抗菌药物防突变浓度与耐药突变选择窗的研究进展

由于抗菌药物的广泛应用,细菌不断产生耐药变异。尽管人们已对细菌的耐药机理做了大量深入的研究,并在抗菌药物治疗及药物开发方面做了很多工作,但是细菌耐药问题依然变得越来越严重。从目前防细菌耐药突变株选择浓度（MPC）理论所研究的药物、针对的致病菌、测定方法。以及其研究的意义等方面出发,总结了近几年MPC理论的研究概况,提出抗菌药物合理应用的新策略和新思路。     

抗菌药物使用对耐药菌富增影响的研究进展

为制定更为合理的给药方案,防止细菌耐药性的过快增长,介绍了近年来给药方案对细菌耐药性选择性富增的影响的研究进展,认为防突变浓度和突变选择窗可能成为制定合理给药方案的新标准以及今后兽用抗菌药物研究的一个方向.     

抗菌药物的防突变浓度（MPC）

随着抗菌药物的广泛应用,细菌不断发生耐药性变异,以求生存。虽然人们对细菌的耐药机制进行了广泛和深入的研究,并从抗菌药物治疗及药物开发方面做了大量工作,但细菌耐药仍变得越来越严重。部分原因是由于目前基于最低抑菌浓度(MIC)的治疗浓度,仅阻止了大部分敏感细菌的生长,而使耐药突变菌株得到选择性富集扩增。为了减少细菌耐药,人们曾试图采用限制抗菌药物使用及抗菌药物轮换使用等手段,但效果并不理想。为了解决这一难题,国外科研人员于1 999年提出了防突变浓度(mutantpreventionconcentration ,MPC)的概念。这一概念为研究耐药机制…     

基于药效和作用机制拮抗的防耐药性新策略研究

1 基于新型PK/PD的MSW理论及其防耐药用药策略 1.1防细菌耐药突变体选择浓度和突变选择窗最小防突变浓度(MPC)是指抑制细菌耐药突变体被选择性富集扩增所需的最低抗菌药物浓度.     

酒石酸泰乐菌素联合阿莫西林钠缩小猪链球菌耐药突变选择窗的研究

为了使临床合理使用抗菌药物,防止细菌耐药性的产生提供理论依据。本研究探讨了在体外初步联合用药缩小猪链球菌(S.suis)的耐药突变选择窗(MSW)。应用肉汤法富集1010CFU/mL细菌,琼脂平板二倍稀释法分别测定酒石酸泰乐菌素(TLST)、阿莫西林(Amoxil)钠及两药联用后对S.suis分离株D10的防突变浓度(MPC)和MSW。TLST、Amoxil钠单药和两药联用对S.suis分离株D10的MSW分别为9.71、5.16和2.81。TLST和Amoxil钠联合用药使TLST对D10的MSW缩小3.5倍;TLST和Amoxil钠联合用药使Amoxil钠对D10的MSW缩小1.8倍。TLST和Amoxil钠联合用药可缩小各自单药对S.suis分离株D10的MSW。     

氟苯尼考联合多西环素缩小猪源大肠杆菌耐药突变选择窗

为使临床合理使用抗菌药物,防止细菌耐药性的产生提供理论依据。本研究探讨在体外初步联合氟苯尼考和多西环素缩小猪源大肠杆菌耐药突变选择窗（MSW）。应用琼脂二倍稀释法,肉汤法富集10^11：FU/mL细菌分别测定了氟苯尼考、多西环素对15株猪源大肠杆菌临床分离株的最小抑菌浓度（MIC）、防耐药突变浓度（MPC）,并计算出它们各自的MSW,对于2种药物MSW均比较大的菌株S8、S11、S12进行氟苯尼考联合多西环素用药,测定其MPC和MSW。结果显示,从吉林省分离的15株猪源大肠杆菌对氟苯尼考、多西环素耐药率分别为33．3％和46．7％;MPC大于100mg／L的分别占到40％和13．3％;MSW大于50的分别占40％和0．067％。对于S8、S11、S12氟苯尼考联合多西环素用药,联合指数分别为0．75、0．09、0．438,均呈现相加或协同作用。联合用药可使氟本尼考对S8、S11、S12菌株的MPC分别由原来的256．0、128．0、460．8mg／L,变为8．0、8．0、16．0mg／L,均关闭了MSW。结果表明,氟苯尼考联合多西环素可以缩小或关闭猪源大肠杆菌耐药选择突变窗。     

三种抗菌药物对大肠杆菌和胸膜肺炎放线杆菌MIC值及FIC指数的测定

最小抑菌浓度(MIC)是抑制细菌生长的最低药物浓度。细菌对抗菌药物的敏感性,通常以药物常用剂量在血清中的浓度大于MIC为敏感,反之则为耐药[1]。各种病原菌对抗菌药物的敏感性不同,同种细菌的不同菌株对同一种药物的敏感性有差异,检测细菌对抗菌药物的敏感性,可筛选最有疗效的药物。同时,探求合适的联合用药可达到协同作用或相加作用,从而增强疗效,在临床上对控制细菌性传染病的流行至关重要。     

