肺炎链球菌大环内酯药物耐药性及耐药基因erm、mef的检测

肺炎链球菌是引起呼吸道感染的常见菌.近年来,耐药型肺炎链球菌出现迅猛,在我国和众多亚洲国家尤为明显~([1]).肺炎链球菌对大环内酯类抗生素的耐药率迅速增加,目前已成为一个全球性问题.其中对红霉素的耐药性已成为一个严重的现象~([2]).因此,我们对52株肺炎链球菌进行了大环内酯药物(红霉素和阿奇霉素)的耐药性情况和耐药基因erm(B)、erm(TR)、mef(A)及mef,(E)的检测分析.     

上海地区儿童化脓性链球菌的红霉素耐药基因研究

目的 了解上海地区化脓性链球菌对红霉素的耐药情况及耐药基因谱的特点.方法 2004年11月至2006年6月经复旦大学附属儿科医院传染病门诊诊断为猩红热的患儿,其咽拭子培养分离获得100株化脓性链球菌.应用PCR及序列分析法检测红霉素耐药基因mefA、ermA、ermB在该批菌株中的分布规律及其与红霉素耐药性的关系.结果上海地区化脓性链球菌的红霉素耐药率为98%,克林霉素耐药率为95%,两者耐药性的一致率为97%.100株菌株中含ermB基因的化脓性链球菌94株,占所有菌株的94%,对红霉索耐药率为100%;含mefA基因的化脓性链球菌16株,占所有菌株的16%,对红霉素耐药率为100%;100株菌株中未发现ermA基因.5株菌株未发现ermB基因及mefA基因,其中2株对红霉素敏感,3株对红霉素耐药.mefA基因单独阳性的菌株仅占1%.结论上海地区化脓性链球菌对红霉奈普遍具有较高耐药率,且与克林霉素存在较严重的交叉耐药.ermB基因是决定上海地区化脓性链球菌对红霉素耐药的重要基因.     

肺炎链球菌对红霉素的耐药表型及耐药基因

目的　研究肺炎链球菌对红霉素的耐药表型及耐药基因。方法　根据美国临床实验室标准化委员会标准使用微量肉汤稀释法 ,检测 192株肺炎链球菌对红霉素、克林霉素、青霉素、喹诺酮类抗菌药物的最低抑菌浓度 (MIC)。应用红霉素、克林霉素、螺旋霉素纸片行三纸片扩散法 ,检测 14 8株红霉素耐药肺炎链球菌的耐药表型。应用PCR检测 14 8株红霉素耐药肺炎链球菌携带的耐药基因。结果　肺炎链球菌对青霉素的耐药率 (中介率 耐药率 )为 4 2 7% ,对红霉素、克林霉素的耐药率分别为 77 6 %、6 6 7%。 14 8株红霉素耐药株中 ,耐药基因以ermB基因 (79 1% )为主 ,耐药表型以内在型耐药 (cMLS) (85 1% )为主。携带ermB基因的肺炎链球菌 ,74 4 %的菌株对红霉素的MIC值 >16 0 μg/ml;而携带mefA基因的肺炎链球菌对红霉素的MIC值在 0 5～ 4 0 μg/ml之间。 结论肺炎链球菌对红霉素的耐药率较高 ;耐药表型以cMLS为主 ,耐药基因以ermB介导的靶位改变多见。     

肺炎链球菌耐药性检测及其对大环内酯类抗生素耐药机制的研究

目的了解浙江地区肺炎链球菌临床菌株对临床常用抗生素耐药性以及肺炎链球菌对大环内酯类抗生素耐药机制。方法从浙江省不同地区病人临床标本中分离并鉴定肺炎链球菌138株。采用K-B纸片法和E-test检测上述肺炎链球菌临床菌株对9种抗生素的敏感性。采用PCR检测上述菌株中与大环内酯类抗生素耐药性密切相关的ermB和mefE基因携带情况。分析ermB和mefE基因携带、分布状况与红霉素耐药性关系。结果138株肺炎链球菌临床菌株中,对红霉素耐药率高达93.5%(129/138),对头孢噻肟、头孢呋辛、阿莫西林和左氧氟沙星的耐药率较低(2.9%~4.3%)。上述菌株er-mB基因检测阳性率(91.3%,126/138)明显高于mefE基因(33.3%,46/138)(P0.05),其中27.5%菌株(38/138)同时携带。不携带ermB和mefE基因菌株均对红霉素敏感,仅携带mefE基因菌株对红霉素耐药率(62.5%)明显低于同时携带两种基因菌株耐药率(100%)及仅携带ermB基因菌株耐药率(97.7%)(P0.05)。结论红霉素已不适合作为治疗肺炎链球菌感染性疾病的药物。ermB基因是肺炎链球菌临床菌株携带的红霉素耐药相关优势基因。ermB基因可使肺炎链球菌产生较mefE基因更强的红霉素耐药性。     

