儿童肺炎链球菌耐药性与pbp2b基因RFLP和PFGE图型分析

目的　监测肺炎链球菌对青霉素和其他抗生素的耐药情况 ;分析pbp2b基因扩增产物图谱与青霉素耐药性的相关性 ;对 3 1株青霉素耐药菌株进行血清分型 ;分析血清型 2 3F和血清型 6的青霉素耐药菌株的脉冲电场凝胶电泳 (PFGE)图型 ,初步了解北京地区耐药菌株分子流行病学上的特点。方法　 ( 1)抗生素药物敏感试验 ;( 2 )用聚合酶链反应 (PCR)和限制性内切酶片段长度多态性分析 (RFLP)方法 ,分析pbp2b基因扩增产物图谱与青霉素耐药性的相关性 ;( 3 )使用乳胶凝集的方法 ,对 3 1株青霉素耐药菌株进行血清分型 ;( 4 )使用PFGE图型方法分析 6株血清型 2 3F和 3株血清型 6的青霉素耐药菌株流行病学上的基本特性。结果　 ( 1)对青霉素的耐药性由 1997年 9 9%上升到 2 0 0 0年 14 6% (P >0 0 5)。对红霉素、复方磺胺甲基异唑和氯霉素耐药性分别由 1997年76 8%、74 7%和 2 2 6%上升到 2 0 0 0年的 87 4%、88 3 %和 40 8% (P <0 0 5) ;( 2 )通过PCR和RFLP方法分析青霉素耐药菌株和青霉素敏感菌株表明 ,pbp2b基因扩增图谱与青霉素耐药性之间有较好的相关性 ;( 3 )对 3 1株青霉素不敏感肺炎链球菌 (PRSP)做了血清型 2 3F和 6分型。前者为 6株 ( 19% ) ;后者为 3株 ( 9% )。 6株血清型 2 3F肺炎链球菌均为低水平     

北京地区上呼吸道感染儿童青霉素不敏感肺炎链球菌的血清型分布研究

目的 了解青霉素不敏感肺炎链球菌（PNSP）的耐药性和血清型分布。方法 以2000～2004年门诊上呼吸道感染的0～5岁患儿鼻咽部分离的肺炎链球菌为研究菌株,采用E-test法检测青霉素最低抑菌浓度,简易棋盘式分型系统进行血清分型。结果 检出PNSP菌株129株,其中中介102株,耐药27株;2000～2004年各年耐药株占PNSP比率分别为19．0％、8．0％、17．6％、30．0％和26．1％;常见血清型共有124株（96．1％）,为19群、23群、不能分型、6群和14型;7价结合疫苗覆盖率从2000年到2004年分别为57．1％、76．0％、88．2％、75．0％和82．6％。结论 北京儿童中分离的PNSP的青霉素耐药性有所增强;7价结合疫苗覆盖率升高,有利于应用疫苗来预防PNSP的携带、感染及耐药性的进一步播散。     

儿童呼吸道感染肺炎链球菌耐药性及pbp2B与TEM基因的研究

目的：了解广州地区儿童呼吸道感染肺炎链球菌(Streptococcus pneumoniae,SP)的耐药情况以及SP中青霉素耐药相关基因TEM与pbp2B的流行分布及突变情况。方法：采用E-test和K-B纸片法对44株SP分离株进行药敏试验;PCR扩增SP中的TEM基因及pbp2B,并对pbp2B基因进行测序,结果与SP青霉素敏感株R6进行序列比对分析。结果：①44株SP对青霉素的敏感率仅为11.4％,不敏感率高达88.6％。对红霉素耐药率已达100％,对克林霉素、复方新诺明的耐药性也在90％以上。但对头孢曲松、阿莫西林、亚胺培南仍敏感,耐药率分别为0,2.6%和3.9%。未发现对氧氟沙星、万古霉素耐药菌株。②44株SP的pbp2B基因扩增序列与R6敏感株相比较,5株青霉素敏感株99%以上的核苷酸序列相同,未发生氨基酸的替换。39株青霉素不敏感株均发生核苷酸序列的改变,核苷酸序列突变率为13.2％～23.1%,约6.5％～10.9%的氨基酸发生了替换。根据氨基酸在Ser391-Thr492片段之间的突变情况,可将39株青霉素不敏感株分为四型,其中Ⅰ型突变30株,Ⅱ型突变7株,Ⅲ型和Ⅳ型各1株。44株SP均未检出TEM型β-内酰胺酶耐药基因。结论：广州地区儿童呼吸道感染SP多重耐药情况较严重,青霉素、红霉素已不适宜作为SP感染的临床一线用药,阿莫西林及第三代头孢菌素可作为SP感染的经验用药。pbp2B基因突变是广州地区儿童SP对青霉素耐药的主要机制之一。［中国当代儿科杂志,2009,11（8）：623-626］     

