肺炎链球菌对大环内酯类抗生素耐药及传播机制研究

目的 研究上海3所医院临床分离肺炎链球菌对大环内酯类抗生素的耐药机制及传播方式。方法收集上海市3所医院临床分离的红霉素耐药肺炎链球菌共118株，用E试验和K-B纸片扩散法检测对12种抗菌药的敏感度；用双纸片法（D试验）确定大环内酯类耐药表型；用PCR扩增检测耐药基因ermB、mefA、mefE、msrD及Tn1545-Tn916家族转座子整合酶基因intTn；用转化试验证实耐药传播方式。结果①118株肺炎链球菌对红霉素的MIC范围为4-256mg／L，其中5．9％对克林霉素敏感，对青霉素不敏感率达72．7％。左氧氟沙星、阿莫西林-克拉维酸对红霉素耐药的肺炎链球菌仍有较好的体外活性；②该组细菌耐药基因ermB检出率为88．1％，mefE、msrD检出率各为50％，未检出，mefA基因，转座子整合酶基因intTn检出率达97．5％。耐药基因组合模式以ermB（＋）reefE（＋）msrD（＋）intTn（＋）和ermB（＋）mefE（-）msrD（-）intTn（＋）为主，两者均为cMLSB型耐药。ermB（-）mefE（＋）msrD（＋）intTn（＋）模式占5．9％，耐药表型为M型。③cMLSB型耐药代表菌株ET37和M型耐药代表菌株RJ324基因组DNA均成功转化敏感株，使之表现红霉素耐药性并可传代。结论上海地区肺炎链球菌对大环内酯抗生素耐药以ermB介导的cMLSB耐药表型为主；大环内酯外排基因有流行趋势，但仅限于起源于肺炎链球菌的，mefE。耐药基因可以转化方式进行传播，转座子可能在本地区肺炎链球菌耐药基因的传播中起重要作用。     

肺炎链球菌大环内酯类耐药表型与相关基因的关系

目的了解肺炎链球菌临床分离株红霉素耐药基因的流行情况和耐药表型的关系。方法对42株肺炎链球菌用E-试验和K-B纸片扩散法检测其对10种抗生素的敏感性;用红霉素和克林霉素双纸片协同试验确定其耐药表型;用PCR扩增这些菌株的耐药基因ermB、mefA和mefE。结果 42株肺炎链球菌中耐药基因ermB总检出率为95.2%(40/42),mefE总检出率为26.1%(11/42),未检出mefA基因。耐药基因组合ermB(+)mefE(-)和ermB(+)mefE(+)占95.2%,两者均呈cMLSB耐药表型。ermB(-)mefE(+)占4.8%(2/42),耐药表型为M型。结论耐药基因ermB导致的cMLSB耐药是大环内酯类耐药的主要原因。大环内酯类抗生素已不是治疗肺炎链球菌的有效药物。     

乐清地区儿童肺炎链球菌耐药性及大环内酯类耐药表型和耐药基因型

目的：了解乐清地区儿童患者分离的肺炎链球菌耐药性及大环内酯类耐药表型和耐药基因型分布情况。方法对2014年乐清地区儿童患者分离的124株肺炎链球菌采用细菌鉴定分析仪进行9种抗菌药物的最低抑菌浓度（MIC）检测,同时对大环内酯类耐药肺炎链球菌用红霉素和克林霉素双纸片协同试验确定其耐药表型,用聚合酶链反应（PCR）扩增这些菌株的耐药基因ermB和mefE。结果124株肺炎链球菌中,红霉素、克林霉素、四环素和复方新诺明的耐药率依次为96.77％、93.55％、84.68％和81.45％;青霉素、氯霉素和左旋氧氟沙星的耐药率较低,分别为20.16％、5.65％和0.81％,未发现对阿莫西林/克拉维酸和万古霉素耐药的菌株。120株大环内酯类耐药肺炎链球菌中,大环内酯类耐药表型cMLS占96.67％、iMLS占0.83％、M型占2.50％;耐药基因ermB检出率为97.50％,mefE的检出率为6.67％。结论乐清地区儿童肺炎链球菌对大环内酯类抗生素的耐药性严重,ermB基因介导的cMLS型耐药是大环内酯类耐药的主要原因,大环内酯类抗生素已不是治疗乐清地区儿童肺炎链球菌感染的有效药物。     

