嗜麦芽窄食单胞菌敏感株的适应性耐药及其初步机制
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摘要
背景
近年来,随着广谱抗生素的大量使用,致病力原本不强的条件致病菌嗜麦芽窄食单胞菌(stenotrophomonas maltophilia,SMA)已然成为医院感染的重要病原菌,致使感染率不断上升,导致免疫力低下或长期大量使用广谱抗生素的人群呼吸道感染、心内膜炎及败血症等危及生命的严重感染。体外药敏试验结果证实,嗜麦芽窄食单胞菌对磺胺类、β内酰胺类、喹诺酮类、氨基糖苷类等几乎所有抗生素均可产生耐药性(Resistance)。因此,目前国内尚无学者对嗜麦芽窄食单胞菌的感染提出统一的治疗方案,一些学者的推荐方案仅基于回顾性研究和病例报告,但嗜麦芽窄食单胞菌耐药突变快速,因此其实用价值有限。国外学者推荐的治疗方案也常有悖于药敏试验结果和临床治疗结果,故对于该菌的治疗,国内外一致提倡根据药敏结果选择敏感抗生素进行治疗。
但临床实践证明,即使根据药敏结果选用抗生素,甚至联合几个同时敏感的抗生素进行治疗也不能使感染完全得到控制,这就是临床上治疗该菌时常见的体外药敏试验结果与临床治疗结果不一致的现象,导致大量患者因不能有效控制感染而死亡。但对于这类常见现象,国内学者长期以来不予重视,只是认为由于该菌生长缓慢而突变迅速造成了该结局,并未对其进行深入研究。试验证明,该菌生长并不缓慢而是生长很快,并且如果发生了耐药遗传物质突变应该能从基因水平得以证实,但至今未见到该菌由于基因突变致使体外敏感性与临床疗效不一致的报道。
国外学者对其他革兰阴性菌研究时发现,不同类型的多种抗生素皆可诱导敏感菌产生暂时性耐药,本文按国外惯用定义称为适应性耐药。这种耐药形式可导致细菌耐药表型暂时转变,并认为这种耐药性使敏感菌疗效不佳,即适应性耐药引起敏感菌治疗效果低下,但该现象一直未得到重视。这种耐药性和有耐药遗传物质介导的耐药性不同,它不涉及基因组突变,因此临床微生物实验室不能检测出来,更不知其发生规律,无法报告临床。在特定条件下,敏感的嗜麦芽窄食单胞菌能否产生适应性耐药,这种耐药有无规律,其发生机制是什么,阐明这些,可为临床治疗嗜麦芽窄食单胞菌敏感株作出提示,避免适应性耐药发生。
嗜麦芽窄食单胞菌中Ⅰ型整合子介导的复方新诺明耐药基因的发现,将导致该抗生素耐药传播迅速,其应用会受到限制。喹诺酮类治疗嗜麦芽窄食单胞菌时易诱发敏感株突变,造成多种抗生素耐药致使治疗失败。可见,该菌本身耐药变迁快速,耐药机制复杂,不同医疗机构根据当地耐药监测结果选择符合当地耐药特点的抗生素进行治疗,所以各地治疗方案不尽相同。而对于由该菌引起的危及生命的严重感染,除了采用国外推荐的复方新诺明和喹诺酮类治疗外,仍有很多地方根据药敏报告采用阿米卡星或庆大霉素联合其他抗生素进行治疗。鉴于上述原因以及中国药品管理局(SDA)规定和参照美国临床实验室标准化委员会(NCCLS)常规药敏实验报告时假单胞菌的选药指南,本文选用对庆大霉素、阿米卡星、左氧氟沙星和复方新诺明皆敏感的嗜麦芽窄食单胞菌,对其诱导处理后,再检测其能否对原本敏感的上述四种抗生素产生耐药性,产生规律及初步探讨其耐药机制。
目的
(1)筛选出左氧氟沙星,复方新诺明,庆大霉素和阿米卡星表型敏感且不含抗庆大霉素和阿米卡星修饰酶基因的敏感菌作为受试菌。
(2)分别测定受试菌对庆大霉素、阿米卡星、复方新诺明和左氧氟沙星的最低抑菌浓度值(MIC)和最低杀菌浓度值(MBC)。
(3)诱导受试菌适应性耐药,检测处于适应性耐药期细菌的广谱耐药性。
(4)测定不同耐药时期菌体内抗生素含量。
(5)测定不同耐药时期菌体外膜蛋白构成,对有差异的蛋白进行序列测定,确定有差异的蛋白类型。
方法
(1)用K-B法对收集到的88株嗜麦芽窄食单胞菌临床分离株做药敏试验,筛选出对庆大霉素、阿米卡星、复方新诺明和左氧氟沙星敏感株。再用7对抗庆大霉素和阿米卡星的修饰酶基因引物对全部株进行扩增,收集不含任何一种氨基糖苷修饰酶基因且4种抗生素全为敏感的菌株作为受试菌。用液体稀释法测定受试菌对上述4种抗生素的MIC值和MBC值;
(2)将受试菌因为处理方法不同分为试验组和对照组,对照组只是在诱导阶段不加抗生素诱导即空白诱导,其余处理与试验组完全相同。以1×MIC庆大霉素/阿米卡星分别诱导试验组细菌1h,再以4×MIC浓度的庆大霉素/阿米卡星杀菌1h,计算杀菌速度,比较试验组平均杀菌速度和对照组平均杀菌速度大小。若抗生素对试验组的杀菌速度小于对对照组的杀菌速度表示诱导使细菌产生了耐药性;如果两组杀菌速度相同则诱导不产生耐药性;如果对试验组杀菌速度大于对照组杀菌速度表示诱导使试验组细菌产生了敏感性。以阿米卡星诱导细菌后,再分别用复方新诺明/左氧氟沙星进行杀菌,证实诱导菌能否使复方新诺明/左氧氟沙星敏感性降低。
(3)鉴于测定抗生素含量费用昂贵,故本试验只检测01号菌诱导后第2、4、6、8、10h吸收庆大霉素和阿米卡星的量来评价诱导后不同时段(第2、4、6、8、10h)抗生素进入菌体的情况。具体操作如下:一边做诱导试验(诱导试验同前),一边同步收集诱导后第2、4、6、8和10h并加入了抗生素至终浓度为10μg/ml,且杀菌1h的细菌。当诱导试验结果证明诱导耐药成功后,所收集的细菌方能用于测定抗生素含量。离心去除培养上清,PBS(pH 7.0)洗涤沉淀2次,去除PBS后再加新的PBS混匀细菌悬液,超声破菌。取超声后的菌液一滴涂片染色,镜下确定全部菌体都破裂。离心收集上清,上清液中的抗生素含量就是不同耐药时期的细菌吸收至体内的抗生素。采用高效液相色谱法测定此液体中的抗生素含量;
(4)Carlone法提取受试菌诱导后第1h～第10h的外膜蛋白(outer membraneprotein,OMP),聚丙烯酰胺凝胶电泳(SDS-PAGE)分析OMP的构成成分;对有变化的外膜蛋白溶液送交蛋白测序公司进行氨基酸序列测定,测序结果在GeneBank中进行序列同源性比对,确定所测外膜蛋白的种类。
结果
(1)K-B法和PCR法筛选出符合条件的受试菌3株,分别命名为01、02、03号菌株。01、02、03号菌对各抗生素的最低抑菌浓度(MIC)值(μg/ml)和最低杀菌浓度(MBC)值(μg/ml)分别如下:庆大霉素的MIC值分别是0.5、1、0.5;庆大霉素的MBC值分别是1.5、2.5、1.5;阿米卡星的MIC值分别是0.25、0.5、0.5;阿米卡星的MBC值分别是0.5、1.5、1;复方新诺明的MIC值分别是0.25、0.25、0.25;复方新诺明的MBC值分别是0.5、0.5、0.5;左氧氟沙星的MIC值分别是0.25、0.25、0.5;左氧氟沙星的MIC值分别是0.5、0.5、1;
(2)诱导后试验组细菌对4种抗生素均产生耐药性,即从诱导后第4h～第8h抗生素杀菌能力减弱甚至失去杀菌力即产生了适应性耐药。从诱导后第9h以后,抗生素开始逐渐恢复杀菌能力即细菌逐渐失去耐药性又变为敏感菌。对照组细菌没有这种规律性变化;
