两种嗜血杆菌在呼吸道感染患者痰培养中的检出情况及耐药分析

目的:通过对呼吸道感染患者痰标本的培养,以建立一种简便、有效的分离嗜血杆菌的方法,为临床提供诊断及用药依据.方法:利用嗜血杆菌分离专用巧克力平板对痰标本进行嗜血杆菌的分离,用HTM平板作药敏试验,用β-内酰胺酶纸片测定β-内酰胺酶.结果:在分离出的236株嗜血杆菌中,流感嗜血杆菌有124株,占52.5%,副流感嗜血杆菌112株,占47.5%,其中流感嗜血杆菌生物型以Ⅰ型(50.8%)、Ⅲ型(29.8%)为主,Ⅳ、Ⅷ型各占9.7%;副流感嗜血杆菌以Ⅰ型(61.6%)、Ⅱ型(25.0%)为主,Ⅲ型占13.4%.β-内酰胺酶产酶率为2.7%,对β-内酰胺类抗生素的敏感率较高.结论:用嗜血杆菌专用巧克力平板分离嗜血杆菌,方法简便、实用,效果较好,能满足临床需要.     

马血巧克力平板分离流感嗜血杆菌及其耐药性分析

目的了解本地区流感嗜血杆菌感染情况及其抗生素耐药性,为临床提供参考.方法用添加万古霉素的马血巧克力平板分离流感嗜血杆菌,选用5种抗生素做K-B法药敏试验.数据处理使用WHONET-5软件.结果 979份标本检出137株嗜血杆菌,检出率14.0%,其中流感嗜血杆菌55株(5.6%),副流感嗜血杆菌82株(8.4%).55株流感嗜血杆菌对氨苄青霉素、头孢呋辛、头孢噻肟、复方磺胺甲NFDA9唑、氯霉素等抗生素的耐药率分别为34.5%,56.4%,3.6%,9.1%,30.9%.结论二代头孢菌素、三代头孢菌素对流感嗜血杆菌有极好的抗菌活性,复方磺胺甲NFDA9唑、氨苄青霉素及氯霉素耐药现象常见.     

副流感嗜血杆菌生物分型与方法评价

目的对临床分离的副流感嗜血杆菌进行生物分型,并对两种不同方法的分型结果进行比较.方法用嗜血杆菌专用平板(HAE)进行分离培养,采用推荐的标准方法,对70株副流感嗜血杆菌进行生物分型,并用Vitek-60微生物自动分析系统对副流感嗜血杆菌进行鉴定和生物分型.结果标准法结果显示副流感嗜血杆菌以生物I型多见,检出45株(64.3%),其次为生物II型11株(15.7%),另检出生物Ⅲ型4株(5.7%),生物Ⅳ型1株(1.4%),生物Ⅷ型1株(1.4%),未能分型有8株(11.4%).标准法与仪器法对副流感嗜血杆菌的生物分型符合率为67.1%.结论Vitek-60微生物自动分析系统对副流感嗜血杆菌的生物分型结果与标准方法存在着一定差异.     

2000～2002年患儿呼吸道致病菌的耐药性研究

目的　研究 2 0 0 0～ 2 0 0 2年上海地区患儿呼吸道流感嗜血杆菌、肺炎链球菌及卡他莫拉菌感染情况 ,肺炎链球菌的血清分型 ,3种致病菌对抗菌药物的耐药情况。方法　细菌的生化鉴定采用API鉴定系统 ,肺炎链球菌血清分型采用荚膜肿胀试验 ,β内酰胺酶测定采用头孢硝噻酚试验 ,抗生素敏感试验采用浓度梯度扩散法及琼脂扩散法。结果　流感嗜血杆菌持续 3年位居呼吸道致病菌首位 ,对氨苄西林素耐药率接近 2 0 %。卡他莫拉菌及肺炎链球菌分离率呈增加趋势 ,其中 17 2 %流感嗜血杆菌及 95 1%卡他莫拉菌 β内酰胺酶阳性。 72 7% β内酰胺酶阳性卡他莫拉菌对氨苄西林耐药。检测到 11种肺炎链球菌血清型 ,19F及 2 3F为主要血清型 ,青霉素耐药菌株 (PRSP)占 4 2 %。结论　流感嗜血杆菌、卡他莫拉菌及肺炎链球菌已成为上海地区患儿呼吸道主要院外感染致病菌。流感嗜血杆菌、肺炎链球菌感染对青霉素类抗生素已产生不同程度的耐药 ,19F、2 3F是本地区肺炎链球菌主要血清型。     