肺炎链球菌对大环内酯类抗生素耐药情况及耐药基因研究

目的调查上海地区肺炎链球菌对红霉素的敏感度，研究肺炎链球菌对大环内酯类抗生素耐药机制。方法对中山医院57株临床分离肺炎链球菌进行红霉素药敏试验；应用聚合酶链反应（PCR）技术对上海4所医院中分离的53株红霉素耐药肺炎链球菌检测耐药基因（ermB，mefA，merE）。结果57株肺炎链球菌中12株（21．0％）敏感，3株（5．3％）中介，42株（73．7％）耐药。53株红霉素耐药肺炎链球菌中，ermB基因、mere基因、mefA基因分别在51株（96．2％）、22株（41．5％）和1株（1．9％）中检测到。其中21株（39．6％）同时检测到ermB基因和mefE基因，1株（1．9％）同时检测到ermB基因和mefA基因，1株（1．9％）未检测到ermB基因、mefE基因或mefA基因。结论上海地区肺炎链球菌对大环内酯类抗生素耐药率较高。ErmB介导的靶位改变是最常见的耐药机制，mef（特别是mefE）介导外排机制引起者也较常见。     

广州地区ermB和mef E基因介导对红霉素耐药的肺炎链球菌及其耐药性分析

目的了解广州地区对红霉素耐药的肺炎链球菌中ermB及mefE基因分布，比较ermB基因与mefE基因对红霉素耐药的肺炎链球菌的耐药性。方法用克林霉素纸片法检测199株对红霉素耐药的肺炎链球菌，并用浓度梯度法检测其耐药性。结果199株对红霉素耐药的肺炎链球菌中，ermB、mefE基因介导的耐药率分别为70．9％(141／199)和29．1％(58／199)。141株ermB基因介导对红霉素耐药的肺炎链球菌，对青霉素(MIC500．19μg／ml、MIC901．5μg／ml)、阿莫西林／克拉维酸(MIC500．19μg／ml、MIC901．0μg／ml)、头孢曲松(MIC500．19μg／ml、MIC900．75μg／ml)、头孢呋辛(MIC500．38μg／ml、MIC902．0μg／ml)、头孢克洛(MIC502．0Vg／ml、MIC9032．0μg／ml)的不敏感率分别为58．2％、0．7％、21．3％、46．1％和51．1％。58株mefE基因介导对红霉素耐药的肺炎链球菌，对青霉素(MIC500．5μg／ml、MIC901．5Vg／ml)、阿莫西林／克拉维酸(MIC500．38μg／ml、MIC901．0μg／ml)、头孢曲松(MIC500．38μg／ml、MIC900．75μg／ml)、头孢呋辛(MIC501．0μg／ml、MIC903．0μg／ml)、头孢克洛(MIC506．0μg／ml、MIC9048．0μg／ml)的不敏感率分别为67．2％、0、19．0％、58．6％和62．1％。结论广州地区对红霉素耐药的肺炎链球菌，其耐药机制以ermB基因介导为主；ermB基因介导的红霉素耐药水平高于mefE基因介导的耐药性。     

肺炎链球菌对抗菌药物的耐药性调查及对大环内酯类抗生素耐药机制研究

目的：调查成都地区肺炎链球菌对抗菌药物的敏感性，研究成都地区肺炎链球菌对大环内酯类抗生素耐药机制。方法：收集2001年9月-2002年9月成都地区临床分离的肺炎链球菌，测定其对13种抗菌药物的耐药性及对大环内酯类抗生素的耐药表型；用聚合酶链反应(PCR)扩增耐药基因ermB和mefA，并对ermB和mefA进行基因序列分析。结果：82株肺炎链球菌中13株对青霉素低度耐药(占15．9％),肺炎链球菌对大环内酯类抗生素和克林霉素表现出较高的耐药率，对红霉素和克林霉素耐药率分别为80．5％(66／82)和68．3％(56／82)。耐大环内酯类肺炎链球菌中,96．4％菌株表现为内在型耐药。标准菌株ATCC49619及16株红霉素敏感菌株均未检测到ermB基因及mefA基因；ermB基因和；mefA基因分别在62和11株耐红霉素肺炎链球菌中检测到，其中7株菌同时检测到ermB基因和mefA基因。所测ermB和mefA基因序列与基因库收录序列高度一致。结论：成都地区临床分离的肺炎链球菌对青霉素耐药率较低，但对大环内酯类抗生素和克林霉素耐药却非常普遍。ermB基因介导的靶位改变是成都地区肺炎链球菌对大环内酯类抗生素的主要耐药机制。     