2000-2002年三家儿童医院分离的肺炎链球菌血清型分布及其对β内酰胺类抗生素敏感性的变化

目的 了解当前我国儿童人群中肺炎链球菌血清型分布,及不同血清型菌株对B内酰胺类抗生素的敏感性变化,评估疫苗在预防肺炎链球菌感染及控制其耐药性流行中的价值。方法 以2000-2002年于北京、上海和广州三家儿童医院门诊分离的625株肺炎链球菌为研究对象,应用简易棋盘式肺炎链球菌分型系统检测血清型,分析肺炎链球菌7价结合疫苗（4、6B、9V、14、18C、19F和23F）覆盖率;采用E—test最小抑菌浓度（MIC）法检测分离菌株对5种B内酰胺类抗生素的敏感性。结果最常见的血清型／群为19群,共121株（19．4％）,其次是23群（15．4％）、6群（13．3％）、14型（6．6％）和15群（4．3％）。140株（22．4％）不能分型,还有117株（18．7％）属于其他28种少见的血清型／群。肺炎链球菌7价结合疫苗覆盖约360株,占57．6％;其中,血清型／群4、9和18分别有1、6和12株,共计占3．0％。常见血清型／群中,19群和23群与青霉素不敏感肺炎链球菌（PNSP）明显有关,其他血清型／群与PNSP无明显相关。结论 常见的血清型为19群、23群、6群、14型和15群,19群和23群与PNSP明显相关。肺炎链球菌7价结合疫苗可覆盖多数分离株。     

流感嗜血杆菌脉冲电场凝胶电脉法分型研究

为了解北京儿童医院流感嗜血杆菌（Hi)抗生素耐药菌株和b型菌株（Hib)的分子流行病学情况，采用脉冲电场凝胶电泳9PFGE）方法，对1999年和2000年分离的氨苄青霉素，头孢菌素耐药株，b型流感嗜血杆菌菌株和随机选取的非b型抗生素敏感菌株进行分型。结果显示，20株Hi菌株得到19种不同PFGE带型。1999年3株氨苄青霉素耐药菌株中有2株仅有一条带的差别；4株Hib菌株中，2株带型完全相同，与另1株有一条带的差别；2株头孢菌素耐药株有各自不同带型。2000年所有菌株带型各异，提示1999年从肺炎病人鼻咽部分离的氨苄青霉素耐药及Hib菌株多为流行病学相关菌株；2000年从上呼吸道感染儿童鼻咽部分离的氨苄青霉素耐药及Hib菌株无流行病学相关性。需要进一步监测方能确定有无耐药及Hib菌株流行，PFGE是对Hi进行流行病学研究的最佳工具之一。     

