EDTA-2Na联合抗菌药物对铜绿假单胞菌的体外抑菌及生物膜抑制作用研究

目的 探讨乙二胺四乙酸二钠(ethylenediaminetetraacetic acid disodium,EDTA-2Na)联合抗菌药物对铜绿假单胞菌的体外抑菌及生物膜抑制作用。方法 对临床分离的10株亚胺培南耐药铜绿假单胞菌、8株黏菌素耐药铜绿假单胞菌和8株阿米卡星耐药铜绿假单胞菌,通过微量肉汤稀释棋盘法检测EDTA-2Na分别与亚胺培南、黏菌素和阿米卡星联用以及各自单用时对相应耐药菌株的最小抑菌浓度(minimal inhibititory concentration,MIC),并通过计算部分抑菌浓度指数(fractional inhibitory concentration index,FICI)评价联合抑菌效果;结晶紫生物膜试验检测EDTA-2Na分别与亚胺培南、黏菌素和阿米卡星联用及各自单用时对相应耐药菌株生物膜的抑制作用,通过测量A595值评价抑制生物膜效果。结果 EDTA-2Na与亚胺培南、黏菌素和阿米卡星联用后对相应耐药菌株的抑菌作用表现为协同或相加,对生物膜也具有明显的协同抑制作用。结论 EDTA-2Na联合抗菌药物对铜绿假单胞菌具有良好的体外抑菌作用及抑制生物膜作用。     

铜绿假单胞菌中群体感应系统的研究进展

群体感应系统(quorum sensing)是一种细菌细胞与细胞间的信号传递系统。细菌通过可扩散的小分子信号分子感知细胞群体的密度,从而引起一些特定基因在细菌群体中的协调表达。铜绿假单胞菌中的QS系统包括LasI/LasR和RhlI/RhlR两条主要的信号系统和喹诺酮信号系统。本文系统地介绍了铜绿假单胞菌中QS系统的研究现状和相关应用。     

铜绿假单胞菌临床菌株生物被膜的定量分析

目的 定量分析铜绿假单胞菌临床分离株生物被膜形成能力.方法 采用平板培养结合结晶紫染色法建立生物被膜定量检测法,用该法对56株铜绿假单胞菌临床分离株的生物被膜形成能力进行分析.结果 平板培养结合结晶紫染色法可对铜绿假单胞菌生物被膜形成能力进行定量检测.在所检测的56株铜绿假单胞菌中,46株(82.1%)具有生物被膜形成能力,其中生物被膜形成能力弱者为16株(28.6%),生物被膜形成能力强者为30株(53.6%);10株菌(17.9%)不能形成生物被膜.结论 绝大多数铜绿假单胞菌临床分离株具有生物被膜形成能力,半数以上菌株具有较强的生物被膜形成能力.     

乳铁蛋白多肽嵌合体对泛耐药铜绿假单胞菌生物被膜形成的作用

目的研究乳铁蛋白多肽嵌合体对泛耐药铜绿假单胞菌生物被膜形成的作用。方法在泛耐药铜绿假单胞菌培养基（OD600=0.05）中分别加入乳铁蛋白多肽（LFcin、LFampin）以及乳铁蛋白多肽嵌合体（LFchimera）溶液干预;结晶紫法定量检测生物被膜;以结晶紫染色法,在光镜下观察生物被膜形态。结果乳铁蛋白多肽以及乳铁蛋白多肽嵌合体均能抑制泛耐药铜绿假单胞菌生物被膜形成（P〈0.05）,生物被膜量与多肽浓度呈负相关（P〈0.05）;乳铁蛋白多肽嵌合体的作用强于乳铁蛋白多肽及其混合物（P〈0.05）。结论乳铁蛋白多肽和乳铁蛋白多肽嵌合体,尤其是LFchimera对泛耐药铜绿假单胞菌生物被膜形成具有显著的抑制作用,有望用于泛耐药铜绿假单胞菌感染的治疗。     

