Molecular Identification Of Coagulase Negative Staphylococcus And The Study Of Staphylococcus Lugdunensis And Staphylococcus Saccharolyticus

Chapter1Rapid molecular identification of Coagulase Negative StaphylococcusObjective To establish the rapid molecular diagnosis of Coagulase NegativeStaphylococcus which were encountered frenquently in clinical infection. MethodsDNA sequencing of15typies of CNS were obtained with gap gene. After the alignmentgap gene sequences, the bacteria were identified with homological alignment comparedwith the16SrRNA gene. Results The sequence similarity of the gap ranged from39%to94%. The sequence similarity of the16SrRNA ranged from96%to99%, at least twospecies of bacteria possessed similar rate of99%and the most four species had similarrate of99%. Phylogenetic homology analysis showed high confidence(99%) in thedetection of S.xylosus, S.lentus, S.chromogenes and S.intermedius, but for other speciesof bacteria,gap homology analysis has less unreliable confidence(42%,56%) and16SrRNA has more unreliable confidence(41%,44%,51%,55%,64%). ConclusionAnalysis of gap sequence could identify15different kinds of CNS timely andaccurately with higher confidence than16SrRNA.Chapter2Experimental study of Staphylococcus lugdunensisObjective To study the prevalence and rate of drug resistance of Staphylococcuslugdunensis. Methods670isolates of CNS were tested with ornithine decarboxylase(ODC) and pyrrolidonyl arylamidase (PYR) firstly. Isolates positive for both wereidentified with API20Staph and VITEK2gram-positive (GP) kits, followed by partialsequencing of gap gene. Susceptibility testing was performed by K-B method and thedrug-resistance genes ermA, ermB, ermC and mecA were amplified by PCR. Amolecular epidemiological analysis was performed using PFGE. Results Five strains ofS.lugdunensis were identified with a detection rate of0.7%. Three isolates were resistant to erythromycin, clindamycin and penicillin and carried the ermC gene, onewas resistant to cefoxitin and penicillin and carried the mecA gene, and only one isolatewas not resistant to any of the tested antimicrobials. A PFGE cluster dendrogramrevealed that two isolates with a similarity of96.6%were obtained from the Departmentof Orthopedics,two isolates with a similarity of96.0%were obtained from a patientwith premature rupture of fetal membranes and a neonatal patient, and one isolate with asimilarity of below87.3%to the other isolates was obtained from an outpatient.Conclusions The detection rate for S.lugdunensis was low. Drug resistance was severeand predominantly presented as multi-drug resistance, PFGE analysis helped revealepidemiological characteristics of S.lugdunensis.Chapter3Staphylococcus saccharolyticus isolated and identified from bone marrowObjective To establish the rapid molecular diagnosis of Staphylococcus saccharolyticus.Methods Wash/alkali/heat lysis methods were used to extract DNA from positive bloodculture bottles, and gap gene was amplified and sequenced. After the alignment of gapgene sequences,this bacteria was identified by phylogenetic analysis. Antimicrobialsusceptibility was done by E-Test. Results gap gene sequencing allowed undoubtedidentification of the isolate as S.saccharolyticus since the maximum of homology wasobserved with S.saccharolyticus99%and the bootstraping was100%. Antimicrobialsusceptibility testing by E-test method showed that the MICs of vancomycin,penicillin,clindamycin,levofloxacin and metronidazole were1.5μg/mL,0.002μg/mL,0.023μg/mL,0.094μg/mL and256μg/mL respectively. Conclusion Analysis of gap sequence andextraction of DNA by using Wash/alkali/heat lysis could identify S.saccharolyticustimely and accurately.