肺炎链球菌临床分离株PBP2b基因序列分析及其应用探索

目的通过分析肺炎链球菌PBP2b基因转肽酶编码区的序列差异，建立可区分对青霉素敏感肺炎链球菌（PSSP）与青霉素不敏感肺炎链球菌（PNSSP）的分子生物学方法。方法检测青霉素对肺炎链球菌的最低抑菌浓度（MIC），对菌株PBP2b转肽酶编码区进行PCR扩增和序列分析，根据序列差异设计相应引物和探针，并采用PCR-反向杂交方法区分PSSP与PNSSP。结果43株肺炎链球菌（42株临床分离株及1株标准株）中8株为PSSP，30株为青霉素中介株（PISP），5株为青霉素耐药株（PRSP）。经PBP2b基因限制性片段长度多态性分析（RFLP）及测序分析，PSSP与PNSSP（PISP＋PRSP）存在谱型及序列差异。根据序列差异建立的检测方法可正确区分PSSP与PNSSP。结论肺炎链球菌PSSP和PNSSP菌株的PBP2b基因转肽酶编码区之间存在可供鉴别的序列差异，据此可建立快速、特异的分子生物学方法，有望为该菌耐药基因的快速诊断提供客观依据。     

红霉素耐药粪肠球菌耐药基因erm(A)、erm(B)和erm(C)分布及耐药性分析

目的了解临床分离株红霉素耐药粪肠球菌耐药特点及耐药基因erm(A)、erm(B)和erm(C)分布特点及耐药机制。方法采用BD Phoenix-100全自动细菌鉴定/药敏系统对2010~2016年临床分离的313株粪肠球菌进行药敏试验,并用微量肉汤稀释法检测红霉素的MIC值,比较红霉素耐药组和红霉素敏感组细菌的耐药性差异,使用PCR方法检测erm(A)、erm(B)和erm(C)基因并分析其分布特点。结果 313株粪肠球菌主要分离自患者的中段尿、血液、胆汁和分泌物,红霉素耐药组和红霉素敏感组细菌对万古霉素、利奈唑胺、氨苄西林、替考拉宁和呋喃妥因的敏感性高,对庆大霉素的耐药率均90%。红霉素耐药基因erm(A)、erm(B)和erm(C)基因检出比例分别为3.51%、66.77%和0.32%;携带红霉素耐药基因erm(A)细菌利奈唑胺(LZD)耐药为81.82%。粪肠球菌对红霉素耐药率为76.04%。结论红霉素耐药株粪肠球菌主要携带erm(B)基因。红霉素耐药基因erm(A)阳性菌株LZD的MIC值偏高。     