(3)01号菌试验组细菌在诱导后第2、4、6、8和10小时的庆大霉素含量(gg/m1)情况分别是:1.69、0.81、0.32、0.71和1.57;对照组细菌诱导后第2、4、6、8和10小时的庆大霉素含量(μg/ml)情况分别是:1.71、1.76、1.69、1.73和1.8。01号菌试验组细菌在诱导后第2,4,6,8和10小时的阿米卡星含量(μg/ml)情况分别是:1.56、0.66、0.29、0.59和1.37;对照组细菌诱导后第2、4、6、8和10小时的阿米卡星含量(μg/ml)情况分别是:1.73、1.75、1.69、1.74和1.79。
(4)收集诱导后不同时段的细菌,分别提取其外膜蛋白(outer membraneprotein,OMP),SDS-PAGE电泳显示耐药高峰期试验菌在45kD处及60kD的OMP表达几乎消失;对45kD处及60kD的OMP进行氨基酸序列测定,测定序列与美国国家生物信息中心的蛋白序列进行比对。结果显示,45kD处的蛋白与提交到生物信息中心的该菌的氨基酸运输跨膜蛋白完全同源(amino-acidtransporter transmembrane protein),全长475aa;60kD处的外膜蛋白测序结果与生物信息中心报道的该菌外膜上的转运蛋白(putative transfer protein)完全同源,全长823aa。
结论
(1)庆大霉素和阿米卡星能诱导嗜麦芽窄食单胞菌产生适应性耐药,试验用的4种抗生素皆对适应性耐药期细菌的杀菌能力减弱。由于受试菌没有耐药基因,可见没有耐药基因的变化,耐药表型也能发生变化,即嗜麦芽窄食单胞菌中,耐药不完全由耐药基因决定;
(2)该菌中,某些耐药表型是动态变化着的,其转变有时限性,即经过一定时间之后,其表型又可回复到始发状态,揭示了该菌耐药的复杂性和传统耐药性检测的粗糙;
(3)诱导后细菌吸收抗生素的变化步调与细菌发生适应性耐药步调基本一致,提示耐药表型的变化与抗生素在菌体内的含量有关;
(4)45kD处及60kD处OMP的表达变化规律与适应性耐药的发生步调基本一致,与抗生素进入菌体内的变化规律也基本一致,其氨基酸序列比对显示,这两个OMP都是转运物质进入细菌体内的相关成分蛋白,而受试菌中不含耐药基因,进一步提示45kD处及60kD处的OMP与适应性耐药的产生有密切关系。
Backgroud
With the extensive application of broad-spectrum antibiotics in recent years, Stenotrophomonas maltophilia(SMA) has become an important nosocomial pathogen associated with infections of compromised individuals including respiratory tract, endocarditis,bacteremia and other serious infections.The susceptible test results in the world prove that SMA is resistant to almost every antibiotic widely used in hospitals,so there has no uniform method for treating the infections caused by SMA in our country.Based on a number of retrospective cases or researches in the past, some methods for treating SMA infections have been suggested,but on the other hand,their effectiveness has not been verified in clinical practices as the result of the fast resistance mutations of SMA.Although there has indeed an advocated method for treating infections caused by SMA by some foreign specialists,it has been proved adverse to the results of susceptibility test and clinical treatment.So almost specialists in the world now advocate methods for treating SMA infections according to its susceptibility test results.
But unfortunately,even the susceptible antibiotics fail in treating infections caused by susceptible SMA,which come to a result that clinical treatment results are inconsistent with their susceptibility test results.Although these cases frequently occurred in their practical work,specialists in our country usually pay little attention to them and just think about that they are caused by SMA slow growth and fast resistance mutations,so no investigations on them.While in the practices SMA grows fast and no resistant genetic materials mutations have been found in discribed above special cases.