肺炎链球菌红霉素相关耐药基因与耐药表型的关系

目的:了解肺炎链球菌(Streptococcuspneum oniae,SP)临床分离株红霉素耐药基因的流行状况及和耐药表型的关系。方法:对住院儿童分离到的43株肺炎链球菌进行红霉素药敏试验,并用PCR法检测与红霉素耐药相关的红霉素核糖体甲基化酶基因(ermB)、主动外排转运基因(mefA)。结果:43株肺炎链球菌红霉素药敏试验40株耐药(占93%),3株敏感。红霉素ermB基因总检出率为76.7%(33/43),mefA基因总检出率为23.3%(10/43)。3株红霉素敏感的肺炎链球菌均未检出ermB基因和mefA基因;40株红霉素耐药肺炎链球菌ermB基因和mefA基因的PCR检出率分别为82.5%(33/40)和25%(10/40)。共有35株肺炎链球菌检出ermB基因或/和mefA基因,其中单独携带ermB基因的耐药表型为25株(占71.4%);单独携带mefA基因的耐药表型2株(占5.7%);同时携带ermB基因和mefA基因的耐药表型8株(占22.9)%。结论:ermB基因和mefA基因同时表达或单独表达均可导致红霉素耐药,ermB基因表达是儿童肺炎链球菌对红霉素耐药的主要原因,mefA基因表达是造成对红霉素耐药的次要原因。红霉素已不是治疗肺炎链球菌的有效药物。     

老年人呼吸道革兰阳性球菌对大环内酯类抗菌药物耐药监测研究

将我院2002年1月至2006年6月间呼吸内科、干部病房及老年病科收治的老年下呼吸道感染患者的痰进行了针对性的病原菌分离培养，共检出优势菌1724株（1658株细菌和66株真菌），对其中的金黄色葡萄球菌和肺炎链球菌进行了大环内酯类抗菌药物耐药性监测和分析，并对肺炎链球菌由ermB和mefE基因介导的红霉素耐药进行调查。     

肺炎链球菌对大环内酯-林可酰胺-链阳菌素的耐药监测及耐药机制研究

目的研究肺炎链球菌对大环内酯-林可酰胺-链阳菌素类抗菌素的耐药机制。方法K-B纸片法测定肺炎链球菌对红霉素、克林霉素、泰利霉素和喹奴普汀/达福普汀的耐药性。对全部红霉素耐药菌株和部分红霉素敏感菌株用聚合酶链反应(PCR)检测ermB和mefA基因。结果97株肺炎链球菌对红霉素、克林霉素、泰利霉素和喹奴普汀/达福普汀的耐药率分别为60.8%、58.8%、0和0。59株红霉素耐药菌株均检出ermB和/或mefA基因,其中34株(57.6%)ermB阳性,18株(30.5%)ermB和mefA同时阳性,7株(11.8%)mefA阳性。5株敏感菌株ermB和mefA基因均为阴性。结论本研究显示肺炎链球菌对泰利霉素和喹奴普汀/达福普汀高度敏感,而对红霉素和林可霉素则表现出较高的耐药性。肺炎链球菌对大环内酯-林可酰胺-链阳菌素的耐药机制以ermB基因介导的靶位改变为主。     