氨苄西林耐药流感嗜血杆菌的基因分型研究

目的了解2000-2003年北京、上海、广州细菌耐药监测项目中,小于5岁呼吸道感染儿童鼻咽部携带氨苄西林（AMP）耐药流感嗜血杆菌（Haemophilus influenzae,Hi）的分子流行病学情况。方法对上述呼吸道感染儿童鼻咽部分离的899株Hi进行AMP敏感性检测,筛选出74株AMP耐药Hi,采用巢式PCR荚膜分型和玻片凝集法,对AMP耐药菌株进行b型Hi（Haemophilus influenzaetypeb,Hib）检测,并用脉冲电场凝胶电泳（pulsed—field gel electrophoresis,PFGE）和多重PCR两种方法,对AMP耐药菌株进行基因分型。结果74株AMP耐药Hi中,有2株Hib（占2．7％）。PFGE分型74株AMP耐药Hi中有38种基因型,具有克隆传播趋势的有5型,包括41株Hi（占55．4％）。其中菌株数最多的为A型,有18株,占24．3％,以2002年上海地区为主。多重PCR分型结果有31型,多重PCR与PFGE分型结果一致率为63．5％。结论北京、上海、广州三地区四年内小于5岁的呼吸道感染儿童鼻咽部携带的AMP耐药Hi有55．4％的菌株存在克隆传播。     

5岁以下健康儿童鼻咽部肺炎链球菌的流行病学研究

目的了解武汉地区儿童肺炎链球菌携带率、耐药性、耐药基因及血清型流行状况。方法采集鼻咽拭子,以琼脂稀释法测定肺炎链球菌对12种抗菌药物的最低抑菌浓度,聚合酶链式反应检测红霉素耐药基因,荚膜肿胀试验进行血清学分型。结果武汉地区儿童肺炎链球菌携带率为22．31％（135／605）。存活的133株细菌中,青霉素不敏感肺炎链球菌（PNSSP）发生率为45．9％（61／133）;头孢菌素第一代（头孢氨苄）、二代（头孢克洛）、三代（头孢克肟、头孢泊肟、头孢曲松）敏感率依次为6．0％、45．1％、54．9％、56．4％、88．7％;除1株环丙沙星低耐株外,未发现氟喹诺酮类耐药株。大环内酯类敏感率仅为14．3％～15．8％。在114株红霉素耐药株中,检出ermB基因76株（66．7％）,2株（1．8％）低耐株含有mefA基因,46株（40．4％）同时具有ermB和mefA基因。血清分型涉及17个血清群,主要分布在19、23、6、15和14血清群,7株细菌未能分群。PNSSP分布在19、23、6和未分型血清群。结论武汉地区肺炎链球菌耐药严重,红霉素耐药主要为核糖体修饰（ermB介导）引起。流行血清群以19、23、6为主。     

住院肺炎患儿不同血清型肺炎链球菌对抗菌药物的耐药性分析

目的 了解当前从我国住院肺炎儿童分离的肺炎链球菌血清型分布和不同血清型菌株对抗菌药物的耐药状况,评估应用疫苗预防儿童肺炎链球菌感染和控制耐药菌传播的价值。方法 肺炎链球菌分离自4家儿童医院呼吸科年龄 ≤ 5岁的住院肺炎患儿,采用荚膜肿胀试验进行血清型分析,E试验法检测菌株对8种抗菌药物的敏感性。结果 279株肺炎链球菌中以19F型为最常见（占60.6%）,其次为19A（9.7%）。23F（9.3%）和6B（5.4%）,7价结合疫苗（PCV7）覆盖率为81.0%,PCV7在青霉素不敏感菌株和敏感菌株中的覆盖率分别为84.2%（202/240）和61.5%（24/39）。超过90%的19F和19A型菌株对青霉素不敏感,19F型以中介株为主（71.6%）,19A型以耐药株为主（55.6%）。结论 住院儿童肺炎病例分离的肺炎链球菌以19F。19A。23F和6B型常见;PCV7覆盖大多数肺炎链球菌和青霉素不敏感菌株,应用该疫苗可有效地预防国内儿童肺炎链球菌感染和阻止耐药菌株传播;非PCV7覆盖的19A型具有较强耐药性。     