Foreign researchers investigated that adaptive resistance frequently could be induced by different classes of antibiotics in different species of susceptible Gram-negative bacteria,which made the antibiotics fail even treating the susceptible bacterium.But this phenomenon of adaptive resistance has not been paid enough attentions by researchers and as a result of no resistance genes mutations involved in, clinical labs of microbiology can not find adaptive resistance occurrence,not even know its regularity,so clinical practitioners know no about it from clinical labs of microbiology.This phenomenon described above causes our desires to know why antibiotics fail in treating susceptible SMA,how they fail and whether susceptible SMA turns to be adaptive resistance in the period of treatment,elucidating these problems would make it avoided and enhance the recovery rates of susceptible SMA.
Class 1 integrons with resistance to trimethoprim-sulfarnethoxazole in SMA will lead to limited uses of trimethoprim-sulfamethoxazole.Susceptible SMA treated by fluoroquinolones are tended to be induced to be a resistant mutants which cause different classes of antibiotics failed.SMA's fast resistant mutations and its complicated resistance mechanisms contribute to different methods applied in different places.While as for the severe infections threatening human lives caused by SMA amikacin/gentamicin,proved to be susceptible,combined with other antibiotics is usually adopted to treat besides the classic regimen of trimethoprim-sulfamethoxazole and fluoroquinolones recommended by foreign experts.Based on above reasons and Chinese SDA and NCCLs' drugs directions on pseudomonas, gentamicin,amikacin,sulfamethoxazolecompound(SMZCO) and levofloxacin in this research are used to test if susceptible SMA can produce temporary resistance to them and its occurring regularities and primary mechanisms.
Objectives
(1) SMA clinical isolates,without aminoglycoside modification enzyme(AMEs) genes,susceptible to gentamicin,amikacin,SMZCO and levofloxacin have been chosen to be used astest objects in the following induction work.
(2) The object isolates'minimal inhibitory concentrations(MIC) and minimal bactericidal concentrations(MBC) of above four antibiotics were determined
(3) Object isolates were induced to be adaptive resistance and to be detected their general resistance.
(4) Detected the antibiotic content in bacteria at different periods of adaptive resistance.
(5) At different periods of adaptive resistance,the outer membrane proteins(OMP) of bacteria were detected to whether they have been changed and sent those changed OMPs to sequence their amino acids for their identification.
Methods
(1) With the method of K-B,88 clinical isolates were used to choose susceptible ones to not only gentamicin,amikacin,SMZCO but also levofloxacin, only those susceptible to all of above four antibiotics and without AMEs genes through PCR screening could be used as object isolates.The MICs and MBCs of 3 object isolates to above four antibiotics were determined by macrodilution in MHB.