亚胺培南联合罗红霉素对铜绿假单胞菌生物被膜的作用

目的本实验研究了罗红霉素对铜绿假单胞菌生物被膜合成的抑制及其与亚胺培南的抗菌增效作用。方法色氨酸法测定多糖蛋白复合物（GLX）;噻唑兰法测定活菌数。结果1／16MIC、1／4MIC的罗红霉素可以显著抑制细菌生物被膜多糖蛋白复合物的合成。1／16MIC、1／4MIC的罗红霉素可以显著增强1／4MIC,1／2MIC及1MIC的亚胺培南对生物被膜细菌的抑菌活性。结论罗红霉素本身对铜绿假单胞菌并无抑菌活性而可以抑制多糖蛋白复合物的合成,显示罗红霉素能提高亚胺培南对生物被膜的渗透从而增强其对铜绿假单胞菌生物被膜的抑菌活性。     

下呼吸道标本铜绿假单胞菌对亚胺培南的耐药性及机制探讨

目的探讨下呼吸道标本铜绿假单胞菌对亚胺培南的耐药性及机制探讨。方法选择下呼吸道标本中培养分离的铜绿假单胞菌75株,从金属β-内酰胺酶,超广谱β-内酰胺酶（ESBLs）和头胞菌素酶（AmpC酶）的产生状况方面来检测其耐药机制。结果 41株对亚胺培南耐药,34株对亚胺培南敏感。耐亚胺培南铜绿假单胞菌中检出产金属β-内酰胺酯酶6株（14.6%）;检出产ESBLs10株（24.4%）,产AmpC酶6株（14.6%）,其中同时产AmpC酶和ESBLs共3株（7.3%）。结论产生金属β-内酰胺酶、ESBLs和AmpC酶是下呼吸道感染患者铜绿假单胞菌对亚胺培能抗生素耐药的主要原因。     

多药耐药铜绿假单胞菌医院感染分布及耐药性分析

目的探讨本院多药耐药铜绿假单胞菌感染分布及其耐药特性特征。方法对126株铜绿假单胞菌的分布特点及药敏结果进行回顾分析。结果 126株铜绿假单胞菌株中痰液分离率最高,占85株（67.46%）;分离菌株中ICU病区所占比率最高,为35.71%;铜绿假单胞菌对环丙沙星耐药率最高（42.9%）,对美罗培南和亚胺培南的耐药率最低,均为7.9%。结论铜绿假单胞菌耐药性较强,临床应依据药敏试验选择合适的抗菌药物治疗。     

铜绿假单胞菌群体感应系统分子受体基因lasR与Ⅰ类整合酶intI1基因表达的相关性分析

目的通过研究铜绿假单胞菌Ⅰ类整合子阳性株群体感应系统基因lasR与Ⅰ类整合子整合酶基因intI1表达水平的相关性,初步探讨群体感应系统对Ⅰ类整合子的调控机制。方法通过荧光定量RT-PCR方法检测铜绿假单胞菌群体QS信号分子受体基因lasR和Ⅰ类整合子整合酶基因intI1在液相菌与生物被膜菌中mRNA的表达水平,分析两基因表达的相关性。结果铜绿假单胞菌在生物被膜中群体感应系统lasR基因及Ⅰ类整合子intI1基因的mRNA表达水平高于液相菌,两者表达水平呈现正相关性(r=0.695,P<0.05)。结论铜绿假单胞菌群lasR和intI1基因表达呈正相关,提示intI1基因表达可能与群体感应系统的调控作用有关。     

革兰阴性杆菌对亚胺培南耐药率的变迁

目的调查近4年革兰阴性杆菌对亚胺培南的耐药率变化。方法收集2003年7月～2006年6月从我院住院患者各种临床标本中分离的革兰阴性杆菌，使用Vitek-60全自动微生物分析仪进行菌种的鉴定和药敏试验，对结果进行回顾性调查。结果共分离出革兰阴性杆菌8714株，97个细菌种，其中肠杆菌科细菌3510株，占40．3％（3510／8714）；非发酵菌5152株，占59．1％（5152／8714）。分离率前5位的细菌分别是铜绿假单胞菌、大肠埃希菌、鲍曼不动杆菌、肺炎克雷伯菌和洋葱伯克霍尔德菌。肠杆菌科细菌对亚胺培南的耐药率非常低，除弗氏柠檬酸杆菌外，低于1％。嗜麦芽寡养假单胞菌、洋葱伯克霍尔德菌、脑膜脓毒性金黄杆菌、铜绿假单胞菌和鲍曼不动杆菌对亚胺培南的耐药率分别为97．8％、98．6％、98．3％、60．7％和45．3％。结论临床分离的革兰阴性杆菌以非发酵菌为主，亚胺培南对肠杆菌科细菌具有非常强的体外抗菌活性，铜绿假单胞菌和鲍曼不动杆菌对亚胺培南的耐药率呈上升趋势。     