杭州地区肺炎链球菌耐药性及遗传背景研究

目的 了解杭州地区肺炎链球菌儿童株的耐药性和流行特征。方法 用Kirby-Bauer法和Etest法对323株肺炎链球菌致病株和携带株进行耐药性分析,并对青霉素耐药株及部分相关菌株用BOX-PCR技术进行遗传背景研究。结果 青霉素敏感株136株,占42．1％,青霉素中介株和耐药株分别占40．2％和17．7％。青霉素的最低抑菌浓度范围为0．012～4．0μg／ml。所有菌株都对头孢噻肟和万古霉素敏感。90．7％的菌株对红霉素耐药,对四环素、甲氨苄啶一磺胺异嗯唑的耐药率也分别高达87．6％和48．6％,对氯霉素的耐药率为14．9％。多重耐药率61．0％,多数对红霉素、四环素和甲氨苄啶一磺胺异嗯唑联合耐药。99．4％的菌株对利福平和氧氟沙星敏感。BOX-PCR分析耐青霉素肺炎链球菌的临床株,未发现高度集中分布的BOX图谱,同一标本中分离到的表型不同的菌株或同一病人多次分离到的菌株BOX图谱可以不完全相同。结论 杭州地区肺炎链球菌耐药现象比较严重,头孢噻肟等第三代头孢菌素是治疗肺炎链球菌感染的理想药物。同一病人可同时或先后受不同肺炎链球菌克隆感染或定植。     

2003-2004年广州地区儿童肺炎链球菌、流感嗜血杆菌抗生素敏感性调查

目的了解2003-2004年广州地区儿童呼吸道感染常见病原肺炎链球菌和流感嗜血杆菌对常用抗生素的敏感性,以便有效指导临床合理用药和预防.方法对2003-2004年在广州市儿童医院就诊的1565例上呼吸道感染儿童鼻咽分泌物进行培养,分离肺炎链球菌和流感嗜血杆菌;采用纸片扩散法及E-test方法对分离株进行常用抗生素敏感性检测.结果从1565例呼吸道感染儿童鼻咽部分离肺炎链菌球共172株,流感嗜血杆菌484株.172株肺炎链球菌对常用抗生素耐药株数（耐药率）分别为：青霉素55（32.0%）、阿莫西林/克拉维酸19（11.1%）、头孢曲松56（32.6%）、头孢呋辛31（18.1%）、头孢克洛68（39.5%）、红霉素142（82.6%）、四环素135（78.5%）、氯霉素42（24.4%）、复方磺胺甲基异噁唑150（87.2%）、克林霉素119（69.2%）、氧氟沙星5（3.1%）;肺炎链球菌的青霉素不敏感株对β-内酰胺类、红霉素、复方磺胺甲基异噁唑耐药率明显高于青霉素敏感株,且多重耐药率在90%以上.484株流感嗜血杆菌中,143株β-内酰胺酶阳性,产酶率为29.5%,484株流感嗜血杆菌的耐药株数（耐药率）分别为：氨苄西林194（40.1%）,阿莫西林/克拉维酸16（3.4%）、头孢曲松20（4.1%）、头孢呋辛9（1.9%）、头孢克洛27（5.6%）、复方磺胺甲基异噁唑272（56.2%）、四环素252（52.1%）、氯霉素84（17.4%）、阿奇霉素10（2.1%）、氧氟沙星3（0.6%）.结论广州地区儿童呼吸道肺炎链球菌和流感嗜血杆菌耐药形势严峻,与该地区前3年资料比较,肺炎链球菌对青霉素敏感性有所增加,但对头孢曲松耐药性增加,青霉素不敏感株的多重耐药率较高,以红霉素、四环素、复方磺胺甲基异噁唑多重耐药为主要特点.流感嗜血杆菌的产酶率上升,导致菌株对氨苄西林耐药性明显增高,对头孢二、三代抗生素、阿莫西林/克拉维酸、阿奇霉素有较高的敏感性.     