(2) Object isolates were divided into test group and control group because of different performings in the pratices.Gentamicin/amikacin of concentration of 1×MIC were used to induce test group isolates for 1 hour,then they were again used to kill the induced test group ones at the concentration of 4×MIC for 1 hour again. The test group mean killing velocity were calculated and compared with those of control one.If the test group mean killing velocity was smaller than that of control one,which indicated the induction has produced adaptive resistance;If the results were on the contrary,induction has made the isolates more susceptible than induced before.If these two groups' killing velocity was same as each other,induction has no effects on them.Compared to the test group,the control group has no antibiotic in its induction periods but the other dealing steps were totally same as test group;The test group bacteria induced by amikacin were later used to detect whether they became resistant to SMZCO and levofloxacin.
(3) Only those gentamicin and amikacin absorbed by 01 isolate at different periods(induced after the second hour,fourth hour,sixth hour,eighth hour and tenth hour) were sent to detect,just only because of high prices paid for detection,to evaluate the absorbtion of gentamicin and amikacin.The detailed operations are as follows:antibiotics were added to the induced cultures to a final concentration of 10μg/ml for 1 hour at 37℃,then the bacteria were harvested by centrifuging at 1500 rpm for 10 min and washed twice with PBS,the pellet was resuspended and sent to ultrasonicate.When bacteria were totally broken proved by microscopic examination, the supernatant was harvested by centrifuging at 15000 rpm for 10 min and the antibiotics in it were the absorbed ones by bacteria.The antibiotics in supernatant were sent to detect by high performance liquid chromatography(HPLC).When we harvested bacteria to ultrasonicate for antibiotics,simultaneously we plated bacteria through ten times dilution for viable accounts in the next morning for the killing rates.
(4) The OMPs of induced bacteria were extracted by the method of Carlones and the OMPs were analyzed their constituents through SDS-PAGE.The changed OMPs constituents were sent to sequenced and the sequenced results alignments were compared to those in GeneBank to identify which kind of protein they individually should be fallen into.
Results
(1) Three object isolates were named for 01,02,and 03 separately,the MIC (μg/ml) and MBC(μg/ml) of them were as follows:the MIC of GEN was 0.5,1,0.5; the MBC of GEN was 1.5,2.5 and 1.5;the MIC of AMK was 0.25,0.5 and 0.5;the MBC of AMK was 0.5,1.5 and 1;the MIC of SXT was 0.25,0.25 and 0.25;the MBC of SXT was 0.5,0.5 and 0.5;the MIC of LVF was 0.25,0.25 and 0.5;the MBC of LVF was 0.5,0.5 and 1.
(2) The adaptive resistance appeared when induced after from 4~(th) to 8~(th) hour examined with those 4 kinds of antibiotics,and from the 9~(th) hour after induction,the tested group bacteria gradually returned to be susceptible to the tested 4 kinds of antibiotics.While the control ones has no such changes in the whole period.
(3) The content changes of GEN uptaken by 01 test group from the 2nd,4th, 6th,8th and 10th hour after induction was respectively as follows:1.69,0.81,0.32, 0.71and 1.57;the control group was:1.71,1.76,1.69,1.73 and 1.8;The content changes of AMK uptaken by 01 test group from the 2nd,4th,6th,8th and 10th hour after induction was respectively as follows:1.56,0.66,0.29,0.59and 1.37;the control group was:1.73,1.75,1.69,1.74and 1.79
(4) Induced bacteria in different resistant stages were harvested and the OMPs were extracted for SDS-PAGE analysis,45kD and 60kD OMPs almost disappeared at the peak of resistance.45kd and 60kd OMPs sequenced results showed that,the 45kD OMP was completely same as amino-acid transporter transmembrane protein existing in outer membrane of SMA reported before in GeneBank containing 475 amino-acids; 60kD OMP was totally same as putative transfer protein reported in outer membrane of SMA containing 823 amino-acids in full length.
Conclusions
(1) Both gentamicin and amikacin can induce adaptive resistance in SMA and when the bacteria in the period of adaptive resistance,the susceptibilities of those 4 tested kinds of antibiotics turned to be declining.Resistance phenotype changes can be taken place in SMA although there is no anti-gentamicin and anti-amikacin genes involved.
(2) Some resistance phenotypes are unstable in SMA which indicates that the resistance of SMA is so complicated and the traditional detecting methods in clinical laboratory of microbiology have to be improved.
(3) The changing regularity of antibiotics absorbed by induced SMA is just consistent with that of adaptive resistance,which indicates that the resistance phenotypes changes are just closed to the antibiotics uptaken by SMA.
(4) The changing regularity of 45kD and 60kD OMPs expression of induced SMA is in agreement with that of antibiotics absorbed in induced SMA,and the amino acids sequences show that the two OMPs are the components of SMA in relation to materials entrance into bacteria.So we have reasons to conclude that the OMPs described above might be closely connected with adaptive resistance in SMA for the absence of anti-antibiotics genes involved in the whole progress.
