| Streptococcus pneumoniae is the leading cause of community-acquired pne-umonia, and also an important cause of otitis media and meningitis. For decad-es, the treatment of pneumococcal infections relied primarily on ?-lactam and macrolide agents. However, with the emergence and spread of ?-lactam-and m-acrolide-resistant strains, new fluoroquinolones, because of their anti-pneumococ-cal activities, have become essential in providing effective treatment options; However, with the increased clinical application, fluoroquinolone-resistant stains have emerged and increased. In recent years, during the study of the resistance mechanisms of S. pneumoniae, biofilm formation has absorbed more and more attention, which is closely related to colonization and repeated infections of S. pneumoniae. Biofilm not only can be tolerated to antibiotics, but also evade th-e host immune system.Our objective was to to investigate the antimicrobial resistance evolution of S. pneumoniae isolates in Nanjing and the resistance mechanisms of fluoroqui-nolones; Morever, we will study of the biofilm formation of S. pneumoniae an-d related gene expression. Three parts of this research are as following.1. Antimicrobial resistance evolution of clinical isolates of St-reptococcus pneumoniae in NanjingObjective To investigate the antimicrobial resistance evolution of S. pneu-moniae isolates in Nanjing and guide the rational use of antimicrobial drugs.Methods Clinical isolates of S. pneumoniae were collected from 7 teachi-ng hospitals in Nanjing from 2010 to 2012.The minimum inhibitory concentrat-ions (MICs)of 9 antimicrobial agents were determined by agar dilution method. Statistical methods were used to compare the antibiotic resistance with S. pne-umoniae isolates collected in 2006-2007.Results Of the 147 S. pneumoniae strains,89.1% were resistant to erythr-omycin. The rate of penicillin non-susceptible S. pneumoniae was 3.4% (MIC ?4?g/ml).26.5%?1.4%?3.4%?1.4% and 0.7% of these isolates were resistant to cefuroxime, ceftriaxone, meropenem, levofloxacin and moxifloxacin respectiv-ely. All strains were sensitive to vancomycin and linezolid. Compared to the is-olates collected in 2006-2007, the rate of resistant isolates collected in 2010-2 012 was lower in penicillin and cefuroxime, similar in erythromycin and ceftria-xone. But isolatees resistant to levofloxacin and moxifloxacin emerged in 2010-2012.Conclusion The S.pneumoniae isolates collected in Nanjing are still highl-y resistant to erythromycin, and the cefuroxime resistance rates also can't be i-gnored. Most of S. pneumoniae isolates are sensitive to penicillin, ceftriaxone and meropenem. There are levofloxacin and moxifloxacin-resistant strains in the study. We find no isolates resistant to vancomycin and linezolid.2. Mutation in QRDR of Streptococcus pneumoniae resistant to fluoroquinolonesObjective To investigate fluoroquinolone resistance of S. pneumoniae isol-ates in Nanjing and detect the gene mutations in QRDR.Methods 147 Streptococcus pneumoniae strains were isolated at 7 teachin-g hospitals in Nanjing from 2010 to 2012, and the MICs of levofloxacin and moxifloxacin were determined by agar dilution method. QRDR in gyrA, gyrB, parE and parC genes of levofloxacin or moxifloxacin-resistant strains were anal-yzed by gene amplification and sequencing.Results Two strains were resistant to levofloxacin (MIC 32pg/ml), one of which was also moxifloxacin-resistant strain (MIC 4?g/ml).The strain resistant to levofloxacin and moxifloxacin had gyrA mutation (Asn 167-Ile) and parE mutation (Ile460-Val); with the other having parC mutation (Ser81-Gly); Asn 94-Asp). the rest gens had some point mutations, without leading to amino ac-id mutations.Conclusion Fluoroquinolone-resistant Streptococcus pneumoniae emerged in Nanjing. The fluoroquinolone-resistant strains had gene mutations in target p-osition, with gyrA, parE and parC mutations in QRDR.3. The relationship between Streptococcus pneumoniae biofil-m formation and MIC of penicillinObjective To investigate the biofilm formation of Streptococcus pneumoni-ae and the relationship with minimum inhibitory concentrations(MIC)of penicill-in.Methods A total of 147 clinical isolates of S. pneumoniae were collected from 7 teaching hospitals in Nanjing from 2010 to 2012. MIC of penicillin w-ere determined by agar dilution method. We selected randomly S. pneumoniae with various penicillin MIC, as follow:MIC?0.065?g/ml?0.5?g/ml?2?g/ml?? 4?g/ml, which would be incubated to form biofilms in 96-well plates and 24-well plates for 24 hours. Then we measured the OD values at 570nm and obs-erved the biofilm through confocal laser scanning microscope.Results The biofilm semi-quantitative detection and CLSM both displayed all strains could form biofilms. The OD value of the group whose MIC was ?0.065?g/ml was higher than the group(MIC=0.5?g/ml) and the group(MIC?4?g /ml) (P<0.05), and there was no difference among the groups which MIC we re 0.5?g/ml?2?g/ml and ?4?g/ml respectively(P>0.05).Conclusion S. pneumoniae can form biofilms. Streptococcus pneumoniae with high penicillin MIC had a trend of weakened biofilm formation.In conclusion, The S.pneumoniae isolates collected in Nanjing are still hig-hly resistant to erythromycin, and the cefuroxime resistance rates also can't be ignored. But most of S. pneumoniae isolates are sensitive to Penicillin, ceftria-xone and Meropenem. There are no isolates resistant to vancomycin and linez-olid. There emerged moxifloxacin and levofloxacin-resistant strains in our study, and gene sequencing in QRDR showed mutations of parE, parC and gyrA, w-hich needs to arouse the attention of the clinicians. S.pneumoniae isolates unde-r the study can form biofilms. Streptococcus pneumoniae with high penicillin MIC had a trend of weakened biofilm formation. |
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