The Molecular Epidemiology And Fitness Of Penicillin-resistant Streptococcus Pneumoniae

BackgroundStreptococcus pneumoniae is a common pathogen of pneumonia, otitis media, meningitis and b. It is estimated thateach year approximately14.5million episodes of serious pneumococcal disease occur in the world, while1.7million episodes happen in China. Antimicrobial resistance of Streptococcus pneumonia is continually increasing. In Shanghai, maximum MIC of penicillin is1μg/ml in1996-1999,4μg/ml in2004-2005and more than8μg/ml in2008-2009. Clonal spread of International resistant Clonesplayed a great role in the increase of resistance of Streptococcus pneumoniae.PurposeTo analyze the prevalence trend of resistant Streptococcus pneumoniae clones by serotyping and multilocus sequence analysis (MLST) of penicillin-resistant Streptococcus pneumonia (PRSP) isolated in2011. To compare the fitness between international resistant clones and Shanghai native resistant clone in the condition with or without antibiotic selective pressure, so as to explain the reason of prevalence trend and deduce the future prevalenct clone.Method1. Molecular epidemiology of PRSP Twenty-six PRSP were from SOAR (Streptococcus pneumonia susceptibility surveillance program) in2011. Among them,22were isolated from Shanghai Children’s Hospital,2isolated from the first affiliated hospital of Zhejiang University,2isolated from Beijing Union Medical College Hospital and1isolatd from people’s hospital of Jiangxi province). All isolates were collected from sputum and defined as PRSP according to the standard for oral penicillin and nonmeningitis isolates of2011the Clinical Laboratory Standards Institute (CLSI)(susceptible≤0.06μg/ml, intermediate0.12-1μg/ml, resistant≥2μg/ml). The strains were reidentified by colony morphology characteristics and bile solubility test method. Susceptibility to penicillin and amoxicillin were measured by microdilution method. Serotyping of strains was performed by polymerase chain reaction (PCR). MLST was performed according to methods previously described (6,10). STs obtained were compared to those at the MLST website. Sibship between clonal complex and strain were analyzed by eBURST software.2. Evaluation of fitness of pneumococcal resistant clones Three Spain23F-1strains,1Taiwan19F-14strains and4Shanghai native resistant clonal strains were selected from the26PRSP. Amoxicillin and amoxicillin/clavulanic acid are commonly-used antibiotics in the treatment of pneumococcal infections, especially paediatric respiratory tract infection. Therefore, amoxicillin was selected to evaluate antibiotic selective pressure. Time-killing curves were drawed with colony count method (including blank control,1/2MIC,1MIC,2MIC,4MIC and8MIC drug concentration). According to growth curves of blank control (in condition of no selective pressure) and1/2MIC drug concentration (in condition of subinhibitory concentration), growth kinetics parameters (generation time) was calculated. Fitness was compared between international resistant clone isolates and Shanghai native resistant clone isolates under no selective pressure and subinhibitory concentration. Fitness under selective pressure of amoxicillin above MIC concentration was compared by the time and concentration to achieve99.9%bactericidal rate.3. Evaluating fitness of Streptococcus pneumonia in pharmacokinetic and pharmcodynamic (PK/PD) model One ST320strain and1ST271strain of Taiwan19F-14clone, and1Shanghai native resistant clone strain were selected. HPLC-MS-MS methodology of amoxicillin concentration was established and validated in order to validate the PK/PD model. Through PK/PD simulation, dynamic time-killing curves was drawed and pharmacodynamic index was calculated. The rate of elimination of bacteria (Ke) and the area above the log NA-t curve and under the baseline drawn at the level of NA equal to N0(AAC) reflected the fitness of Streptococcus pneumoniae under antibiotic selective pressure, and difference