葡萄球菌对大环内酯类抗生素耐药及诱导耐药表型与基因型的相关性研究

目的了解葡萄球菌对大环内酯类抗生素生物耐药表型与基因型的符合情况,分析耐药基因检出与生物耐药诱导的关系,并预测细菌耐药流行趋势,从基因水平指导临床合理用药。方法用KB纸片法检测本院2004-2005年分离的136株葡萄球菌对红霉素和克林霉素的耐药性,以聚合酶链反应（PCR）法检测葡萄球菌MsrA、vgb、Sat4、ermA、ermB、ermC、mphA、MefA／E、ereA、ereB10种耐药基因,并将其耐药表型与基因型进行比较。将对红霉素表型敏感而耐药基因阳性的菌株进行诱导,对比诱导前后药敏结果。结果136株葡萄球菌对红霉素耐药的占80．88％,对红霉素和克林霉素同时耐药占43．38％,结果表明葡萄球菌对大环内酯类有较高的耐药率。耐药基因检测结果表明,共检出MsrA、Sat4、ermA、ermB、ermC、mphA、ereB7种耐药基因,耐药基因检出率为83．82％。其中核糖体甲基化酶基因ermC的阳性率最高,占检测基因的67．54％;其次为Sat4、MsrA、ereB、ermA、ermB、mphA,分别占50．00％、28．95％、22．81％、15．80％、9．65％、1．75％。MRSA、MRCNS、MSSA、MSCNS菌株耐药基因的检出率分别为100．00％、90．78％、73．08％、55．55％。11株对红霉素敏感而耐药基因阳性的菌株经红霉素诱导后有9株转为耐药,诱导阳性率为81．82％。结论葡萄球菌对大环内酯类抗生素有较高的耐药性,在部分敏感株中检测出耐药基因,经诱导可产生耐药,提示菌株有潜在耐药性,需特别引起临床的重视。临床微生物实验室应同时开展表型及基因型检测,指导临床更加合理使用抗生素。     

High proportion of pharyngeal carriers of commensal streptococci resistant to erythromycin in Spanish adults

The presence of erythromycin-resistant (ErR) commensal streptococci in the throat of 110 healthy subjects and 87 patients with pharyngitis was investigated. The resistance determinants were studied by PCR using the primers for mef and erm genes, followed by hybridization and sequencing analysis. Overall, 94.4% of the subjects carried one or more ErR strains in their pharynx. A total of 253 ErR strains was studied: 127 (50.2%) showed constitutive or inducible resistance to clindamycin (MLS(B) phenotype) and 126 (49.8%) were susceptible to clindamycin (M phenotype). In 50 subjects (25.4%) both phenotypes were detected. The ermB gene was predominant among the MLS(B) phenotype strains (97.6%). The mefA (mefA/mefE) gene was detected in 100% of the strains with the M phenotype. One Streptococcus oralis strain bearing the MLS(B) phenotype carried both mefA and ermB genes. The mefA gene from clinical isolates of Streptococcus mitis and S. oralis was transferred by conjugation to an erythromycin-susceptible Streptococcus pneumoniae strain.     

mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae.

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.     

Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates

Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.     

儿童鼻咽部肺炎链球菌携带株研究

目的了解武汉地区健康儿童肺炎链球菌带菌状况、耐药性、耐药基因及血清型流行情况。方法收集武汉地区2所幼儿园469名健康儿童的鼻咽拭子标本,分离鉴定肺炎链球菌,琼脂稀释法测定其对12种抗菌药物的MIC;PCR检测红霉素耐药基因ermB和mefA;“荚膜肿胀”试验进行血清学分型。结果469份鼻咽拭子标本共分离出116株肺炎链球菌,分离率为24.7%。存活的114株中,肺炎链球菌对青霉素的敏感率为51.8%(59/114),对红霉素的敏感率为13.2%(15/114)。99株红霉素耐药肺炎链球菌中,ermB基因总检出率为98.0%(97/99),其中30株(31.6%)同时具有ermB和mefA基因,2株红霉素低耐株仅检出mefA基因。血清分型涉及16个血清型、群,主要分布在19、23、6和14血清群。结论武汉地区肺炎链球菌耐药性高,多为多重耐药菌株,红霉素耐药基因主要为ermB,19、23、6血清群多重耐药株分布广泛。     

Distribution of mefE and ermB genes in macrolide-resistant strains of Streptococcus pneumoniae and their variable susceptibility to various antibiotics.

