特地唑胺对利奈唑胺不敏感革兰阳性球菌的体外抗菌活性研究

摘要:目的检测利奈唑胺不敏感革兰阳性球菌对新型噫唑烷酮类抗菌药物特地唑胺的敏感性，并探讨特地唑胺不敏感菌株 的耐药机制。方法收集临床分离非重复革兰阳性球菌 170株,包括利奈唑胺耐药头状葡萄球菌46株、利奈唑胺敏感头状葡 萄球菌19株、利奈唑胺不敏感肠球菌55株、利奈唑胺敏感肠球菌12株、甲氧西林耐药金黄色葡萄球菌19株、甲氧西林敏感 金黄色葡萄球菌18株、利奈唑胺耐药金黄色葡萄球菌1株。采用微量肉汤稀释法检测所有菌株对特地唑胺和利奈唑胺的最 小抑茵浓度(MIC),并比较两种药物的抗菌活性。采用PCR结合Sanger测序技术分析特地唑胺不敏感革兰阳性球茵fr、optrA 基因携带情况及23S rRNA V区突变。结果利奈唑胺耐药头状葡萄球茵(MICg0>256 μg/mL)对特地唑胺的MIC 值为4~ 32 ug/mL; 利奈唑胺不敏感肠球菌(MIC值4~ 16 μg/mL)中,特地唑胺的敏感率为10.% ,其MIC值为0.5~2 μg/mL;1株利奈 唑胺耐药金黄色葡萄球菌对特地唑胺敏感，MIC值为0.5μg/mL。特地唑胺对利奈唑胺敏感的金黄色葡萄球茵和肠球茵的 MIC值均为0.5 μg/mL,对利奈唑胺敏感头状葡萄球菌的MIC值为0. 125 μg/mL。耐药基因分析显示，特地唑胺耐药头状葡萄 球菌gfr基因携带率为87.0%(40/46) ,23S rRNA V区G2576T的突变率为100% ;特地唑胺不敏感肠球菌optrA 基因携带率为 85.7% (42/49) ,显著高于特地唑胺敏感株的22.2%( P<0.001);1株利奈唑胺耐药特地唑胺敏感的金黄色葡萄球菌携帶cfr基 因。结论对于利奈唑胺不敏感的革兰阳性球菌,特地唑胺的抗茵活性是利奈唑胺的8~32倍,其耐药机制可能与携带optrA 基因及23S rRNA V区G2576T突变有关。     

战创伤涂膜剂对厌氧菌体外抗菌实验研究

目的测定战创伤涂膜剂对产气荚膜梭菌（有芽孢菌）和脆弱拟杆菌（无芽孢菌）标准菌株的最低抑菌浓度（minimum inhibitory concentration,MIC）和最低杀菌浓度（minimum bactericidal concentration,MBC）。方法应用培养基将涂膜剂、氯已定甲硝唑乳膏稀释至不同浓度,分别加入产气荚膜梭菌和脆弱拟杆菌悬液,控制菌液浓度为10^5 efu／ml,厌氧培养后观察细菌生长情况,确定两药的MIC和MBC并进行比较。结果涂膜剂体外对脆弱拟杆菌、产气荚膜梭菌MIC均为1μg／ml,MBC分别为1μg／ml、2μg/ml。氯已定甲硝唑乳膏对脆弱拟杆菌、产气荚膜梭菌MIC分别为4μg／ml、1μg／ml,MBC为8μg／ml、2μg／ml。两药杀菌率均〉99％。结论涂膜剂对有芽孢和无芽孢类厌氧菌有良好的抑落、杀菌作用。     