碳青霉烯类药物对铜绿假单胞菌生物膜形成及其基因表达的影响

目的 研究碳青霉烯类药物对铜绿假单胞菌生物膜形成和基因表达的影响,为治疗铜绿假单胞菌引起的感染提供分子基础和理论依据。方法 选取3株临床分离的对碳青霉烯类药物敏感,但对其他常用抗菌药物耐药的铜绿假单胞菌(TL1552、TL1728和TL947),采用琼脂稀释法检测3株菌对临床常用抗菌药物的最低抑菌浓度(mininal inhibotory concentration, MIC);结晶紫染色法及扫描电子显微镜观察不同浓度碳青霉烯类药物对铜绿假单胞菌生物膜形成能力和形态的影响;通过转录组测序分析生物膜相关的差异表达基因,并用qRT-PCR进行确认。结果 亚胺培南和美罗培南均能有效抑制铜绿假单胞菌生物膜的形成,但对已经形成的生物膜影响较小;扫描电子显微镜显示美罗培南处理后的生物膜细胞数量、大小和形态均发生了显著改变,继续在不含抗菌药物的培养基上孵育6h后发现生物膜细胞的整体形态基本恢复正常;转录组测序分析及qRT-PCR表明碳青霉烯类药物可导致涉及蛋白质运输、代谢、生物膜形成等相关基因的改变,并能触发生物膜细胞的应激反应。结论 碳青霉烯类药物对铜绿假单胞菌的生物膜形成有抑制作用,但对已经形成的生物膜影响甚微,去除药物后生物膜细胞的形态基本得到恢复,说明铜绿假单胞菌感染后早期治疗和足疗程治疗的重要性。     

Comparative Activity of Carbapenem Testing (COMPACT) study in Germany

The aim of this study was to determine the current susceptibility of hospital isolates of contemporary Gram-negative pathogens to the carbapenems doripenem, imipenem and meropenem. Between May and October 2008, seven centres in Germany were invited to collect and submit Pseudomonas aeruginosa, Enterobacteriaceae and other Gram-negative bacterial Intensive Care Unit (ICU)/non-ICU isolates from patients with complicated intra-abdominal infections (cIAIs), bloodstream infections (BSIs) or nosocomial pneumonia (NP). Susceptibility was determined at each centre by Etest. A central laboratory performed species confirmation as well as limited susceptibility and quality control testing. In total, 363 isolates were collected, comprising 46.0% Enterobacteriaceae, 45.2% P. aeruginosa, 4.7% Acinetobacter spp. and 4.1% other Gram-negatives. Most isolates (47.9%) were collected from NP, 32.8% were from cIAIs and 19.3% from BSIs; 57.3% were obtained from ICU patients. The MIC(90) values (minimum inhibitory concentration for 90% of the isolates) for doripenem, meropenem and imipenem were, respectively, 4, 16 and 32 mg/L against P. aeruginosa, 0.06, 0.06 and 0.5mg/L against Enterobacteriaceae and ≥ 64 mg/L for each carbapenem against other Gram-negative isolates. Using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 81.1%, 75.6% and 79.3% of P. aeruginosa were susceptible to doripenem, imipenem and meropenem, respectively. Against all pathogens combined, MIC(90) values for ICU versus non-ICU isolates, respectively, were 4 mg/L vs. 1mg/L for doripenem, 8 mg/L vs. 1mg/L for meropenem and ≥ 64 mg/L vs. 8 mg/L for imipenem. Doripenem showed comparable activity against P. aeruginosa from patients with BSIs, cIAIs or NP. Similar findings were observed for Enterobacteriaceae and other Gram-negatives, including Acinetobacter spp. Doripenem generally showed similar or slightly better activity than meropenem and better activity than imipenem against Gram-negative pathogens collected in Germany.     