医院内气载耐甲氧西林金黄葡萄球菌（MRSA）耐药性及耐药基因分析

目的监测医院中气载耐甲氧西林金黄葡萄球菌（MRsA）对临床常用抗生素的耐药性及相关耐药基因。方法用FA-1型六级筛孔撞击式空气微生物采样器采集5所医院的大厅、ICU及病房等场所的空气,分离金黄葡萄球菌,采用头孢两丁纸片法检测MRSA,应用K-B法测定MRSA药敏情况,应用多重PCR扩增MRSA甲氧西林耐药基因rrlecA,氨基糖苷修饰酶基因aacA-aphD,大环内酯类23srRNA甲基化酶基因ermA、errnC,四环素类核糖体保护蛋白基因tetM、tetK以及金黄葡萄球菌属特异性基因16srDNA。结果共采集空气样品250份,分离气载金黄葡萄球菌219株,其中MRSA88株。所有气载MRSA均对青霉素及头孢曲松耐药,对庆大霉素、红霉素及四环素耐药率〉90％,但均对万古霉及素替考拉宁敏感。所有MRSA菌株iTIeCA基因和16srDNA基因均阳性,vat（A）、vat（B）、vat（C）基因均阴性。96％以上MRSA菌株可同时检出氨基糖苷类、红霉素类和四环素类耐药基因,呈现多重耐药;携带aacA—aphD、erm（A）或（和）erm（C）、tetM或（和）tetK耐药基因的MRSA菌株总检出率分别为96．59％、100％和96．59％。结论5所医院空气中均存在MRSA分布且呈多重耐药性,相关耐药基因检出率高。因此应加强对医院空气中致病菌的监测与消毒,预防和控制医院气源病原微生物感染的发生。     

114株耐碳氢酶烯肺炎克雷伯杆菌耐药表型及基因分析

目的:分析114株耐碳氢酶烯肺炎克雷伯杆菌（CRKP）的耐药表型与基因型,为控制耐碳氢酶烯肺炎克雷伯杆菌传播及临床治疗提供依据。方法： 选取住院患者痰样本分离的114株CRKP,采用VITECK2全自动细菌鉴定仪进行菌种鉴定并验证其对碳青霉烯的药敏性。采用改良碳青霉烯灭活法（mCIM法）检测CRKP的耐药表型;采用PCR法检测碳氢酶烯酶blaKPC、blaNDM、blaIMP、blaVIM、blaOXA 4耐药基因。结果： 114株CRKP经mCIM法检测阳性率为95.61%,耐药表型均为KPC型。耐药菌对青霉素类、头孢菌素类和碳青霉烯类药物的耐药率均在95%~100%;对氨基糖苷类、喹诺酮类、四环素类和氯霉素类药物的耐药率分别为70%~75%、82%~87%、90%~95%和80%~84%（均P<0.05）。经PCR检测,blaKPC基因阳性检出率为100%。脉冲凝胶电泳（PFGE）分子分型共分为13个谱型,优势型别为KPN01.005型（35株）,各菌株间相似性系数达92.0％以上。结论：宁乡市人民医院CRKP的耐药十分严重,与其携带blaKPC耐药基因密切相关。应加强细菌耐药性监测和医院感染的监控。     

长春地区猪链球菌对大环内酯和林克酰胺类耐药的分子机制研究

目的　分析长春地区猪链球菌耐大环内酯类和林克酰胺类的分子机制。方法　采用微量稀释法和双纸片扩散法分别测定相关抗生素的耐药谱和红霉素耐药型 ,并以猪链球菌的染色体DNA为模板 ,PCR扩增ermB基因和mefA/E基因 ,然后将其克隆到pMD18-T载体中 ,用双脱氧链末端终止法测定DNA序列后进行序列分析。结果　 2 2株猪链球菌的临床分离菌株均扩增出ermB基因 ,而未扩增出mefA/E基因 ;对四环素、环丙沙星和大环内酯和克林霉素有高的耐药率和耐药水平 ;红霉素的耐药型以CR型为主。同源性分析显示 ,ermB基因的差异为 36 %～ 10 0 % ,与GenBank中的肺炎链球菌、粪肠球菌等的erm基因有 98%～ 10 0 %的同源性。结论　长春地区猪链球菌对MLSB 的耐药是红霉素甲基化转移酶所介导的 ,以CR型为主的耐药 ,编码该类耐药的是ermB基因 ,并与人源及动物源性的耐药基因可能存在着广泛的交换。     