value between ABBC0-24h and AAC0-24h reflected growing ability of Streptococcus pneumoniae without antibiotic selective pressure.Result1. Molecular epidemiology of PRSP The main serotypes of26PRSP were19F(26.9%),19A(23.1%),14(15.4%) and23F(11.5%), while6isolates weren’t typed by PCR method. It indicated that46.2%(12/26) of tested strains can not be covered by7-valent protein conjugate vaccine (PCV7). Ten MLST STs (ST81, ST243, ST271, ST320, ST876,, ST1464, ST2971, ST7963, ST7962and ST7964) were identified, among which ST7962, ST7963and ST7964were new assignments. Eighteen of26isolates of tested strains belong to4international resistant clones (Taiwan19F-14/CC320, Taiwan23F-15/CC242, Spain23F-1/CC81and Netherlands15B-37/CC199). A Shanghai native resistant clone is discovered (2ST2971and4ST7964strains).2. Evaluation of fitness of international resistant clone and Shang-hai native resistant of streptococcus pneumoniae. The mean generation time without antibiotic selective pressure was spain23F-1(0.2524h)<Taiwan19F-14(0.2821h)<Shanghai native resistant clone(0.3834h). Generation time in1/2MIC amoxicillin concentration was spain23F-1(0.3504h)<Taiwan19F-14(0.3990h)<Shanghai native resistant clone(0.5115h). The time to achieve99.9%bactericidal rate of Shanghai native clone was4h or6h, while that of international resistant clones was2h or4h.3. Evaluating fitness of Streptococcus pneumonia in pharmacokinetic and pharmcodynamic (PK/PD) model In amoxillin regimen of500mg po q8h, ST2971were not inhibited, and Ke of ST320(0.0677/h)<Ke of ST271(0.117/h). In amoxillin regimen of1000mg po q8h, Ke of ST2971(0.0418/h)<Ke of ST320(0.0872/h)<Ke of ST271(0.0890/h). In both regimens, the AAC0-24h of ST271, ST320and ST2971decreased successively.Conclusion1. Clonal spread of resistant clones played a great role in the increase of resistance of Streptococcus pneumoniae. Accompanying the spread, resistant clones were constantly evolving through recombination of DNA. Serotype19A accounted for a high proportion in PRSP, therefore in China pneumococcal vaccine should cover serotype19A.2. The fitness of spain23F-1, Taiwan19F-14and Shanghai native resistant clone decresed successivelv in the condition without antibiotic selective pressure or under selective pressure of subinhibitory concentration of amoxicillin (1/2MIC). The fitness of Shanghai native clone is better than that of spain23F-1and Taiwan19F-14under selective pressure of2MIC amoxicillin concentratiom, which implied that the Shanghai native resistant clone prevailed by obtaining growth advantage under antibacterial selective pressure and.3. PK/PD model which simulate pharmacokinetics of amoxicillin in regimen500mg po q8h and1000mg po q8h was established to the fitness of Taiwan19F-14(ST320and ST271) and Shanghai native resistant clone(ST2971) in both regimens. According to the elimination rate and AAC0-24h, the fitness of Shanghai native resistant clone was better than that of Taiwan19F-14. Among Taiwan19F-14, The fitness of ST320was higher than that of its Single Locus Variant (SLV) ST271under selection pressure in both regimens. In amoxicillin regimen of500mg q8h, T>MICs of3strains were all less than30%; In amoxicillin regimen of1000mg q8h, T>MIC of the2Taiwanl9F-14clone strains were close to40%. However, Shanghai native resistant clone (ST2971) was less than20%. To inhibit the spread of Taiwan19F-14clone, the amoxicillin regimen of1000mg q8h should be recommended.4. Shanghai native resistant clones were all isolated from children and high-level resistant to penicillin (MIC4～12μg/ml). In the amoxicillin regimen of both500mg q8h and1000mg q8h, the fitness of Shanghai native resistant clone was better than that of international resistant clone spain23F-1and Taiwan19F-14. Its serotype was uncovered by now-available pneumococcal polysaccharide or conjugate vaccine. Therefore, it is likely to obtain advantage of prevalence under dual selective pressure of antibiotics and vaccine, and should be closely monitored.