泰地唑胺对革兰阳性球菌的体外抗菌活性研究

目的研究革兰阳性球菌对新型噁唑烷酮类抗菌药物——泰地唑胺的敏感性,并探讨泰地唑胺不敏感菌株的耐药机制。方法收集1 069株革兰阳性球菌[耐甲氧西林金黄色葡萄球菌(MRSA)202株、甲氧西林敏感金黄色葡萄球菌(MSSA)294株、凝固酶阴性葡萄球菌(CoNS)115株、粪肠球菌206株、屎肠球菌55株、无乳链球菌159株和咽峡炎链球菌群菌株38株]非重复临床分离株。采用微量肉汤稀释法检测所有菌株对泰地唑胺和利奈唑胺的最小抑菌浓度(MIC),分析泰地唑胺和利奈唑胺MIC值的差异,比较2种抗菌药物的抗菌活性;采用聚合酶链反应(PCR)检测泰地唑胺/利奈唑胺不敏感菌株的耐药基因。结果所有葡萄球菌(MIC≤0.5μg/mL)、屎肠球菌(MIC≤0.5μg/mL)和链球菌(MIC≤0.25μg/mL)均对泰地唑胺敏感,粪肠球菌对泰地唑胺的敏感率为94.7%,检测出11株泰地唑胺和利奈唑胺均不敏感粪肠球菌(泰地唑胺MIC=1μg/mL,利奈唑胺MIC=8μg/mL)。泰地唑胺的抗菌活性是利奈唑胺的4～8倍。泰地唑胺/利奈唑胺不敏感菌株只携带optrA基因。结论泰地唑胺作为治疗革兰阳性球菌感染的一种新型抗菌药物,具有较大的应用价值,但携带optrA基因的泰地唑胺不敏感肠球菌值得关注。     

替硝唑对厌氧菌体外抗菌活性的观察研究

目的 测定替硝唑对产气荚膜梭菌（有芽孢菌）和脆弱拟杆菌（无芽孢菌）标准菌株的最低抑菌浓度（mininmm inhibitory concentration,M1C）和最低杀菌浓度（minimum bactericidal concentration,MBC）。方法应用培养基稀释替硝唑至不同浓度,然后分别加入产气荚膜梭菌及脆弱拟杆菌悬液,菌液浓度为10^5cfu／ml,厌氧培养后观察细菌生长情况,确定MIC和MBC。结果替硝唑对脆弱拟杆菌、产气荚膜梭菌的MIC均为1μg／ml,MBC均为2μg／ml。结论替硝唑对有芽孢和无芽孢类厌氧菌都有良好的抑菌、杀菌作用。     

革兰阳性球菌对利奈唑胺耐药机制的研究

目的 对利奈唑胺耐药革兰阳性球菌主要耐药分子机制进行初步研究。方法 采用KB法及E-test法对6株利奈唑胺耐药革兰阳性球菌进行药敏试验,并应用脉冲场凝胶电泳(PFGE),PCR及测序技术对菌株主要分子流行病学及耐药分子机制进行研究。结果 5株柯氏葡萄球菌PFGE分为2型,对利奈唑胺MIC介于16～64 mg/L,菌株均呈现cfr基因阳性、L3存在双位点突变; 1株粪肠球菌对利奈唑胺MIC为8 mg/L,耐药与23SrRNA存在G2576T点突变相关。结论 利奈唑胺耐药革兰阳性球菌在临床的出现应引起广泛关注,为有效进行抗感染治疗应重视对利奈唑胺耐药菌株的临床监测。     

利奈唑胺在大鼠血、脑组织和脑脊液中的药代动力学研究

目的 探讨利奈唑胺在大鼠血、脑组织和脑脊液中药代动力学特性,为临床治疗中枢神经系统感染提供参考.方法 78只清洁型SD大鼠,尾静脉单次注射利奈唑胺54 mg/kg后,采用高效液相色谱法测定大鼠不同时间点的血、脑组织和脑脊液中利奈唑胺的药物浓度,计算其药代动力学参数.结果 注射后0～19h,利奈唑胺在大鼠的血、脑组织和脑脊液的峰浓度（Cmax）分别为31.39、5.11、23.97 μg/ml;消除半衰期（t1/2）分别为2.67、2.42、1.99 h;药物浓度-时间曲线下面积（AUC 0～∞）分别为134.55、28.23、136.43 h·μg/ml,血脑屏障穿透率为76.36％.结论 利奈唑胺能很好地进入脑脊液达到有效的抑菌浓度,具有良好的药代动力学特征和组织穿透性.     

脑脊液与血清利奈唑胺浓度监测指导颅内感染临床治疗的价值

目的监测神经外科手术后颅内感染患者应用利奈唑胺抗感染治疗时脑脊液利奈唑胺药物浓度及血脑屏障通透率,指导利奈唑胺临床应用。 方法选取2019年6月至2019年11月入住青岛大学附属医院神经外科监护室的颅脑术后感染患者6例,静脉应用利奈唑胺抗感染治疗。连续监测患者脑脊液及血液利奈唑胺浓度、脑脊液常规、脑脊液生化及一般生命体征,分析利奈唑胺血脑屏障通透率。 结果利奈唑胺给药前0.5 h的血清和脑脊液药物谷浓度分别为（4.65±2.72）μg/ml和（3.78±1.53）μg/ml。在开始用药后2 h,血清中利奈唑胺的最大平均浓度为（12.53±3.79）μg/ml,而脑脊液中最大平均浓度为（5.55±2.00）μg/ml。脑脊液利奈唑胺曲线下面积/血清曲线下面积约为45%。所有患者应用利奈唑胺抗感染治疗后,颅内感染均得到治愈。 结论利奈唑胺具有满意的血脑屏障通透率,临床治疗效果良好。静脉应用利奈唑胺脑脊液浓度个体间差异大,推荐临床监测脑脊液浓度指导应用。     