青霉素结合蛋白PBP1a的克隆表达和对肺炎链球菌耐药性的影响

目的克隆并表达肺炎链球菌青霉素结合蛋白PBP1a,探讨其对青霉素耐药的影响。方法化学合成肺炎链球菌含STMK区的pbp1a基因片段,PCR扩增后进行1.0%琼脂糖凝胶电泳鉴定。将扩增的pbp1a基因连接pUC19载体,构建重组质粒pUC19-pbp1a,转化DH5α,PCR扩增后进行1.5%琼脂糖电泳分析和DNA测序鉴定。将pUC19-pbp1a转化对青霉素敏感、中度敏感和耐药,并存在不同的pbp1a、pbp2b突变的肺炎链球菌,SDS-PAGE分析PBP1a的表达,并测定青霉素对转化菌的MIC。结果 pbp1a基因扩增产物经琼脂糖凝胶电泳为330bp,大小与预期一致。重组质粒基因全长为360bp,扩增产物约330bp,测序无碱基缺失、插入等突变。重组质粒转化存在不同的pbp1a、pbp2b突变和对青霉素不同敏感性的肺炎链球菌后,大量表达约110ku蛋白,其中对青霉素敏感和中度敏感的无pbp1a突变株MIC无变化,对中度敏感菌中的pbp1a突变株MIC略有变化,对存在pbp1a突变的耐药菌株MIC显著降低(P<0.01)。结论在肺炎链球菌中成功表达了青霉素结合蛋白PBP1a,可使其对青霉素的耐药性显著降低。     

Evolution of erythromycin-resistant Streptococcus pneumoniae from Asian countries that contains erm(B) and mef(A) genes

To investigate the genetic characteristics of erythromycin-resistant Streptococcus pneumoniae in Asian countries, 110 pneumococcal isolates were analyzed by multilocus sequence typing (MLST). MLST analysis showed that 2 clonal complexes--CC236 (Taiwan19F-14 clone) and CC81 (Spain23F-1 clone)--are the major lineages of erythromycin-resistant S. pneumoniae in the Asian region. Pneumococcal isolates containing both the erm(B) and the mef(A) genes are thought to have originated from the Taiwan19F-14 clone containing the mef(A) gene, after introduction of the erm(B) gene. Further evolution of this variant clone has generated resistant strains with different sequence types. Dissemination of these variant clones of the Taiwan19F-14 could be the main reason for the high frequency of pneumococcal isolates containing both erm(B) and mef(A) in some Asian countries. Data suggest that the high prevalence of erythromycin-resistant S. pneumoniae in the Asian region is partly due to the clonal spread of a few multidrug-resistant clones.     

Antimicrobial use and colonization with erythromycin-resistant Streptococcus pneumoniae in Greece during the first 2 years of life.

We evaluated nasopharyngeal colonization with erythromycin-resistant Streptococcus pneumoniae during the first 2 years of life in central and southern Greece. Of 2448 children studied from February 1997 to February 1999, 766 (31%) carried 781 pneumococcal isolates. Ninety-five (3.9%) of the children attended day care centers. Eighteen percent of the pneumococci were resistant to erythromycin (minimal inhibitory concentration 1 to >128 microg/mL), with 67.9% of them carrying the erm(B) gene and 29.2% mef(A) gene products. Four strains possessed neither the erm(B) nor the mef(A) gene. Multidrug resistance occurred in 97% and 40% of isolates carrying the erm(B) and mef(A) gene, respectively. An association was found between the erm(B) gene and serotypes 6B and 23F and between the mef(A) gene and serotypes 14 and 19F. A significant relationship existed between carriage of erythromycin-resistant pneumococci and use of macrolides or beta-lactams in the previous 3 months; the association was strongest when macrolide therapy was administered during the last month (odds ratio, 5.92; P=.0001). The findings indicate the necessity of a judicious use of both macrolides and beta-lactams in young children to reduce the colonization with erythromycin-resistant pneumococci and the subsequent spread of such strains to the community.     

