods:In this study, two molecular techniques—fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR)—were compared. We considered1300clinical specimens in this study. After smear test, the specimens containing gram-positive cocci in clusters were investigated. These specimens had subjected to FISH test and PCR examination simultaneously. In all,131specimens to be members of the Staphylococcus genus were taken into statistics. We compared the effectiveness, efficiency and costliness of the two methods.Results:Comparing the culture determination methods revealed that the identification sensitivity, specificity, and the positive and negative predictive values were100.0%,100.0%,100.0%and100.0%, respectively, for the FISH method and98.5%,100.0%,100.0%and98.5%for the PCR method, respectively. The FISH method took approximately3h, while the PCR method took approximately4h. Therefore, S. aureus was differentiated from CoNS by the FISH method1h faster than by PCR identification. FISH is more appropriate for testing a few specimens, while PCR is more appropriate for testing a large amount of specimens at a lower cost.Conclusions:The FISH and PCR methods both allow for the rapid and reliable identification of S. aureus in clinical specimens. Part3Drug-resistance of Staphylococcus aureus strains and analysis for the resistance spectrum phenotype and related genesBackgroud and Objective:To investigate the clinical distribution and drug-resistance characteristics of staphylococcus aureus strains isolated from patients.Methods:We investgated the the drug resistance rate of Staphylococcus aureus isolates from2000to2011. We analysed the drug resistance trend of Staphylococcus aureus through retrospective analysis method.Saphylococcus aureus isolated from clinical specimens was identified and antimicrobial susceptibility tested by BD all-automatic microbiology analysis system. Resistance genes were detected by PCR method.Results:Totally1066Staphylococcus aureus strains were isolated. The infection is mainly distributed in brain surgery and respiratory medicine, especially the two department of intensive care unit; The mainly types of infection specimens were sputum, urine, wound secretion and blood specimen.The resistant rate of penicillin, ampicillin, erythromycin, oxacillin, cipro and gentamicin are more than50%. We have not yet found Staphylococcus aureus strains resistan to tetracycline, linezolid and vancomycin. To chloramphenicol and tetracycline the drug resistant reduced. But in recent years, the drug resistant to cipro and gentamicin were relative increase; The resistant rate of azole schering rose to58.5%in2011. There was little change in other antimicrobial drugs.For the resistance gene detection of76cases of staphylococcus aureus。The number of penicillin resistance gene (blaZ) positive is68. The number of oxacillin gene (mecA) positive is68.The number of erythromycin and clindamycin resistance genes (ermA, ermB, ermC and msrA) is31; The number of tetracycline resistance genes (tetK and tetM) is27. The number of gentamicin resistance gene (aac (6’)/aph (2")) is22.Conclusions:The drug resistance rate to oxacillin, macrolides, and aminoglycoside resistant were high, and offen present multiple drug resistance; The resistant genes (mecA, ermA, ermB, ermC, tetK, tetM, msrA, blaZ, aac(6’)-aph(2")) detected in this study showed high coincidence rates with resistant spectrum of Staphylococcus aureus (>60%). |
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