近年来,随着广谱抗生素的大量使用,致病力原本不强的条件致病菌嗜麦芽窄食单胞菌(stenotrophomonas maltophilia,SMA)已然成为医院感染的重要病原菌,致使感染率不断上升,导致免疫力低下或长期大量使用广谱抗生素的人群呼吸道感染、心内膜炎及败血症等危及生命的严重感染。体外药敏试验结果证实,嗜麦芽窄食单胞菌对磺胺类、β内酰胺类、喹诺酮类、氨基糖苷类等几乎所有抗生素均可产生耐药性(Resistance)。因此,目前国内尚无学者对嗜麦芽窄食单胞菌的感染提出统一的治疗方案,一些学者的推荐方案仅基于回顾性研究和病例报告,但嗜麦芽窄食单胞菌耐药突变快速,因此其实用价值有限。国外学者推荐的治疗方案也常有悖于药敏试验结果和临床治疗结果,故对于该菌的治疗,国内外一致提倡根据药敏结果选择敏感抗生素进行治疗。
但临床实践证明,即使根据药敏结果选用抗生素,甚至联合几个同时敏感的抗生素进行治疗也不能使感染完全得到控制,这就是临床上治疗该菌时常见的体外药敏试验结果与临床治疗结果不一致的现象,导致大量患者因不能有效控制感染而死亡。但对于这类常见现象,国内学者长期以来不予重视,只是认为由于该菌生长缓慢而突变迅速造成了该结局,并未对其进行深入研究。试验证明,该菌生长并不缓慢而是生长很快,并且如果发生了耐药遗传物质突变应该能从基因水平得以证实,但至今未见到该菌由于基因突变致使体外敏感性与临床疗效不一致的报道。
国外学者对其他革兰阴性菌研究时发现,不同类型的多种抗生素皆可诱导敏感菌产生暂时性耐药,本文按国外惯用定义称为适应性耐药。这种耐药形式可导致细菌耐药表型暂时转变,并认为这种耐药性使敏感菌疗效不佳,即适应性耐药引起敏感菌治疗效果低下,但该现象一直未得到重视。这种耐药性和有耐药遗传物质介导的耐药性不同,它不涉及基因组突变,因此临床微生物实验室不能检测出来,更不知其发生规律,无法报告临床。在特定条件下,敏感的嗜麦芽窄食单胞菌能否产生适应性耐药,这种耐药有无规律,其发生机制是什么,阐明这些,可为临床治疗嗜麦芽窄食单胞菌敏感株作出提示,避免适应性耐药发生。
嗜麦芽窄食单胞菌中Ⅰ型整合子介导的复方新诺明耐药基因的发现,将导致该抗生素耐药传播迅速,其应用会受到限制。喹诺酮类治疗嗜麦芽窄食单胞菌时易诱发敏感株突变,造成多种抗生素耐药致使治疗失败。可见,该菌本身耐药变迁快速,耐药机制复杂,不同医疗机构根据当地耐药监测结果选择符合当地耐药特点的抗生素进行治疗,所以各地治疗方案不尽相同。而对于由该菌引起的危及生命的严重感染,除了采用国外推荐的复方新诺明和喹诺酮类治疗外,仍有很多地方根据药敏报告采用阿米卡星或庆大霉素联合其他抗生素进行治疗。鉴于上述原因以及中国药品管理局(SDA)规定和参照美国临床实验室标准化委员会(NCCLS)常规药敏实验报告时假单胞菌的选药指南,本文选用对庆大霉素、阿米卡星、左氧氟沙星和复方新诺明皆敏感的嗜麦芽窄食单胞菌,对其诱导处理后,再检测其能否对原本敏感的上述四种抗生素产生耐药性,产生规律及初步探讨其耐药机制。
目的
(1)筛选出左氧氟沙星,复方新诺明,庆大霉素和阿米卡星表型敏感且不含抗庆大霉素和阿米卡星修饰酶基因的敏感菌作为受试菌。
(2)分别测定受试菌对庆大霉素、阿米卡星、复方新诺明和左氧氟沙星的最低抑菌浓度值(MIC)和最低杀菌浓度值(MBC)。
(3)诱导受试菌适应性耐药,检测处于适应性耐药期细菌的广谱耐药性。
(4)测定不同耐药时期菌体内抗生素含量。
(5)测定不同耐药时期菌体外膜蛋白构成,对有差异的蛋白进行序列测定,确定有差异的蛋白类型。
方法
(1)用K-B法对收集到的88株嗜麦芽窄食单胞菌临床分离株做药敏试验,筛选出对庆大霉素、阿米卡星、复方新诺明和左氧氟沙星敏感株。再用7对抗庆大霉素和阿米卡星的修饰酶基因引物对全部株进行扩增,收集不含任何一种氨基糖苷修饰酶基因且4种抗生素全为敏感的菌株作为受试菌。用液体稀释法测定受试菌对上述4种抗生素的MIC值和MBC值;
(2)将受试菌因为处理方法不同分为试验组和对照组,对照组只是在诱导阶段不加抗生素诱导即空白诱导,其余处理与试验组完全相同。以1×MIC庆大霉素/阿米卡星分别诱导试验组细菌1h,再以4×MIC浓度的庆大霉素/阿米卡星杀菌1h,计算杀菌速度,比较试验组平均杀菌速度和对照组平均杀菌速度大小。若抗生素对试验组的杀菌速度小于对对照组的杀菌速度表示诱导使细菌产生了耐药性;如果两组杀菌速度相同则诱导不产生耐药性;如果对试验组杀菌速度大于对照组杀菌速度表示诱导使试验组细菌产生了敏感性。以阿米卡星诱导细菌后,再分别用复方新诺明/左氧氟沙星进行杀菌,证实诱导菌能否使复方新诺明/左氧氟沙星敏感性降低。
(3)鉴于测定抗生素含量费用昂贵,故本试验只检测01号菌诱导后第2、4、6、8、10h吸收庆大霉素和阿米卡星的量来评价诱导后不同时段(第2、4、6、8、10h)抗生素进入菌体的情况。具体操作如下:一边做诱导试验(诱导试验同前),一边同步收集诱导后第2、4、6、8和10h并加入了抗生素至终浓度为10μg/ml,且杀菌1h的细菌。当诱导试验结果证明诱导耐药成功后,所收集的细菌方能用于测定抗生素含量。离心去除培养上清,PBS(pH 7.0)洗涤沉淀2次,去除PBS后再加新的PBS混匀细菌悬液,超声破菌。取超声后的菌液一滴涂片染色,镜下确定全部菌体都破裂。离心收集上清,上清液中的抗生素含量就是不同耐药时期的细菌吸收至体内的抗生素。采用高效液相色谱法测定此液体中的抗生素含量;
(4)Carlone法提取受试菌诱导后第1h～第10h的外膜蛋白(outer membraneprotein,OMP),聚丙烯酰胺凝胶电泳(SDS-PAGE)分析OMP的构成成分;对有变化的外膜蛋白溶液送交蛋白测序公司进行氨基酸序列测定,测序结果在GeneBank中进行序列同源性比对,确定所测外膜蛋白的种类。
结果
(1)K-B法和PCR法筛选出符合条件的受试菌3株,分别命名为01、02、03号菌株。01、02、03号菌对各抗生素的最低抑菌浓度(MIC)值(μg/ml)和最低杀菌浓度(MBC)值(μg/ml)分别如下:庆大霉素的MIC值分别是0.5、1、0.5;庆大霉素的MBC值分别是1.5、2.5、1.5;阿米卡星的MIC值分别是0.25、0.5、0.5;阿米卡星的MBC值分别是0.5、1.5、1;复方新诺明的MIC值分别是0.25、0.25、0.25;复方新诺明的MBC值分别是0.5、0.5、0.5;左氧氟沙星的MIC值分别是0.25、0.25、0.5;左氧氟沙星的MIC值分别是0.5、0.5、1;
(2)诱导后试验组细菌对4种抗生素均产生耐药性,即从诱导后第4h～第8h抗生素杀菌能力减弱甚至失去杀菌力即产生了适应性耐药。从诱导后第9h以后,抗生素开始逐渐恢复杀菌能力即细菌逐渐失去耐药性又变为敏感菌。对照组细菌没有这种规律性变化;