解脲支原体临床分离株生物被膜药物敏感性分析

目的 检测解脲支原体(Ureaplasma urealytium, Uu)临床分离株生物被膜的形成及其对抗菌药物的敏感性,为Uu临床治疗提供参考。方法 通过结晶紫染色半定量法检测Uu临床菌株生物被膜的形成能力;采用微量肉汤稀释法检测并分析Uu临床菌株在浮游状态与生物被膜状态下对四环素、红霉素、阿奇霉素、左氧氟沙星的敏感性。结果 在所检测的25株Uu中64%(16/25)菌株能形成生物被膜,9株不能形成生物被膜。16株Uu形成生物被膜后对四环素和左氧氟沙星的最小生物被膜药物抑制浓度(minimal biofilm inhibitory concentration, MBIC)比浮游菌的最小抑菌浓度(minimal inhibitory concentration, MIC)高4~8倍或以上,差异有统计学意义(P＜0.05);对红霉素和阿奇霉素MBICs增加的0~2倍,差异有统计学意义(P＜0.05)。Uu形成生物被膜后对四环素和左氧氟沙星的耐药率显著提高,差异有统计学意义(P＜0.05);对红霉素和阿奇霉素的耐药率无显著性差异(P＞0.05)。9株不能形成生物被膜Uu菌株,在浮游状态和生物被膜状态培养下对所选4种抗生素体外药敏试验的MIC和MBIC的差异无统计学意义(P＞0.05);对所选4种抗生素耐药率的差异亦无统计学意义(P＞0.05)。结论 Uu临床菌株多数可形成生物被膜,生物被膜形成后降低对四环素和喹诺酮类抗菌药物的敏感性,对阿奇霉素和红霉素的敏感性影响较小。阿奇霉素和红霉素可用于Uu生物被膜临床菌株的治疗。     

Doripenem

Doripenem is a new member of the carbapenem class of beta-lactam antibiotics with broad-spectrum coverage of Gram-positive, Gram-negative and anaerobic pathogens similar to imipenem and, especially, meropenem. This parenteral antibiotic may offer slightly more activity than meropenem against selected pathogens, including coverage of some strains of Pseudomonas aeruginosa strains not susceptible to the other antipseudomonal carbapenems. Empiric therapy with doripenem, as with other antipseudomonal carbapenems, may be useful in hospital and intensive care unit settings where multidrug resistance has emerged, especially in Gram-negative enteric pathogens. When P. aeruginosa or multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci are involved, doripenem must be used in combination. Doripenem has a broad spectrum of activity against many common hospital pathogens, including P. aeruginosa and Burkholderia cepacia, which is similar to meropenem.     

Comparative in vitro activity of carbapenems against major Gram-negative pathogens: results of Asia-Pacific surveillance from the COMPACT II study

Resistance rates amongst Gram-negative pathogens are increasing in the Asia-Pacific region. The Comparative Activity of Carbapenem Testing (COMPACT) II study surveyed the carbapenem susceptibility and minimum inhibitory concentrations (MICs) of doripenem, imipenem and meropenem against 1260 major Gram-negative pathogens isolated from hospitalised patients at 20 centres in five Asia-Pacific countries (New Zealand, the Philippines, Singapore, Thailand and Vietnam) during 2010. Pseudomonas aeruginosa (n=625), Enterobacteriaceae (n=500), and other Gram-negative pathogens including Acinetobacter baumannii (n=135) were collected from patients with bloodstream infection (32.2%), nosocomial pneumonia including ventilator-associated pneumonia (58.1%), and complicated intra-abdominal infection (9.7%), with 36.7% being isolated from patients in an Intensive Care Unit. As high as 29.8% of P. aeruginosa and 73.0% of A. baumannii isolates were not susceptible to at least a carbapenem, whereas the majority of Enterobacteriaceae (97.2%) were susceptible to all carbapenems. Respective MIC(50)/MIC(90) values (MICs for 50% and 90% of the organisms, respectively) of doripenem, imipenem and meropenem were: 0.38/8, 1.5/32 and 0.38/16 mg/L for P. aeruginosa; 0.023/0.094, 0.25/0.5 and 0.032/0.094 mg/L for Enterobacteriaceae; and 32/64, 32/128 and 32/64 mg/L for A. baumannii. Doripenem and meropenem had comparable activity against P. aeruginosa, both being more active than imipenem. All carbapenems were highly potent against Enterobacteriaceae, although imipenem demonstrated higher MIC values than doripenem and meropenem. The three carbapenems showed less activity against A. baumannii. The high prevalence of carbapenem resistance amongst important nosocomial pathogens (P. aeruginosa and A. baumannii) warrants rigorous infection control measures and appropriate antimicrobial use in the Asia-Pacific region.     