儿童肺炎链球菌分离株青霉素、红霉素耐药性与耐药相关基因研究

目的了解苏州地区儿童肺炎链球菌(Streptococcuspneumoniae,Sp)临床分离株青霉素、红霉素耐药的状况。方法对呼吸道感染患儿痰标本中分离到的26株Sp进行青霉素、头孢呋辛、头孢曲松、头孢噻肟、红霉素E test药敏试验,和与青霉素、红霉素耐药相关的pbp2B、ermB、ermA/B、mefA基因PCR检测,测得pbp2B基因产物进行测序并与SpR6株(青霉素敏感株,登录号:NC003098)序列比较。结果(1)pbp2B基因突变15株(58%),均为点突变,突变类型有A型11株(73%)、B型2株(13%)、C型1株(7%)、D型1株(7%);(2)11株无突变菌中青霉素、头孢呋辛、头孢曲松、头孢噻肟敏感株分别为9株(82%)、10株(91%)、11株(100%)、11株(100%);15株突变菌中青霉素、头孢呋辛、头孢曲松、头孢噻肟不敏感株分别为13株(87%)、11株(73%)、1株(7%)、1株(7%);11株A型突变株对青霉素均不敏感,10株对二代头孢不敏感;(3)ermB基因阳性9株(35%)、ermA/B基因阳性16株(62%)、mefA基因阳性7株(27%)、erm或(和)mef基因阳性21株(81%);(4)erm或(和)mef基因阳性株红霉素最低抑菌浓度为2～>256mg/L。结论pbp2B基因突变是本地区Sp青霉素耐药机制的主要遗传学基础之一;突变株中,A类突变率最高,表现对青霉素、二代头孢不敏感;ermA、ermB和mefA3种红霉素耐药基因单独或共同表达     

Pneumococcal serotypes from acute otitis media in rural Kentucky

OBJECTIVE: Acute otitis media (AOM) accounts for most infections caused by, but few data are available regarding the incidence of pneumococcal serotypes recovered from children with AOM in the United States. METHODS: Between January 1992 and March 1998, 777 middle ear pathogens from AOM were obtained from 701 patients by tympanocentesis (84.6%) or by culture of otorrhea (15.4%) from spontaneous perforation or draining tubes. The ambulatory patient population was mostly white and cared for by a sole private pediatric practice in rural Kentucky. RESULTS: Penicillin-nonsusceptible (penicillin MIC > or = 0.1 microg/ml) (PNSP) isolates accounted for 18% [6% resistant PNSP (rPNSP) and 12% intermediate resistant PNSP], and penicillin-susceptible strains accounted for 35% of the pathogens recovered from children with culture-proved AOM. Comparing the frequency of isolates between 1992 and 1993 with those between 1994 and 1998, overall rates of PNSP strains remained remarkably stable (32.2% 37.3%), but intermediate resistant PNSP strains doubled from 14% to 27% ( < 0.01), whereas rPNSP strains fell by one-third. Serotypes 19F (34%), 23F (30%), 6B (26%) and 14 (8%) accounted for nearly all rPNSP isolates. Two cross-reactive serotypes (6A and 19A) not included in the available pneumococcal conjugate vaccine comprised 8.4 and 15% of all serotypes and PNSP serotypes, respectively. Nearly all PNSP strains recovered in children < or =24 months are included in the vaccine serogroups. CONCLUSION: Depending on rates of efficacy and serotype cross-protection, the current pneumococcal conjugate vaccine could potentially protect against most PNSP strains in all ages, particularly in those < or =24 months.     