探讨全自动快速微生物检测系统在鉴定流感嗜血杆菌的应用

目的探讨全自动快速微生物检测系统(VITEK MS)在鉴定流感嗜血杆菌的应用价值。方法收集广州市黄埔区红十字会医院与广东省中医院2014年3月至2015年1月从痰液或咽拭子标本中分离的流感嗜血杆菌35株,用16SrDNA测序确证。并且用VITEK MS质谱仪,API NH常规生化鉴定与MH琼脂平板上V+X因子需求性试验3种方法对上述菌株鉴定分析。结果在35株流感嗜血杆菌细菌中,VITEK MS质谱仪与API NH常规生化鉴定差异无统计学意义(P0.05),而VITEK MS与MH琼脂平板上V+X因子需求性试验差异有统计学意义(P0.05)。结论对于API NH常规生化鉴定与MH琼脂平板上V+X因子需求性试验,VITEK MS能快速、准确地鉴定流感嗜血杆菌,对临床诊治提供很大的帮助,有非常好的应用前景。     

纸片法卫星试验对流感嗜血杆菌鉴定的影响探讨

目的探讨如何正确鉴定流感嗜血杆菌,提高流感嗜血杆菌的检出率。方法收集经全自动细菌鉴定仪鉴定为流感嗜血杆菌,但脑心琼脂平板纸片法卫星实验阴性的18株流感嗜血杆菌,分别进行纸片法卫星试验。结果 1)0.5麦氏浓度组中48 h试验组与24 h试验组进行比较,差异有统计学意义(P0.05);72 h试验组与24 h试验组进行比较,差异有统计学意义(P0.01);72 h试验组与48 h试验组进行比较,差异无统计学意义(P0.05)。2)1个麦氏浓度24 h试验组与标准方法试验组相比较,差异无统计学意义(P0.05);3个麦氏浓度24 h试验组与标准方法试验组相比较,差异有统计学意义(P0.01)。3)3个麦氏浓度48 h试验组与3个麦氏浓度24 h试验组相比较,差异无统计学意义(P0.05)。结论 1)当纸片法卫星试验孵育时间为24 h时,将待检菌浓度由0.5麦氏单位调整为3麦氏单位,可提高试验阳性率。2)当纸片法卫星试验待检菌浓度为0.5麦氏单位时,孵育时间延长为48 h,可提高试验阳性率。3)当纸片法卫星试验待检菌浓度为3麦氏单位,孵育时间分别为24、48及72h时,试验阳性率无差异。     

732株嗜血杆菌对阿奇霉素敏感性分析

1999年 3月至 2 0 0 0年 12月 ,对南京军区南京总医院住院病人 ,门诊病人送检的痰液及咽拭标本中分离的 732株嗜血杆菌 ,采用Ｋ Ｂ纸片扩散法 ,对阿奇霉素做了药敏试验 ,并与其他 5种抗菌药物作了比较 ,报告如下。1　材料与方法1 1　菌种嗜血杆菌 732株 ,其中流感嗜血杆菌 30 2株 ,占41 3% ;副流感嗜血杆菌 430株 ,占 5 8 7%。1 2　培养基　巧克力培养基按《全国临床检验操作规程》(1997年第 2版 )由本室自制 ;血琼脂和Ｍ Ｈ琼脂 (Ｏｘｏｉｄ)作鉴定用 ;ＨＴＭ培养基 (Ｏｘｏｉｄ)作药敏用。1 3　药敏纸片　阿奇霉素、头孢噻肟、头孢呋辛、…     