利奈唑胺耐药金黄色葡萄球菌耐药机制分析

目的调查临床分离金黄色葡萄球菌对利奈唑胺的耐药情况及耐药机制。方法用Vitek 2系统GP67卡对2019年1月至12月南京鼓楼医院临床分离905株金黄色葡萄球菌进行药敏试验,对检出的利奈唑胺耐药菌株用E-test法复测利奈唑胺最低抑菌浓度(MIC);用PCR扩增及测序技术分析利奈唑胺耐药决定基因cfr、optr A及23S rRNA基因V区;对菌株基因组DNA进行全基因组测序分析,对耐药基因、毒力基因进行生物信息学分析;用多位点序列分型(MLST)技术获得菌株ST分型。结果 905株金黄色葡萄球菌检出1株(0.1%)利奈唑胺耐药株(MIC为16 mg/L),该菌株除对万古霉素和复方磺胺甲噁唑敏感外,对其余常用抗菌药物均耐药。PCR及测序技术显示该菌株携带cfr基因,23S rRNA V区存在T2337G和C2370G核苷酸突变位点;全基因组序列分析显示基因组包含碱基数为2 949 411 bp,总基因数为3 023个,包含59个tRNA编码基因以及7个完整的rRNA基因编码操纵子,获得Gen Bank登录号JAANYO000000000,且该菌株携带包括cfr在内的多种耐药决定基因和毒力基因; MLST分型为新型ST5985。结论利奈唑胺耐药金黄色葡萄球菌临床分离率较低。检出由cfr基因和23S rRNA V区突变介导的新ST型利奈唑胺耐药株。     

利奈唑胺非敏感粪肠球菌相关耐药机制及同源性分析

目的探讨利奈唑胺非敏感粪肠球菌相关耐药机制及同源性。方法收集2014年5月至2015年9月临床分离利奈唑胺非敏感粪肠球菌11株,采用E-test复核菌株对利奈唑胺、万古霉素、替考拉宁的最低抑菌浓度(MIC);PCR扩增和测序检测cfr基因、23S r RNA第5功能区以及L3、L4核糖体位点基因;脉冲场凝胶电泳(PFGE)分析菌株同源性。结果 11株粪肠球菌对利奈唑胺MIC范围为4~48 mg/L,其中耐药7株,中介4株,对万古霉素和替考拉宁均敏感;均未检出cfr基因,在23S r RNA第5功能区及核糖体位点均未检出突变;PFGE显示各菌株电泳条带差异较大,以A克隆最多。结论造成我院粪肠球菌对利奈唑胺敏感性降低机制不明,不存在克隆株的播散。     

肠球菌的临床分布特征及对利奈唑胺等新型抗菌药物的耐药性分析

目的:研究肠球菌属的分布特征及对利奈唑胺、奎奴普丁/达福普丁等新型抗菌药物的耐药性,为临床合理用药提供依据。方法:使用VITEK2-compact全自动微生物分析仪进行细菌鉴定。药敏实验采用最小抑菌浓度(MIC)法,运用统计学软件EpiInfor2000、WHONE5.4分析实验数据。结果:在所有170株受检肠球菌中,屎肠球菌、粪肠球菌、其他肠球菌分别占52.9%、41.2%和5.9%。检出肠球菌株数最多的标本是尿液,其余为胆汁和血液等。药敏结果表明,肠球菌属总体耐药水平较高,仅对万古霉素和利奈唑胺极度敏感,屎肠球菌除对氯霉素、四环素、奎奴普丁/达福普丁的耐药率低于粪肠球菌以外,对其他多数抗生素的耐药率均高于粪肠球菌。利奈唑胺对屎肠球菌和粪肠球菌的MIC值大多为1和2μg/L。72%粪肠球菌对奎奴普丁/达福普丁的MIC值大于2μg/L,而80%的屎肠球菌对其的MIC值则小于2 mg/L。结论:临床肠球菌属感染以屎肠球菌、粪肠球菌为主,屎肠球菌对大多数抗生素的耐药率均高于粪肠球菌。利奈唑胺对屎肠球菌和粪肠球菌均显示出了强大的抗菌活性,而奎奴普丁/达福普丁只对屎肠球菌有较好的抗菌活性。临床治疗应根据药敏试验合理应用抗菌药物,或选用利奈唑胺治疗耐万古霉素的肠球菌所引起的感染,同时密切关注抗菌药物的MIC值变化,以此延长药物的使用寿命。     