(3)01号菌试验组细菌在诱导后第2、4、6、8和10小时的庆大霉素含量(gg/m1)情况分别是:1.69、0.81、0.32、0.71和1.57;对照组细菌诱导后第2、4、6、8和10小时的庆大霉素含量(μg/ml)情况分别是:1.71、1.76、1.69、1.73和1.8。01号菌试验组细菌在诱导后第2,4,6,8和10小时的阿米卡星含量(μg/ml)情况分别是:1.56、0.66、0.29、0.59和1.37;对照组细菌诱导后第2、4、6、8和10小时的阿米卡星含量(μg/ml)情况分别是:1.73、1.75、1.69、1.74和1.79。
(4)收集诱导后不同时段的细菌,分别提取其外膜蛋白(outer membraneprotein,OMP),SDS-PAGE电泳显示耐药高峰期试验菌在45kD处及60kD的OMP表达几乎消失;对45kD处及60kD的OMP进行氨基酸序列测定,测定序列与美国国家生物信息中心的蛋白序列进行比对。结果显示,45kD处的蛋白与提交到生物信息中心的该菌的氨基酸运输跨膜蛋白完全同源(amino-acidtransporter transmembrane protein),全长475aa;60kD处的外膜蛋白测序结果与生物信息中心报道的该菌外膜上的转运蛋白(putative transfer protein)完全同源,全长823aa。
结论
(1)庆大霉素和阿米卡星能诱导嗜麦芽窄食单胞菌产生适应性耐药,试验用的4种抗生素皆对适应性耐药期细菌的杀菌能力减弱。由于受试菌没有耐药基因,可见没有耐药基因的变化,耐药表型也能发生变化,即嗜麦芽窄食单胞菌中,耐药不完全由耐药基因决定;
(2)该菌中,某些耐药表型是动态变化着的,其转变有时限性,即经过一定时间之后,其表型又可回复到始发状态,揭示了该菌耐药的复杂性和传统耐药性检测的粗糙;
(3)诱导后细菌吸收抗生素的变化步调与细菌发生适应性耐药步调基本一致,提示耐药表型的变化与抗生素在菌体内的含量有关;
(4)45kD处及60kD处OMP的表达变化规律与适应性耐药的发生步调基本一致,与抗生素进入菌体内的变化规律也基本一致,其氨基酸序列比对显示,这两个OMP都是转运物质进入细菌体内的相关成分蛋白,而受试菌中不含耐药基因,进一步提示45kD处及60kD处的OMP与适应性耐药的产生有密切关系。
Backgroud
With the extensive application of broad-spectrum antibiotics in recent years, Stenotrophomonas maltophilia(SMA) has become an important nosocomial pathogen associated with infections of compromised individuals including respiratory tract, endocarditis,bacteremia and other serious infections.The susceptible test results in the world prove that SMA is resistant to almost every antibiotic widely used in hospitals,so there has no uniform method for treating the infections caused by SMA in our country.Based on a number of retrospective cases or researches in the past, some methods for treating SMA infections have been suggested,but on the other hand,their effectiveness has not been verified in clinical practices as the result of the fast resistance mutations of SMA.Although there has indeed an advocated method for treating infections caused by SMA by some foreign specialists,it has been proved adverse to the results of susceptibility test and clinical treatment.So almost specialists in the world now advocate methods for treating SMA infections according to its susceptibility test results.
But unfortunately,even the susceptible antibiotics fail in treating infections caused by susceptible SMA,which come to a result that clinical treatment results are inconsistent with their susceptibility test results.Although these cases frequently occurred in their practical work,specialists in our country usually pay little attention to them and just think about that they are caused by SMA slow growth and fast resistance mutations,so no investigations on them.While in the practices SMA grows fast and no resistant genetic materials mutations have been found in discribed above special cases.