不同培养基与前处理方法对副溶血弧菌MALDI-TOF MS鉴定结果的影响评估

目的评估不同培养基及前处理方法在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对副溶血弧菌鉴定中的影响,并寻求最优化条件组合。方法收集副溶血弧菌40株,分别用血琼脂平板、TCBS琼脂平板和双洗琼脂平板进行培养;用直涂法、扩展法(直涂后裂解)和萃取(裂解、萃取后涂靶)法进行质谱前处理。用SAS软件建立不同培养基及前处理方法对该菌质谱检出率影响的logistic回归分析模型,并进行卡方检验统计分析。结果 "血琼脂平板+萃取法"条件组合检出率最高(100%鉴定到属,67.5%鉴定到种);在不使用血琼脂平板的前提下,"双洗琼脂平板+萃取法"条件组合检出率最高(70%鉴定到属,37.5%鉴定到种)。在"属"鉴定水平,前处理方法相同时,不同培养基的检出率差异有统计学意义(P0.01);而培养基相同时,不同前处理方法的检出率差异无统计学意义(P0.05);直涂法和扩展法相对萃取法的相对危险度(OR)分别为0.66和0.95,检出率差异无统计学意义(P0.05);TCBS琼脂平板和双洗琼脂平板相对血琼脂平板的OR分别为0.06和0.10,对检出率差异有统计学意义(P0.05),logistic回归方程Y=2.95-0.41a-0.05b-2.83c-2.35d(a:直涂法;b:扩展法;c:TCBS琼脂平板;d:双洗琼脂平板)。在"种"鉴定水平,采用扩展法或萃取法,不同培养基的检出率差异有统计学意义(P0.05);采用血琼脂平板,不同前处理方法的检出率差异有统计学意义(P0.01);直涂法和扩展法相对萃取法的OR分别为0.32和0.55,检出率差异有统计学意义(P0.05);TCBS琼脂平板和双洗琼脂平板相对血琼脂平板的OR分别为0.18和0.49,检出率差异有统计学意义(P0.05),logistic回归方程Y=0.20-1.15a-0.61b-1.72c-0.72d(a:直涂法;b:扩展法;c:TCBS琼脂平板;d:双洗琼脂平板)。结论欲提升副溶血弧菌MALDI-TOF MS鉴定效果,合适培养基的选择优于改善蛋白质提取的前处理操作。推荐萃取法作为常规前处理方法,培养基的选择建议依次为血琼脂平板、双洗琼脂平板和TCBS琼脂平板。     

呼吸道感染患儿流感嗜血杆菌的生物分型与耐药性研究

目的 了解呼吸道感染患儿流感嗜血杆菌的生物分型及其对常用抗菌药的耐药情况.方法 对320例呼吸道感染患儿的鼻咽分泌物进行流感嗜血杆菌检测,采用流感嗜血杆菌选择性培养基进行分离培养,通过卫星试验进行菌株鉴定,采用吲哚试验、脲酶试验及鸟氨酸脱羧酶试验进行流感嗜血杆菌的生物分型,采用纸片扩散抗菌试验进行微生物敏感性试验,采用头孢硝噻吩纸片检测流感嗜血杆菌β-内酰胺酶.结果 呼吸道感染患儿鼻咽分泌物的流感嗜血杆菌检出率为47.50%（152/320）,共检出Ⅰ、Ⅲ、Ⅳ、Ⅴ、Ⅵ 5型,分别占3.29%（5/152）、5.26%（8/152）、33.55%（51/152）、54.61%（83/152）、3.29%（5/152）.产β-内酰胺酶的流感嗜血杆菌占42.76%（65/152）.流感嗜血杆菌对四环素、利福平和美罗培南敏感;对阿奇霉素和左氟沙星的耐药率均小于10.00%;对氨苄西林、氯霉素和复方磺胺甲噁唑的耐药率为87.50%～100.0%.结论 该院流感嗜血杆菌Ⅳ、Ⅴ型是儿童呼吸道感染的主要生物型,产β内酰胺酶是其耐药的重要机制.     

流感嗜血杆菌对三种培养基的试验与评价

目的建立一种简便、有效的分离嗜血杆菌的方法.方法利用万古霉素巧克力法、血平板点种金黄色葡萄球菌法和传统巧克力法,对合正常菌群的痰标本进行嗜血杆菌的分离.对120份痰标本用以上三种方法进行分离,结果加万古霉素巧克力平板上流感嗜血杆菌阳性率高,为16%,传统巧克力法为4.1%,而血平板点种金黄葡萄球菌法仅为2.5%.结论利用万古霉素巧克力平板分离嗜血杆菌方法简便、实用、效果好.     

改良GCYSB培养基分离流感嗜血杆菌的应用评价

用自配的改良ＧＣＹＳＢ－Ｖ选择培养基从２２５份临床标本中分离到嗜血杆菌属细菌９７件，而用加万古霉素的普通巧克力选择培养基从相同标本中只分离到嗜血杆菌属细菌２９株，分离率相差显著（Ｐ＜０．０１）。流感嗜血杆菌在两种培养基上的平均生长指数分别为７．７２和５．９４。结果表明，ＧＣＹＳＢ培养基比Ｃｈｏｃ培养基更适合流感嗜血杆菌生长。     

