| BackgroudWith 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 4th to 8th hour examined with those 4 kinds of antibiotics,and from the 9th 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.
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