替米考星对胞内金黄色葡萄球菌的抗菌活性

为探究大环内酯类药物替米考星对胞内金黄色葡萄球菌的抗菌活性,本试验以金黄色葡萄球菌ATCC 29213为靶细菌,以小鼠单核巨噬细胞(RAW264.7)为载体细胞,构建金黄色葡萄球菌胞内感染体系,绘制胞内金黄色葡萄球菌的生长曲线,测定替米考星对胞内金黄色葡萄球菌的最低抑菌浓度、最低杀菌浓度、防突变浓度、耐药突变选择窗、抗菌后效应和杀菌曲线。结果显示,胞内金黄色葡萄球菌在0～4、4～18、18～24和24～48 h分别处于迟缓期、对数期、稳定期和衰亡期。最低抑菌浓度、最低杀菌浓度、防突变浓度和耐药突变选择窗分别为4、8、12.8和4～12.8μg/mL。抗菌后效应和杀菌曲线结果显示,替米考星对胞内金黄色葡萄球菌的杀菌特点表现出明显的时间依赖性。结果表明,替米考星对胞内金黄色葡萄球菌表现出较强的抗菌活性,可为治疗由胞内金黄色葡萄球菌引起的奶牛乳腺炎制定合理给药方案提供参考依据。     

临床用药要关注抗菌后效应

细菌与敏感抗菌药物接触生长繁殖会受到抑制，但当抗菌药物在体内消除后，其效能并不是随着药物的消失而马上消失，在一定时间内，细菌的生长繁殖仍然受到持续的抑制，这种特性就是所谓的抗菌后效应（PAE）。抗菌后效应几乎是所有抗菌药物的共性，也是细菌对药物敏感性的结构特征性指标。临床上使用抗菌药物，大多参考的药效学指标主要是最低抗菌浓度（MIC）和最低杀菌浓度（MBC），这种给药方式忽视了药物与细菌之间的作用过程．忽视了药物对细菌持续抑制的潜力，在药效、毒性、费用上并不是最合理的。虽然目前抗菌后效应在兽医临床上研究还较少．但关注抗菌后效应．对于科学设计用药方案，合理使用各种抗菌药物，以期达到优化使用目标，同样具有重要的临床指导意义。     

抗菌药物的合理应用研究进展

自从抗菌药物在医学、兽医临诊实践中被广泛应用以来,已取得良好的疗效。但抗菌药既能发挥防治疾病的有利作用,也会对动物产生损害作用。盲目、滥用抗菌药物带来的危害值得人们关注和反思。抗菌药物的不良反应和滥用抗菌药物的危害,主要表现为细菌产生耐药性、动物体内菌群平衡失调等,其次是药物残留对公共卫生和环境的负面影响。论文强调在了解药动学和药效学原理(峰时间、峰浓度、药时曲线下面积、防突变浓度、抗菌后效应、最小抑菌浓度等)的基础上,指出应该坚持病因学诊断,制定科学的治疗方案,同时要遵守国家相关的法规和条例,做到依法防控疾病。     

Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations

This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 μg/mL.     

Antimicrobial susceptibility testing for bovine respiratory disease: Getting more from diagnostic results

Bovine respiratory disease (BRD) is one of the most common diseases of cattle worldwide. Given the significant bacterial component of this disease, antimicrobial agents remain one of the mainstays of therapy. However, the potential welfare and economic impact resulting from the selection of inappropriate antimicrobial therapy for BRD poses significant risks to both animal and animal owner. To determine the ‘best’ antimicrobial agent for a specific case, the decision-making process needs to incorporate all available evidence, often including the results of bacterial culture and antimicrobial susceptibility testing. While antimicrobial susceptibility testing can be a valuable diagnostic tool, integrating the test results into the clinical decision making process can be a challenging experience. This review details the process by which interpretive criteria for susceptibility tests are developed. Principles for how to best integrate antimicrobial susceptibility testing, both at the individual animal test and aggregate test levels, into the clinical decision making process are discussed. Non-traditional testing methodologies and how they may improve susceptibility testing in the future are also reviewed.     

Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs

The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo.     

Treatment of clinical mastitis. Using antimicrobial susceptibility profiles for treatment decisions.