北京上海广州深圳4家儿童医院肺炎住院患儿肺炎链球菌分离株的血清型分布

目的了解目前从中国住院治疗肺炎患儿分离到的肺炎链球菌的血清型分布，及几种蛋白多糖结合疫苗的覆盖率，评估应用蛋白多糖结合疫苗预防肺炎链球菌感染的价值。方法选择2006年2月16日至2007年2月16日在首都医科大学附属北京儿童医院、复旦大学附属儿科医院、广州市儿童医院和深圳市儿童医院呼吸科住院治疗的肺炎患儿为研究对象，采用一次性吸痰管收集全部病例的呼吸道分泌物标本分离肺炎链球菌，部分患儿进行脑脊液、血液和胸腔积液中肺炎链球菌的分离。采用荚膜肿胀实验进行血清型分析。对4家儿童医院肺炎链球菌分离率和血清型进行分析，率的比较采用χ2检验或Fisher精确概率法。结果 研究期间共纳入2 865例肺炎患儿，2 865例呼吸道吸取物标本中分离到肺炎链球菌279株，其中有2株不同血清型菌株分离自同一病例，分离阳性率为9.7％（278/2 865）。3/8例胸腔积液中分离到肺炎链球菌，其中2例同时从呼吸道分泌物分离到肺炎链球菌，取其一进行血清分型，另1株从胸腔积液中分离的肺炎链球菌复苏失败，未进行血清分型。脑脊液和血液标本中未分离到肺炎链球菌。共有279株肺炎链球菌进行了血清型分析，以19F型最常见（60.6％，169/279），其次为19A（9.7％，27/279）、23F（9.3％，26/279）和6B（5.4％，15/279），上述4种血清型占全部菌株的84.9％（237/279）。肺炎链球菌7价结合疫苗（PCV7）覆盖率为81.0％，但在北京仅为46.0％，明显低于上海（80.0%）、广州(98.4%)和深圳（94.4%）。9价、10价和11价疫苗的覆盖率与PCV7相比并没有明显增加。13价疫苗的覆盖率（92.8％）较PCV7明显升高。结论4家儿童医院肺炎住院患儿分离的肺炎链球菌以19F、19A、23F和6B型常见。PCV7覆盖率为87％     

Community-wide vaccination with the heptavalent pneumococcal conjugate significantly alters the microbiology of acute otitis media

BACKGROUND: Community-wide use of conjugated heptavalent pneumococcal vaccine (PCV7) in children <2 years of age could affect the microbiology of acute otitis media (AOM) in vaccinees, particularly for penicillin-nonsusceptible Streptococcus pneumoniae (PNSP). SETTING: Since Summer 2000, 94% of young children cared for by this 7-clinician, pediatric practice in rural central Kentucky received 3 or 4 doses of PCV7 in the first 18 months of life. OBJECTIVE: To determine changes in microbiology of AOM before and after community-wide routine implementation of PCV7. METHODS: Among children 7-24 months old with severe or refractory AOM, we compared 336 AOM isolates from 1992-1998 with 83 AOM isolates from 2000-2003 in children who had received 3 or 4 doses of PCV7. RESULTS: Comparing each cohort (1992-1998 versus 2000-2003), the proportion of S. pneumoniae decreased from 48% to 31% (P = 0.009; relative risk, 0.754; 95% confidence interval, 0.628-0.906), and nontypable Haemophilus influenzae increased from 41% to 56% (P = 0.01; relative risk, 1.87; 95% confidence interval, 1.15-3.04; beta-lactamase-positive, 56% versus 64%, not significant). The proportions of intermediate PNSP and resistant PNSP, respectively, were 16% and 9% versus 13% and 6% pre- and post-PCV7, respectively. Vaccine and vaccine-related serotypes, respectively, comprised 70% and 8% versus 36% and 32% of S. pneumoniae strains (P = 0.003). Post-PCV7, Gram-negative bacteria and beta-lactamase-producing organisms accounted for two-thirds and one-half of all AOM isolates, respectively. DISCUSSION: The overall proportion of S. pneumoniae isolates and vaccine serotypes in AOM were significantly reduced by community-wide use of PCV7 vaccine in our practice. The proportion of Gram-negative bacteria became 2-fold more frequent than S. pneumoniae in AOM in PCV7-vaccinated young children where PCV7 uptake was community-wide and supply was adequate.     