醋酸氯己定体外抗菌作用的实验观察

目的观察醋酸氯己定体外抗菌作用。方法采用肉汤稀释法,测定了醋酸氯己定对4种条件致病菌和一种厌氧菌的抗菌效果。结果醋酸氯己定对脆弱拟杆菌MIC为4μg/ml,MBC为8μg/ml;对产气荚膜梭菌的MIC为1μg/ml,MBC为2μg/ml。醋酸氯己定对金黄色葡萄球菌MIC为0.0625μg/ml,MBC为0.125μg/ml;对大肠埃希菌MIC为2μg/ml,MBC为4μg/ml;对铜绿假单胞菌MIC为4μg/ml,MBC为4μg/ml;奇异变形杆菌MIC为8μg/ml,MBC为8μg/ml。结论醋酸氯己定对常见条件致病菌和厌氧菌都有良好的体外抗菌作用。     

醋酸氯己定与替硝唑联合药敏试验观察

目的研究醋酸氯己定与替硝唑协同抗菌效果。方法采用棋盘方阵稀释法,对醋酸氯己定与替硝唑联合应用体外抗菌作用进行了观察。结果测定替硝唑和醋酸氯己定对金黄色葡萄球菌、铜绿假单胞菌、产气荚膜梭菌和脆弱拟杆菌的MIC值,替硝唑分别为200、200、1、1μg/ml,醋酸氯己定分别为0.06、4、1、4μg/ml。替硝唑与醋酸氯己定联合应用对上述4种试验菌的MIC值与各自的单方相比较无变化,两药相加无相关作用,但相互也不干扰对方的抗菌作用。结论应用于创伤涂膜剂中的醋酸氯己定和替硝唑两种主要抗菌成分既无协同作用或相加作用,但也不互相干扰。     

In Vitro and In Vivo Antibacterial Evaluation of Cadazolid,a New Antibiotic for Treatment of Clostridium difficile Infections

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.     

In vitro activities of daptomycin, vancomycin, and penicillin against Clostridium difficile, C. perfringens, Finegoldia magna, and Propionibacterium acnes

Daptomycin has in vitro activity against gram-positive anaerobic bacteria, although limited numbers of species have been tested. We studied the in vitro activities of daptomycin, vancomycin, and penicillin against more than 100 strains each of Clostridium difficile, C. perfringens, Finegoldia magna, and Propionibacterium acnes. Daptomycin Etest MICs and results from time-kill studies were determined for selected strains. For 392 of 421 strains (93%), daptomycin was inhibitory at < or =1 microg/ml, including 15 of 16 strains of C. difficile with elevated linezolid MICs of 8 and 16 microg/ml, all 32 strains with moxifloxacin MICs of > or =4 microg/ml, and all 16 strains resistant to clindamycin. Daptomycin MICs were also < or =1 microg/ml for all 16 F. magna strains resistant to clindamycin and all 32 strains resistant to tetracycline. Only one strain, a C. perfringens strain, had a MIC of >2 microg/ml to daptomycin. Eighty-five and 92.5% of the Etest MICs were within 1 dilution of the agar dilution method for all drugs at 24 and 48 h, respectively. In time-kill studies, a C. difficile strain was inhibited by both daptomycin and vancomycin at 1, 2, 4, 8, and 24 h; colony counts were decreased by 2.3 to 2.9 log at 24 h. Vancomycin was not bactericidal for C. perfringens; however, daptomycin showed bactericidal activity as early as 1 h at four and eight times the MIC and at 2 and 4 h at two and four times the MIC.     