用于嗜血杆菌鉴定的卫星试验影响因素研究

目的　了解嗜血杆菌鉴定中X、V因子测定的各种影响因素及可用于嗜血杆菌鉴定的卫星试验的细菌种类。方法　用 4种不同的琼脂平板 ,比较不同培养时间和CO2 环境对嗜血杆菌X、V因子需求试验结果的影响 ,同时比较不同菌种在各种琼脂培养基上对嗜血杆菌琼脂卫星试验结果的影响。结果　X、V因子需求试验结果表明 ,脑心琼脂效果最好 ,其次为营养琼脂 ,M H琼脂结果与脑心琼脂相比差异有显著性 (P <0 .0 1) ;CO2对试验结果有影响 ;培养 4 8h较 2 4h的菌落大 ,易于辨认 ;不同菌种在不同琼脂培养基上对嗜血杆菌琼脂卫星试验的结果有所不同。结论　卫星试验应在CO2 环境中进行 ,用脑心琼脂平板测试嗜血杆菌对X、V因子的需求结果较好 ,多数葡萄球菌及部分革兰阴性杆菌对嗜血杆菌的琼脂卫星试验结果较好     

改良流感嗜血杆菌培养基的实验研究与评价

目的评价自制改良哥伦比亚巧克力培养基（ＩＣＣＡ）对流感嗜血杆菌（Ｈｉｎ）的分离培养效果。方法将ＨｉｎＡＴＣＣ４２９４７接种于ＩＣＣＡ和其他４种培养基上，计算和比较５种培养基Ｈｉｎ的平均生长指数；用ＩＣＣＡ对４４８份痰标本进行检测，比较其与血琼脂加金葡菌划线（ＢＡＳＳ）方法的Ｈｉｎ检出率。结果与其他４种培养基比较，ＩＣＣＡ的Ｈｉｎ平均生长指数为１７．７９，痰标本中的Ｈｉｎ的检出率为２０．１％，ＩＣＣＡ明显高于ＢＡＳＳ（检出率１２％），经χ２检验Ｐ＜００１。结论ＩＣＣＡ是一种较好的Ｈｉｎ选择性培养基，值得推广应用     

Incidence of penicillin-resistantstreptococcus pneumoniae in Japan, 1993–1995

The Working Group for PRSP was organized through the participation of 40 institutions to investigate the incidence of penicillin (Pc)-resistantStreptococcus pneumoniae (PRSP) in Japan. We collected 2410S. pneumoniae clinical isolates between October 1994 and March 1995. The susceptibility to Pc, erythromycin, and minocycline was determined by an agar dilution method using Mueller-Hinton agar supplemented with 5% sheep blood. Pc-susceptibleS. pneumoniae (PSSP) were defined as bacteria for which the minimum inhibitory concentration (MIC) was ≤0.06 μg/mL; Pc-intermediateS. pneumoniae (PISP) as those for which the MIC ranged from 0.125 to 0.25 μg/mL; PRSP, as those with a MIC≥0.5μg/mL. The incidence of resistant strains including PISP and PRSP was 41.8% in 1994 and 40.8% in 1995. Logistic regression analysis showed that PRSP was significantly more frequent in infants aged 0 to 2 years old than in the general population and PSSP was significantly more frequent in elderly patients aged 60 or older. The rate of PRSP was significantly higher in the throat than in the sputum. Among 10 regions studied nationwide, PRSP was detected less frequently in the areas of Hokkaido and Hokuriku and more frequently in the areas of Chugoku, Shikoku, and Kyushu. Most PRSP were resistant to erythromycin and minocycline. PSSP serotyping using the capsule-quellung reaction indicated a number of types. In contrast, most PISP and PRSP were serotyped to types 19, 23, and 6.     

嗜血杆菌分离培养基的评价与应用

目的：选择合适的嗜血杆菌分离培养提高嗜血杆菌分离率。方法：将嗜血杆菌接种于7种培养基上,计算和比较7种培养基上嗜血杆菌的平均生长指数（GI）;对325份呼吸道标本进行检测,比较巧克力琼脂（BRchoc）和加万古霉素巧克力琼脂(BRchoc-V）的嗜血杆菌分离率。结果：添加2％鲜酵母浸液和50％鲜牛肉浸液的各种培养基,较未加者的GI值明显增高;兔血巧克力琼脂培养基较羊血巧克力琼脂培养基的GI值为高（P＜0.001）;加万古霉素（50μg/ml）的兔血巧克力琼脂培养基,较未加者对嗜血杆菌的分离率（77.2％／32.0％）明显提高（P＜0.001）。结论：培养基中增加鲜酵母浸液等营养物质可促进流感嗜血杆菌的生长,制培养基所用血源以兔血最佳,其次为羊血;分离培养基加入50μg/ml的万古霉素,可显著提高嗜血杆菌的分离率。     

Problems with current recommendations for susceptibility testing of Haemophilus influenzae

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.     