外科住院患者感染耐甲氧西林凝固酶阴性葡萄球菌的耐药基因检测及耐药性分析

目的对南京市外科住院患者感染的耐甲氧西林凝固酶阴性葡萄球菌的耐药相关基因进行检测,以更好地控制病原菌耐药性,减少外科感染。方法从南京市多家医院外科住院患者各类临床标本分离耐甲氧西林凝固酶阴性葡萄球菌70株,经纯培养获单菌落后进行鉴定,并进行药敏试验,然后对耐药基因进行PCR测序,对PCR扩增产物进行电泳检测并记录。结果耐甲氧西林凝固酶阴性葡萄球菌对于头孢噻肟和红霉素100%耐药,对万古霉素耐药率为0,对其他抗菌药物的耐药率大小依次为:左氧氟沙星>诺氟沙星>氨苄西林>妥布霉素>环丙沙星>克林霉素>头孢曲松>庆大霉素>四环素>利福平。PCR检测耐药基因,3株只携带mecA 1种耐药基因。15株携带2种耐药基因,分别为:ermA+mecA、aac(6′)/aph(2′′)+mecA和aph(3′)-III+mecA和tetM+mecA。29株携带3种耐药基因,携带方式分别为:ermA+aac(6′)/aph(2′′)+mecA、aph(3′)-III+ermA+mecA、ermA+tetM+mecA、aac(6′)/aph(2′′)+aph(3′)-III+mecA、aac(6′)/aph(2′′)+tetM+mecA和tetM+aph(3′)-III+mecA。20株携带4种耐药基因,携带方式分别为:aac(6′)/aph(2′′)+tetM+ermA+mecA、aph(3′)-III+tetM+aac(6′)/aph(2′′)+mecA、aph(3′)-III+tetM+ermA+mecA和aph(3′)-III+aac(6′)/aph(2′′)+ermA+mecA。有3株携带5种耐药基因,即ermA+aph(3′)-III+tetM+aac(6′)/aph(2′′)+mecA。结论耐甲氧西林凝固酶阴性葡萄球菌对常用抗菌药物的耐药性普遍较高,5种耐药基因在菌株中出现的频率不同,但所有病原菌都存在mecA基因,因而该菌的耐药性可能与mecA基因有关。     

The emergence of drug-resistant Streptococcus pneumoniae and host risk factors for carriage of drug-resistant genes in northeastern Japan

Our 2-year study includes research into the occurrence, molecular characteristics, and host risk factors for the carriage of drug-resistant strains of Streptococcus pneumoniae as a continuation of our previous report. From September 2001 to June 2003, strains of S. pneumoniae were isolated from the nasopharynx of children with respiratory tract infection in Soma General Hospital. Of the total of 949 strains, 761 (81%) had a decreased susceptibility to penicillin (MIC > 0.12 microg/ml), while 818 (86%) were resistant to erythromycin (MIC > 1 microg/ml) and 789 (83%) were resistant to clarithromycin (MIC > 1 microg/ml). More than half of the strains had decreased susceptibility to meropenem. Gene analysis of 226 randomly selected strains showed that 200 strains (88.5%) had one or more altered pbp genes and 191 strains (84.5%) had mef(A) and/or erm(B) genes. We reviewed the patient backgrounds for previous antibiotic use, age, daycare attendance, and siblings. Previous use of oral beta-lactams has shown a strong relationship with the carriage of altered pbp genes (P value < 0.01), and previous oral macrolide use has been related to the carriage of macrolide-resistant genes (P value < 0.01). The controlled use of antibiotics might be an important factor in preventing the emergence of S. pneumoniae with antibiotic-resistant genes.     

Characterization of macrolide resistance in Gram-positive cocci from Colombian hospitals: a countrywide surveillance.

OBJECTIVE: The characterization of macrolide resistance in Gram-positive cocci recovered from Colombian hospitals. METHODS: The resistance profiles and mechanism of macrolide resistance were investigated in isolates of Streptococcus pneumoniae (1679), Staphylococcus aureus (348), coagulase-negative staphylococci (CoNS) (175), and Enterococcus spp (123). Minimum inhibitory concentrations (MICs) for erythromycin (ERY) and clindamycin (CLI), detection of macrolide resistance genes, phenotypic characterization, and pulsed field gel electrophoresis (PFGE) of macrolide-resistant pneumococci were performed. RESULTS: Resistance to ERY and CLI was 3.3% and 2.3% for S. pneumoniae, 58% and 57% for S. aureus (94% for both compounds in methicillin-resistant Staphylococcus aureus (MRSA)), and 78.6% and 60.7% in methicillin-resistant Staphylococcus epidermidis, respectively. ERY resistance was 62% in Enterococcus faecalis and 82% in Enterococcus faecium. The MLS(B)-type accounted for 71% of S. pneumoniae and 100% of MRSA. The erm(A) gene was prevalent in MRSA, erm(B) in S. pneumoniae and enterococci, and erm(C) in CoNS isolates. Efflux pump genes (mef(A) genes) were mostly identified in S. pneumoniae (24%). The most common genotype amongst ERY-resistant pneumococci was the Spain(6B)-2 clone. CONCLUSIONS: The prevalence of macrolide resistance is low in Colombian pneumococci and high in MRSA (cMLS(B)-type).     