Foreign researchers investigated that adaptive resistance frequently could be induced by different classes of antibiotics in different species of susceptible Gram-negative bacteria,which made the antibiotics fail even treating the susceptible bacterium.But this phenomenon of adaptive resistance has not been paid enough attentions by researchers and as a result of no resistance genes mutations involved in, clinical labs of microbiology can not find adaptive resistance occurrence,not even know its regularity,so clinical practitioners know no about it from clinical labs of microbiology.This phenomenon described above causes our desires to know why antibiotics fail in treating susceptible SMA,how they fail and whether susceptible SMA turns to be adaptive resistance in the period of treatment,elucidating these problems would make it avoided and enhance the recovery rates of susceptible SMA.
Class 1 integrons with resistance to trimethoprim-sulfarnethoxazole in SMA will lead to limited uses of trimethoprim-sulfamethoxazole.Susceptible SMA treated by fluoroquinolones are tended to be induced to be a resistant mutants which cause different classes of antibiotics failed.SMA's fast resistant mutations and its complicated resistance mechanisms contribute to different methods applied in different places.While as for the severe infections threatening human lives caused by SMA amikacin/gentamicin,proved to be susceptible,combined with other antibiotics is usually adopted to treat besides the classic regimen of trimethoprim-sulfamethoxazole and fluoroquinolones recommended by foreign experts.Based on above reasons and Chinese SDA and NCCLs' drugs directions on pseudomonas, gentamicin,amikacin,sulfamethoxazolecompound(SMZCO) and levofloxacin in this research are used to test if susceptible SMA can produce temporary resistance to them and its occurring regularities and primary mechanisms.
Objectives
(1) SMA clinical isolates,without aminoglycoside modification enzyme(AMEs) genes,susceptible to gentamicin,amikacin,SMZCO and levofloxacin have been chosen to be used astest objects in the following induction work.
(2) The object isolates'minimal inhibitory concentrations(MIC) and minimal bactericidal concentrations(MBC) of above four antibiotics were determined
(3) Object isolates were induced to be adaptive resistance and to be detected their general resistance.
(4) Detected the antibiotic content in bacteria at different periods of adaptive resistance.
(5) At different periods of adaptive resistance,the outer membrane proteins(OMP) of bacteria were detected to whether they have been changed and sent those changed OMPs to sequence their amino acids for their identification.
Methods
(1) With the method of K-B,88 clinical isolates were used to choose susceptible ones to not only gentamicin,amikacin,SMZCO but also levofloxacin, only those susceptible to all of above four antibiotics and without AMEs genes through PCR screening could be used as object isolates.The MICs and MBCs of 3 object isolates to above four antibiotics were determined by macrodilution in MHB.