Comparative in vitro activities of LFF571 against Clostridium difficile and 630 other intestinal strains of aerobic and anaerobic bacteria

The in vitro activities of LFF571, a novel analog of GE2270A that inhibits bacterial growth by binding with high affinity for protein synthesis elongation factor Tu, fidaxomicin, and 10 other antimicrobial agents were determined against 50 strains of Clostridium difficile and 630 other anaerobic and aerobic organisms of intestinal origin. LFF571 possesses potent activity against C. difficile and most other Gram-positive anaerobes (MIC(90), ≤ 0.25 μg/ml), with the exception of bifidobacteria and lactobacilli. The MIC(90)s for aerobes, including enterococci, Staphylococcus aureus (as well as methicillin-resistant S. aureus [MRSA] isolates), Streptococcus pyogenes, and other streptococci were 0.06, 0.125, 2, and 8 μg/ml, respectively. Comparatively, fidaxomicin showed variable activity against Gram-positive organisms: MIC(90)s against C. difficile, Clostridium perfringens, and Bifidobacterium spp. were 0.5, ≤ 0.015, and 0.125 μg/ml, respectively, but >32 μg/ml against Clostridium ramosum and Clostridium innocuum. MIC(90) for S. pyogenes and other streptococci was 16 and >32 μg/ml, respectively. LFF571 and fidaxomicin were generally less active against Gram-negative anaerobes.     

In vitro activities of ramoplanin,teicoplanin, vancomycin,linezolid, bacitracin,and four other antimicrobials against intestinal anaerobic bacteria

By using an agar dilution method, the in vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, and five other agents were determined against 300 gram-positive and 54 gram-negative strains of intestinal anaerobes. Ramoplanin was active at or=256 microg/ml. Ramoplanin displays excellent activity against C. difficile and other gram-positive enteric anaerobes, including vancomycin-resistant strains; however, it has poor activity against most gram-negative anaerobes and thus potentially has a lesser effect on the ecological balance of normal fecal flora.     

Comparative susceptibilities to fidaxomicin (OPT-80) of isolates collected at baseline, recurrence, and failure from patients in two phase III trials of fidaxomicin against Clostridium difficile infection

A 10-day course of oral fidaxomicin (200 mg twice a day [b.i.d.]), a potent new macrocyclic drug, was compared to vancomycin (125 mg four times a day [q.i.d.]) in 1,164 adults (1,105 in the modified intent-to-treat [mITT] population) with Clostridium difficile infection in two phase III randomized, double-blind trials at sites in North America and 7 European countries. Of 1,105 mITT patients, 792 (71.7%), including 719/999 (72.0%) in the per-protocol (PP) population, provided a C. difficile strain at baseline, of whom 356 received fidaxomicin with 330 cures (92.7%) and 363 received vancomycin with 329 cures (90.6%). The susceptibilities (MIC(90)) of baseline isolates did not predict clinical cure, failure, or recurrence for fidaxomicin (MIC(90), 0.25 μg/ml for both; range, ≤ 0.007 to 1 μg/ml), but there was a one-dilution difference in the MIC(90) (but not the MIC(50)) for vancomycin (MIC(90), 2 μg/ml [range, 0.25 to 8 μg/ml] for cure and 4.0 μg/ml [range, 0.5 to 4 μg/ml] for failures). A total of 65 (7.9%) "rifaximin-resistant" (MIC > 256 μg/ml) strains were isolated in both treatment groups on enrollment, which increased to 25% for failures at the end of therapy. No resistance to either fidaxomicin or vancomycin developed during treatment in either of the phase III studies, although a single strain isolated from a cured patient had an elevated fidaxomicin MIC of 16 μg/ml at the time of recurrence. All isolates were susceptible to ≤ 4 μg/ml of metronidazole. When analyzed by restriction endonuclease analysis (REA) type, 247/719 (34.4%) isolates were BI group isolates, and the MICs were generally higher for all four drugs tested (MIC(90)s: fidaxomicin, 0.5; vancomycin, 2.0; metronidazole, 2.0; and rifaximin, >256 μg/ml) than for the other REA types. There was no correlation between the MIC of a baseline clinical isolate and clinical outcome. MIC(90)s were generally low for fidaxomicin and vancomycin, but BI isolates had higher MICs than other REA group isolates.     

Characterizations of clinical isolates of clostridium difficile by toxin genotypes and by susceptibility to 12 antimicrobial agents, including fidaxomicin (OPT-80) and rifaximin: a multicenter study in Taiwan

A total of 403 nonduplicate isolates of Clostridium difficile were collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC(90) of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤ 0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤ 0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC(90) of 0.5 μg/ml; range, ≤ 0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤ 2 μg/ml). Nonsusceptibility to moxifloxacin (n = 81, 20.1%) was accompanied by single or multiple mutations in gyrA and gyrB genes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreported gyrB mutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains of C. difficile in the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potent in vitro activity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥ 128 μg/ml raises concerns.