Antibiotic susceptibility of clinical mastitis pathogens has traditionally been determined using the agar diffusion method that was designed to reflect the antibiotic concentration in serum and interstitial fluid of human patients after receiving oral or intravenous administration. The validity of applying agar diffusion susceptibility breakpoints derived from humans to the treatment of bovine mastitis has not been established and is extremely questionable because (1) bovine milk pH and electrolyte, fat, protein, and leukocyte concentrations, growth factor composition, and pharmacokinetic profiles are different than those for human plasma and (2) human bacterial pathogens are often different from bovine mastitis pathogens. Also, antibiotics are distributed unevenly in an inflamed gland, and high antibiotic concentrations can alter neutrophil morphology or function in vitro and thereby inhibit bacterial clearance in vivo. The current cost of antibiotic susceptibility testing is $12 to $20 per test. Because the dairy industry is economically driven, any diagnostic test should be validated, have appropriate sensitivity and specificity, and have an acceptable economic return on the cost of testing before it can be routinely recommended. In the authors' opinion, antimicrobial susceptibility testing of mastitis pathogens has not been adequately validated for most mastitis pathogens and antibiotics; therefore, the authors do not currently recommend the use of susceptibility testing to guide treatment decisions for individual cows. Additional research is needed to further define the role, if any, that antimicrobial susceptibility testing should play in the treatment of clinical mastitis.     

Antimicrobial susceptibility testing of bacteria isolated from animals: methods for in-vitro susceptibility testing and their suitability with regard to the generation of the most useful data for therapeutic applications

In-vitro susceptibility testing provides valuable informations for choosing the most suitable antimicrobial agent for the control of bacterial infections in animals. Different diffusion and dilution methods, as conducted according to various approved performance standards, can be used to determine the in-vitro susceptibility of bacterial pathogens. In the present article, problems are discussed which arise from the use of different methods and the difficulty to interpret such results. While most approved performance standards were designed for testing of bacteria from human sources, the NCCLS document M31-A2 exclusively focusses on susceptibility testing of bacteria isolated from animals and--in contrast to all other standards--includes veterinary specific breakpoints for a number of antimicrobial agents used in veterinary medicine. Therefore, performance of in-vitro susceptibility testing of veterinary pathogens should follow the recommendations given in the NCCLS document M31-A2. The microdilution method is recommended as the method of choice for susceptibility testing. The result of a microdilution test is given as the minimum inhibitory concentration (MIC). This value provides a quantitative result which precisely indicates the degree of susceptibility of the tested bacterial strain and in return gives the veterinarian a clear guidance whether therapeutic intervention with the antibiotic in question will be successful.     

Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.     

Pharmacokinetic and pharmacodynamic testing of marbofloxacin administered as a single injection for the treatment of bovine respiratory disease

Vallé, M., Schneider, M., Galland, D., Giboin, H., Woehrlé, F. Pharmacokinetic and pharmacodynamic testing of marbofloxacin administered as a single injection for the treatment of bovine respiratory disease. J. vet. Pharmacol. Therap. 35, 519–528. New approaches in Pharmacokinetic/Pharmacodynamic (PK/PD) integration suggested that marbofloxacin, a fluoroquinolone already licensed for the treatment of bovine respiratory disease at a daily dosage of 2 mg/kg for 3–5 days, would be equally clinically effective at 10 mg/kg once (Forcyl^®), whilst also reducing the risk of resistance. This marbofloxacin dosage regimen was studied using mutant prevention concentration (MPC), PK simulation, PK/PD integration and an in vitro dynamic system. This system simulated the concentration–time profile of marbofloxacin in bovine plasma established in vivo after a single 10 mg/kg intramuscular dose and killing curves of field isolated Pasteurellaceae strains of high (minimum inhibitory concentration (MIC) MIC ≤0.03 μg/mL), average (MIC of 0.12–0.25 μg/mL) and low (MIC of 1 μg/mL) susceptibility to marbofloxacin. The marbofloxacin MPC values were 2‐ to 4‐fold the MIC values for all Mannheimia haemolytica, Pasteurella multocida tested. Marbofloxacin demonstrated a concentration‐dependant killing profile with bactericidal activity observed within 1 h for most strains. No resistance development (MIC ≥4 μg/mL) was detected in the dynamic tests. Target values for risk of resistance PK/PD surrogates (area under the curve (AUC) AUC_{24 h}/MPC and T_>MPC/T_MSW ratio) were achieved for all clinically susceptible pathogens. The new proposed dosing regimen was validated in vitro and by PK/PD integration confirming the single‐injection short‐acting antibiotic concept.     

Comparative evaluation of a commercial microdilution method and a conventional disk diffusion method for determination of antibiograms of gram-positive cocci

A commercial broth microdilution system for testing the antimicrobial susceptibility of gram-positive cocci was compared with the standardized disk agar-diffusion method by testing 254 clinical strains of staphylococci and streptococci using both methods. A total of 2,794 parallel determinations were made with 92.3% complete agreement between the 2 methods; of the discrepancies encountered, 3.0% were minor, 2.5% were major, and 2.1% were very major. The results indicate that the commercial microdilution system may provide a reliable quantitative method for antimicrobial susceptibility testing of clinical isolates from animals.     

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.