Streptococcus pneumoniae and Streptococcus pyogenes isolated from a paediatric population in Great Britain and Ireland: the in vitro activity of telithromcycin versus comparators

OBJECTIVES: To investigate the antimicrobial susceptibility profiles of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes, isolated from children within Great Britain and Ireland (Northern Ireland and Eire), with particular reference to the new oral ketolide telithromycin. To determine the distribution of macrolide resistance genes within the erythromycin resistant population. METHODS: MICs were determined using NCCLS microbroth dilution methodology and macrolide resistance mechanisms were investigated using PCR. RESULTS: Penicillin susceptibility was found to be 92.6% in S. pneumoniae isolates ( n=831; 3.7% intermediate, MIC 0.12-1 mg/l, 3.7% resistant, MIC >2.0 mg/l) and 100% in S. pyogenes isolates (n=1333) 8.8% of S. pneumoniae and 2.5% of S. pyogenes isolates demonstrated erythromycin-A resistance (EryA(R)). One hundred percent of S. pneumoniae and 99.8% of S. pyogenes isolates were susceptible to telithromycin (MIC相似文献   

Characterization of erythromycin resistance of Streptococcus pyogenes isolated from pharyngitis patients in Korea

Six hundred fifteen isolates of Streptococcus pyogenes were collected over a 6-year period from patients with pharyngitis in Korea. All isolates were characterized in terms of their antibiotic resistance, the phenotypes of erythromycin resistance, the frequencies of erm(B), erm(A), and mef(A) genes, and the emm genotype. The prevalent emm genotypes were emm12 and emm4. Moreover, the emm12 genotype was found to be the most resistant strain to erythromycin. Among the 126 strains demonstrating resistance to erythromycin, those with erm(B) were the most prevalent, accounting for 64.3% of the total. In summary, it is suggested that the S. pyogenes pathogen isolated from pharyngitis patients in Korea developed resistant gene acquisition, as well as a resistant phenotype, according to the annual prevailing emm type. It is also suggested that the emm genotype distribution of erythromycin-resistant strains is correlated to the acquisition of resistant genes.     

Antibiotic resistance genes detected by multiplex PCR assays in Staphylococcus epidermidis strains isolated from dialysis fluid and needles in a dialysis service

The rate of the onset of methicillin-resistant Staphylococcus epidermidis infections is increasing in Tunisia. We have isolated 32 S. epidermidis strains from dialysis fluid and needle cultures in dialysis service. The strains were identified by classic methods (colonial morphology, Gram staining, catalase test, coagulase test, and DNase test) as well as by API ID32 Staph. Susceptibilities to 18 antibiotics were tested with the ATB Staph kit. Most of the tested strains were resistant to penicillin. In addition, the presence of multidrug resistant strains that showed resistance to different antibiotics was recorded. We have characterized these strains by multiplex PCR assay to identify intercellular adhesion genes icaA/icaD associated with the adhesiveness of staphylococci in biomaterials, and to identify representative resistant genes: oxacillin resistance, mecA; erythromycin methylase (ermA, ermB, and ermC), and macrolide efflux gene (msrA and mef). The frequency of the carriage of these genes was icaA/icaD (71.9%), mecA (78.1%), ermA (12.5%), ermB (31.3%), ermC (53.1%), msrA (68.8%), and mef (O%). Although the carriage of the genes and the results of susceptibility testing did not match exactly, it could be judged that the PCR identification of antibiotic resistance genes is rapid and supplementary methods for identifying staphylococci or epidemiological study used for the control of nosocomial infection.     

Bacteremic pneumonia due to multidrug-resistant pneumococci in 3 patients treated unsuccessfully with azithromycin and successfully with levofloxacin.

Three patients with bacteremic pneumonia caused by multidrug-resistant Streptococcus pneumoniae were treated unsuccessfully with azithromycin. One S. pneumoniae isolate carried a mef determinant for an efflux pump; a second isolate had an erm determinant. All 3 patients were successfully treated with levofloxacin, an antipneumococcal fluoroquinolone.