D-环丝氨酸抑制铜绿假单胞菌群体感应活性研究

摘要：目的 以铜绿假单胞菌模式菌株PAO1为研究对象,研究抗结核药物D-环丝氨酸通过靶向抑制病原菌群体感应 (quorum-sensing,QS)系统实现抑制病原菌毒力发挥的新应用潜力。方法 用不同浓度的D-环丝氨酸处理铜绿假单胞菌,通过 系列表型实验结合荧光定量PCR以评估D-环丝氨酸对群体感应所调节的毒力因子和相应基因表达的影响,并利用秀丽隐杆线虫 感染模型评估D-环丝氨酸对铜绿假单胞菌毒力抑制的体内活性。结果 D-环丝氨酸表现出良好的铜绿假单胞菌生物膜、绿脓菌 素以及蛋白水解酶抑制活性,显著抑制了QS系统调控基因和下游功能基因的表达,并且可以显著提高秀丽隐杆线虫在铜绿假单 胞菌感染过程的存活率。结论 D-环丝氨酸可以有效抑制铜绿假单胞菌群体感应系统,有望开发成功的抗生素替代药物。     

黄芩苷、黄芩素抑制铜绿假单胞菌生物膜形成的研究

目的:研究黄芩苷、黄芩素对铜绿假单胞菌生物膜形成的影响。方法:选用铜绿假单胞菌X140为研究对象,用32μg·mL-1的黄芩苷、2μg·mL-1的黄芩素干预X140生物膜的培养。以阿奇霉素为对照,采用倍比稀释法对生物膜内活菌菌落计数。扫描电子显微镜(SEM)观察不同培养条件下生物膜的变化。结果:32μg·mL-1的黄芩苷、2μg·mL-1的黄芩素可以显著抑制铜绿假单胞菌的黏附性和抑制生物膜的形成(P<0.01)。结论:黄芩苷、黄芩素可抑制细菌生物膜的形成,影响铜绿假单胞菌的黏附性。     

Synthesis of imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one derivatives as inhibitors of virulence factors production in Pseudomonas aeruginosa

In an attempt to counteract bacterial pathogenicity, a set of novel imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one derivatives was synthesized and evaluated as inhibitors of bacterial virulence. The new compounds were characterized and screened for their effects on the expression of virulence factors of Pseudomonas aeruginosa, including protease, hemolysin, and pyocyanin. Imidazolidine-2,4-diones 4c , 4j , and 12a showed complete inhibition of the protease enzyme, and they almost completely inhibited the production of hemolysin at 1/4 MIC (1/4 minimum inhibitory concentration; 1, 0.5, and 0.5 mg/ml, respectively). 2-Thioxoimidazolidin-4-one derivative 7a exhibited the best inhibitory activity (96.4%) against pyocyanin production at 1 mg/ml (1/4 MIC). A docking study was preformed to explore the potential binding interactions with quorum-sensing receptors (LasR and RhlR), which are responsible for the expression of virulence genes.     