(2) Object isolates were divided into test group and control group because of different performings in the pratices.Gentamicin/amikacin of concentration of 1×MIC were used to induce test group isolates for 1 hour,then they were again used to kill the induced test group ones at the concentration of 4×MIC for 1 hour again. The test group mean killing velocity were calculated and compared with those of control one.If the test group mean killing velocity was smaller than that of control one,which indicated the induction has produced adaptive resistance;If the results were on the contrary,induction has made the isolates more susceptible than induced before.If these two groups' killing velocity was same as each other,induction has no effects on them.Compared to the test group,the control group has no antibiotic in its induction periods but the other dealing steps were totally same as test group;The test group bacteria induced by amikacin were later used to detect whether they became resistant to SMZCO and levofloxacin.
(3) Only those gentamicin and amikacin absorbed by 01 isolate at different periods(induced after the second hour,fourth hour,sixth hour,eighth hour and tenth hour) were sent to detect,just only because of high prices paid for detection,to evaluate the absorbtion of gentamicin and amikacin.The detailed operations are as follows:antibiotics were added to the induced cultures to a final concentration of 10μg/ml for 1 hour at 37℃,then the bacteria were harvested by centrifuging at 1500 rpm for 10 min and washed twice with PBS,the pellet was resuspended and sent to ultrasonicate.When bacteria were totally broken proved by microscopic examination, the supernatant was harvested by centrifuging at 15000 rpm for 10 min and the antibiotics in it were the absorbed ones by bacteria.The antibiotics in supernatant were sent to detect by high performance liquid chromatography(HPLC).When we harvested bacteria to ultrasonicate for antibiotics,simultaneously we plated bacteria through ten times dilution for viable accounts in the next morning for the killing rates.
(4) The OMPs of induced bacteria were extracted by the method of Carlones and the OMPs were analyzed their constituents through SDS-PAGE.The changed OMPs constituents were sent to sequenced and the sequenced results alignments were compared to those in GeneBank to identify which kind of protein they individually should be fallen into.
Results
(1) Three object isolates were named for 01,02,and 03 separately,the MIC (μg/ml) and MBC(μg/ml) of them were as follows:the MIC of GEN was 0.5,1,0.5; the MBC of GEN was 1.5,2.5 and 1.5;the MIC of AMK was 0.25,0.5 and 0.5;the MBC of AMK was 0.5,1.5 and 1;the MIC of SXT was 0.25,0.25 and 0.25;the MBC of SXT was 0.5,0.5 and 0.5;the MIC of LVF was 0.25,0.25 and 0.5;the MBC of LVF was 0.5,0.5 and 1.
(2) The adaptive resistance appeared when induced after from 4~(th) to 8~(th) hour examined with those 4 kinds of antibiotics,and from the 9~(th) hour after induction,the tested group bacteria gradually returned to be susceptible to the tested 4 kinds of antibiotics.While the control ones has no such changes in the whole period.
(3) The content changes of GEN uptaken by 01 test group from the 2nd,4th, 6th,8th and 10th hour after induction was respectively as follows:1.69,0.81,0.32, 0.71and 1.57;the control group was:1.71,1.76,1.69,1.73 and 1.8;The content changes of AMK uptaken by 01 test group from the 2nd,4th,6th,8th and 10th hour after induction was respectively as follows:1.56,0.66,0.29,0.59and 1.37;the control group was:1.73,1.75,1.69,1.74and 1.79
(4) Induced bacteria in different resistant stages were harvested and the OMPs were extracted for SDS-PAGE analysis,45kD and 60kD OMPs almost disappeared at the peak of resistance.45kd and 60kd OMPs sequenced results showed that,the 45kD OMP was completely same as amino-acid transporter transmembrane protein existing in outer membrane of SMA reported before in GeneBank containing 475 amino-acids; 60kD OMP was totally same as putative transfer protein reported in outer membrane of SMA containing 823 amino-acids in full length.
Conclusions
(1) Both gentamicin and amikacin can induce adaptive resistance in SMA and when the bacteria in the period of adaptive resistance,the susceptibilities of those 4 tested kinds of antibiotics turned to be declining.Resistance phenotype changes can be taken place in SMA although there is no anti-gentamicin and anti-amikacin genes involved.
(2) Some resistance phenotypes are unstable in SMA which indicates that the resistance of SMA is so complicated and the traditional detecting methods in clinical laboratory of microbiology have to be improved.
(3) The changing regularity of antibiotics absorbed by induced SMA is just consistent with that of adaptive resistance,which indicates that the resistance phenotypes changes are just closed to the antibiotics uptaken by SMA.
(4) The changing regularity of 45kD and 60kD OMPs expression of induced SMA is in agreement with that of antibiotics absorbed in induced SMA,and the amino acids sequences show that the two OMPs are the components of SMA in relation to materials entrance into bacteria.So we have reasons to conclude that the OMPs described above might be closely connected with adaptive resistance in SMA for the absence of anti-antibiotics genes involved in the whole progress.
引文
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