Antimicrobial activities of cefditoren against respiratory pathogens isolated from children in Japan

There is an increasing spread and incidence of penicillin-resistant bacteria that are becoming less susceptible to commonly prescribed oral antimicrobials, including extended-spectrum cephalosporins. Against this background, we undertook this study to determine the prevalence of penicillin resistance in Streptococcus pneumoniae and the in-vitro activity of oral antimitrobials. Between April 1996 and December 1997, in 245 children with respiratory tract infections (bronchitis in 61, pharyngitis in 115, and tonsillitis in 69), 119 strains of Haemophilus influenzae, 89 strains of Streptococcus pyogenes, 61 strains of Streptococcus pneumoniae, 36 strains of Staphylococcus aureus, and 34 strains of Moraxella catarrhalis were isolated from the pharynx. The antimicrobial susceptibility of these isolates was assessed by a broth microdilution method. The isolation incidence of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-highly resistant S. pneumoniae (PRSP) was 59.0% and 13.1%, respectively. Most strains of PISP and PRSP were highly resistant to cefaclor, cefpodoxime, cefteram, cefdinir, clarithromycin, ampicillin, and minocycline, but susceptibile to ofloxacin and cefditoren (CDTR). The in-vitro activity of CDTR was superior to that of other cephalosporins, such as cefaclor, cefdinir, and cefpodoxime, when tested against both the β-lactamase-positive and -negative H. influenzae isolated. CDTR was also active against all the other strains, including methicillin-sensitive S. aureus, S. pyogenes, and M. catarrhalis. This study suggested that CDTR was a useful oral antibiotic for pediatric respiratory tract infections. Received: June 11, 1998 / Accepted: September 7, 1998     

肺炎链球菌和流感嗜血杆菌分布及耐药性分析

目的：了解临床分离流感嗜血杆菌和肺炎链球菌的分布及耐药情况,为临床合理使用抗菌药物、预防和控制感染提供依据。方法：收集医院2005-2012年各类临床标本分离流感嗜血杆菌92株、肺炎链球菌83株,均经全自动细菌鉴定仪鉴定,用K—B法检测流感嗜血杆菌对常用的15种抗生素及肺炎链球菌对常见的13种抗生素的敏感性,并用头孢硝噻吩纸片法检测流感嗜血杆菌产β-内酰胺酶情况。结果：流感嗜血杆菌和肺炎链球菌主要来源于呼吸道标本,不同季节流感嗜血杆菌的感染率不同,冬春两季为高发季节,肺炎链球菌感染患者的年龄呈双峰分布,以年龄〈5岁和〉50岁的感染者最多。流感嗜血杆菌对氨苄西林、复方新诺明和头孢呋辛的耐药率较高,产β-内酰胺酶检出率为64．17％。肺炎链球菌对红霉素、氯霉素、四环素、克林霉素耐药性非常严重,其中47株（56．63％）为青霉素不敏感肺炎链球菌（PNSP）。结论：流感嗜血杆菌和肺炎链球菌耐药情况较为严重,对流感嗜血杆菌和肺炎链球菌的耐药性进行严密监测具有重要意义。     

反向斑点杂交法检测流感嗜血杆菌核酸

目的　探讨早期、快速检测流感嗜血杆菌的方法。方法　应用流感嗜血杆菌编码外膜蛋白特异的P6基因设计引物 ,通过聚合酶链反应合成特异探针 ,采用反向斑点杂交法检测生物素标记DNA ,并应用于痰标本的检测。结果　只有流感嗜血杆菌扩增出 35 1bp的DNA片段 ,该探针能检测出 10ng的细菌DNA ,与其他细菌、病毒、真菌无交叉反应。该方法和培养法分别检测 5 0份痰标本 ,两者的阳性率分别为 30 %和 2 2 %。结论　该方法快速、特异 ,对流感嗜血杆菌感染有早期诊断价值     

Identification of beta-lactamase-negative, ampicillin-resistant strains of Haemophilus influenzae with four methods and eight media

A challenge set of 143 non-beta-lactamase-producing strains of Haemophilus influenzae was tested for ampicillin susceptibility on two broth media and six agar media, using broth microdilution, agar dilution, disk diffusion, and E-test procedures. When beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were defined as those for which the ampicillin MIC was > or = 4.0 microg/ml, 5 to 44% of our selected strains were BLNAR depending on the medium and/or test method used. If nonsusceptible strains for which ampicillin MICs were intermediate were included in the BLNAR category, 32 to 50% of our isolates would be considered BLNAR. These data emphasize the need for a standardized testing procedure and a universal definition of BLNAR strains before the clinical relevance of such strains can be evaluated. NCCLS dilution tests with haemophilus test medium broth or agar are preferred for testing ampicillin against H. influenzae.