Analysis on the effective dosage regimens for meropenem, biapenem and doripenem against P. aeruginosa infection based on pharmacokinetics and pharmacodynamics theory

Recently, PK/PD (pharmacokinetics/pharmacodynamics) analysis for the antimicrobial dosage method became one of the popular categories in chemotherapy and infectious disease societies world wide. Carbapenems are often used for empiric therapy because of its broad-spectrum and activities against microorganisms. PK/PD analysis is well studied in some antibiotics including carbapenems and it is necessary also from the point of view of prevention for emergence of resistant strains. We report the result of the analysis for the effective dosage regimens of meropenem, biapenem and doripenem against Pseudomonas aeruginosa infection based on PK/PD theory with the MIC distributions against the strains isolated from the patients blood at Keio University in 2004 and 2006. The highest target attainment rate for the free drug 40% time above the MIC (40%T > or = MIC) in traditional infusion with the MIC distribution against P. aeruginosa isolated from the patients blood at Keio University Hospital in 2004 was as follows: 90.89% in 500 mg every 6 hours regimen for meropenem, 83.25% in 300 mg every 6 hours regimen for biapenem, 81.73% in 250 mg every 6 hours regimen for doripenem in the approved maximum daily dose for each agent. The highest target attainment rate for the free drug 40%T > or = MIC in prolonged infusion with the MIC distribution against P. aeruginosa isolated from the patients blood at Keio University Hospital in 2004 was as follows: 100% in 500 mg every 6 hours regimen for meropenem, 83.97% in 300 mg every 8 hours regimen for biapenem, 99.98% in 500 mg every 8 hours regimen for doripenem in the maximum daily dose for each agents. The highest target attainment rate for the free drug 40%T > or = MIC in traditional infusion with the MIC distribution against P. aeruginosa isolated from the patients blood at Keio University Hospital in 2006 was as follows: 80.57% in 500 mg dose in every 6 hours regimen for meropenem, 56.70% in 300 mg every 6 hours regimen for biapenem, 69.44% in 250 mg every 6 hours regimen for doripenem in the maximum daily dose for each agent. The highest target attainment rate for the free drug 40%T > or = MIC in prolonged infusion with the MIC distribution against P. aeruginosa isolated from the patients blood at Keio University Hospital in 2006 was as follows: 89.35% in 500 mg every 6 hours regimen for meropenem, 60.84% in 300 mg every 6 hours regimen for biapenem, 82.78% in 500 mg every 8 hours regimen for doripenem in the maximum daily dose for each agent. The target attainment rates for the free drug Css/MIC > or = 1 with continuous infusion were lower than the target attainment rates for the free drug 40%T > or = MIC in the regimens of prolonged infusion intermittent dose regimens in all three agents using both of the MIC distributions against P. aeruginosa in 2004 and 2006. The result of the PK/PD analysis for meropenem, biapenem and doripenem indicated that intermittent dose with prolonged infusion was the best method to obtain higher target attainment rate.     

Doripenem: S 4661

Doripenem [S 4661] is a new parenteral carbapenem antibacterial undergoing clinical trials in Japan and North America. It is characterised by a 1beta-methyl group and a sulfamoylaminomethyl substituted pyrrolidylthio group at the C2 position. The compound has good activity against both Gram-positive and Gram-negative bacteria and superior activity to meropenem and imipenem against Pseudomonas aeruginosa. It possesses higher stability than imipenem or meropenem against animal dehydropeptidase I. The compound is stable to most beta-lactamases produced by Gram-negative bacteria. Shionogi has stated that doripenem is expected to be effective at low doses. In May 2003, Shionogi and Peninsula Pharmaceuticals (USA) entered a licensing agreement for doripenem under which Peninsula obtained exclusive rights (including sublicense rights) to market and develop the product in North America. Shionogi will manufacture doripenem for worldwide distribution. Doripenem is currently undergoing phase III trials in Japan for pneumonia, chronic respiratory tract infections (RTIs) and urinary tract infections (UTIs). In its November 2002 pipeline, Shionogi stated that it plans to file a NDA in Q4 of 2003. Doripenem is also in phase II studies in hospitalised patients with complicated urinary tract infections including pyelonephritis. In September 2003, Peninsula announced it had completed enrolment in this trial, and results are expected later in the year. Doripenem has also completed two phase I studies in healthy volunteers with a number of different dosing regimens.