From February to October 1995, 62 erythromycin-resistant strains of Streptococcus pneumoniae isolated at Yamanashi Red Cross Hospital were tested to determine their susceptibility to various macrolides, subjected to resistance induction tests by the disc diffusion method and analysed for genes encoding resistance to macrolides (ermB and mefE). On the basis of resistance induction testing, the isolates were classified as having either inducible (59.7%) or non-inducible (40.3%) macrolide resistance. The ermB gene was always detected in resistance-inducible type isolates, either alone or in combination with mefE. The mefE gene alone was found only in non-inducible type isolates. Isolates with non-inducible resistance (those with only the mefE gene) had an intermediate level of resistance to 14-membered macrolides, and were susceptible to rokitamycin, a 16-membered macrolide. According to NCCLS guidelines, 9.6% of S. pneumoniae strains were judged to be susceptible to penicillin, 62.9% of reduced susceptibility and 27.4% penicillin resistant. No correlation was detected between the presence of particular macrolide-resistance genes (ermB, ermB + mefE, or mefE) and resistance to penicillin G.     

Rise of Streptococcus pneumoniae isolates containing both erm(B) and mef(E) genes from an adult tertiary care community hospital system

The emergence of macrolide- and lincosamide-resistant Streptococcus pneumoniae is a worldwide concern. Of particular interest is the increasing prevalence of erythromycin and clindamycin-resistant isolates containing both erm(B) and mef genes. This study determined the prevalence of erythromycin and clindamycin resistance in 596 clinical S. pneumoniae isolates from 2 adult tertiary care hospitals over a 4-year period (2001-2004). Erythromycin resistance increased from 24% to 34%, but S. pneumoniae isolates resistant to clindamycin as well as to erythromycin increased from 3% in 2001 to 15.5% in 2004 (5-fold increase). Among erythromycin-resistant isolates, those also resistant to clindamycin (MLS(B) phenotype) increased 3-fold (12.8-45%). Of forty-one erythromycin/clindamycin-resistant S. pneumoniae isolates tested, 29 (71%) contained both erm(B) and mef(E) genes. Pulsed-field gel electrophoresis performed on 28 erm(B) + mef(E) positive isolates identified 2 predominant and possibly related clones, which made up 64% of the isolates.     

某地区部分医院肺炎链球菌对红霉素的耐药性与基因分析

目的 了解某地区临床分离的肺炎链球菌(SPN)对红霉素的耐药基因流行情况及与耐药表型的关系.方法 采用微量琼脂稀释法,对2008年1月至2009年12月某地区部分医院临床分离到的98株SPN对红霉素的耐药状况进行分析,并用PCR法检测与红霉素耐药基因的关系.结果 98株SPN对红霉素的药敏结果显示,耐药87株,中敏2株,敏感9株;在红霉素耐药菌株中,含有ermB基因70株,含有Mef基因18株,含有ermA基因9株.9株红霉素敏感SPN均未检出ermB基因.结论 ermB基因表达是SPN对红霉素耐药的主要原因,并同时使克林霉素耐药.红霉素已不是治疗SPN的有效药物.     

Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005

The aim of this study was to describe the genetic characteristics of Streptococcus pyogenes showing the MLSB phenotype of macrolide resistance from 1999 to 2005 in Spain and to highlight the substantial increase in these isolates in the last few years. The antimicrobial susceptibilities of 17,232 group A streptococci isolated from Madrid and Gipuzkoa from 1999 to 2005 were studied. The presence of the resistance genes ermA, ermB, mef, tetM, and tetO and the presence of the intTn and xis genes of the Tn916-Tn1545 transposon family were studied in a sample of 739 MLSB-resistant isolates. The epidemiological relationships among these isolates were analyzed by emm typing, T typing, and multilocus sequence typing. Erythromycin resistance was found in 21.3% of the isolates analyzed (annual variation of 14.3% to 28.9%). Until 2003, most erythromycin-resistant isolates showed the M phenotype, but in 2004 and 2005, about 50% of isolates showed the MLSB phenotype. Among the MLSB-resistant isolates studied, 16 clones were identified. The most prevalent clone was a strange emm11/T11/ST403 clone with a null yqiL allele. All but one of the 463 emm11/T11/ST403 isolates carried the ermB, tetM, intTn, and xis genes. The second most prevalent MLSB-resistant clone was emm28/T28/ST52, which comprised two subclones: one bacitracin-resistant, tetracycline-susceptible subclone carrying the ermB gene (n=115) and another bacitracin-susceptible, tetracycline-resistant subclone carrying the ermB and tetM genes (n=33). The rapid diffusion of these two clones, and especially of emm11/T11/ST403, caused the large increase in MLSB-resistant S. pyogenes isolates in Spain, suggesting a potential ability for international dissemination.