������ͯҽԺ19F�ͷ����������ҩ�Է������������в�ѧ�о�

??Abstract??Objective To investigate the genetic structure of Streptococcus pneumoniae serotype 19F isolates from children in Beijing. Methods A total of 130 isolates were identified as serotype 19F among 1033 S. pneumoniae strains collected from 1997 to 2006 and 2010. There were 120 isolates characterized by antibiotic susceptibility?? macrolide resistance gene and multilocus sequence typing??MLST??. Results Among the 120 strains?? only five strains were nonsusceptible to penicilln and the nonsusceptibility rate to cephalosporins increased from 1997 to 2010. There were 119??99.2%?? strains resistant to erythromycin?? 115??96.6%?? carried the ermB gene and 64??53.8%?? carried mefA gene?? 60??50.4%?? carried both genes. All isolates belonged to 31 sequence types?? ST983 was the most prevalent ST?? followed by ST271. From 1997 to 2010?? the percentage of CC271 increased from 14.3% in 1997-1998 to 92% in 2010?? whereas CC983 decreased from 64.3% to 0%. CC271 showed higher nonsuscetibility rate to β-lactam antibiotics than CC983. Conclusion The prevalence of serotype 19F of S.pneumoniae increases from 1997 to 2010 in Beijing. The increase of nonsusceptibility rate to β-lactam antibiotics is associated with the spread of international resistance clone CC271 due to the
selection of antibiotics overuse.     

������pbp2b ply��lytA��϶�ͯ�����������Ĥ��Ӧ�ü�ֵ�о�

??Objective To compare pbp2b??ply and lytA genes PCR with cerebral spinal fluid culture in diagnosis of Streptococcus pneumoniae meningitis and prediction of its susceptibility. Methods A nested PCR targeting pbp2b and another two S.pneumoniae specific PCR targeting the genes of pneumolysin??ply?? and autolysin??lytA?? were developed for detection of S.pneumoniae in cerebral spinal fluid from bacterial meningitis patients. The three PCR results and culture were compared. The consistency of penicillin susceptibility PCR ??using resistant and susceptible primers respectively????sequencing and culture-based phenotypic penicillin resistant results were compared. Results Of the 161 specimens studied??there were 25 cases of S.pneumoniae infection confirmed by different methods??16 by pbp2b PCR??16 by lytA PCR??14 by ply PCR and 9 by cerebrospinal fluid culture??. Of the 16 pbp2b positive specimens??penicillin sensitive and resistant sequence types accounted for half??respectively. Four of the 16 pbp2b positive specimens had culture-based phenotypic penicillin-resistant result. Three of 4 were consistent with penicillin susceptibility PCR result. The results of susceptibility PCR targeting pbp2b was consistent with sequencing result. New sequence types were found but there were no new point mutations in these strains when compared with GenBank ??http??//blast.ncbi.nlm.nih.gov/Blast.cgi??. Penicillin resistance in pneumococcal meningitis was 66.67%??6/9?? by culture phenotype and 50%??8/16?? by PCR and sequencing when culture was negative. Conclusion Pbp2b can serve as a good target gene to detect S.pneumoniae and predict its penicillin susceptibility??which is especially important when culture is negative.     

Pathogens causing recurrent and difficult-to-treat acute otitis media, 2003-2006

This study sought to determine the microbiology of recurrent acute otitis media (AOM) and AOM treatment failure (AOMTF) in the context of widespread use of heptavalent pneumococcal conjugate vaccine (PCV7). In this retrospective cohort study, 244 AOM isolates obtained by tympanocentesis during 3 respiratory seasons--2003-2004 (n = 126), 2004-2005 (n = 52), 2005-2006 (n = 66)--from three geographically diverse pediatric populations were compared. Most isolates were from children less than 2 years old, who had received PCV7. For the 3 seasons the proportion of Streptococcus pneumoniae isolates was 35%, 35%, and 46% and for Haemophilus influenzae was 55%, 58%, and 39%, respectively (change in trend, P = .09). A total of 37%, 39%, and 50% of S. pneumoniae were penicillin nonsusceptible (PNSP) and 48%, 67%, and 50% of H. influenzae produced beta-lactamase, respectively. Although H. influenzae remains the most frequently isolated pathogen in children with AOMTF or recurrent AOM, S. pneumoniae that are PNSP are reemerging as important organisms.