Mutant Prevention Concentration And Resistance Mechanism Of Fluoroquinolones For Stenotrophomonas Maltophilia

PrefaceFollowing the use of broad - spectrum antimicrobial agents, imunosup-pressed drugs, corticosteroid and invasive procedures, Stenotrophomomas maltophilia has risen to prominence over the last decade as an important nosocomial pathogen, which mobidity is increasing every year. Because outbreaks in hospitals are reported, molecular epidemiological study on the S. maltophilia is the basis of preventing and controling nosocomial infection.S. malltophilia is inherently resistant to most antimicrobials due to a low outer membrane permeability and/or natural MDR efflux systems, coupled with specific resistance mechanisms, such as the production of two inducible chromo-somally encoded β - lactamases, L1 and L2, and an aminoglycoside acetyltrans-ferase. The few available antibiotics that are naturally active against S. maltophilia include trimoxazole - sulfamethoxazole, fluoroquinolones, β - lactam β lactamase inhibitor combinations such as piperacillin - tazobactam and a few extended - spectrum cephalosporins. The new generation fluoroquinolones such as moxifloxacin shows more active against S. maltophilia than the older agents such as ciprofloxacin. In order to lengthen the useful lifespan of antimicrobials, especially the newer fluoroquinolones, Drlica proposed the concept of mutant prevention concentration ( MPC) , which is the lowest concentration that inhibit first — step mutants growth. MPC can be used to evaluate antimicrobials not only the efficacy but also the potency of restricting resistant mutants and it provides a rationale for deciding whether a compound should be administered as part of acombination regimen rather than being used alone.The mechanism of bacteria acquiring fluoroquinoloes resistance is including three aspects;amino acid mutations in the quinolone - resistance determining regions(QRDR);increased expression of efflux pumps;permeability defects. The target gene of fluoroquinoloes is topoisomerases II (gyrA and gyrB) and to-poisomerases IV ( parC and parE ) , which are involved in DNA replication, recombination , and transcription and in the partitioning of the replicated chromosome. Fluoroquinolones combine with the two subunits respectively and DNA to interfere DNA replication, leading to bacteria dead. Recent evidence indicates that antibiotic efflux may be a contributing factor to the intrinsic and acquired multidrug resistance of S. maltophilia, including two pumps SmeABC and SmeDEF. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and reserpine are pump inhibitors that can inhibit the efflux pumps activities.In our study, we have determined the genetic relatedness and epidemiologi-cal links among 92 S. maltophilia isolates recovered in four hospitals in Shenyang by using profiles generated by pulsed - field gel electrophoresis ( PFGE). MPC of four fluoroquinoloes are also determined to guide clinical antimicrobials choice. Furthermore, the resistance mechanism is researched by pump inhibitors and target genes.MethodsExperiment Materials1.1 Organisms sourceWe collected 92 clinical strains of S. maltophilia in four hosipitals in Shenyang during September 2003 to December 2005.ReagentMuller - Hinton broth, Muller - Hinton agar, LB broth, tryptic soy agar, ciprofloxacin powder, levofloxacin powder, lomefloxacin powder, moxifloxacin powder, gatifloxacin powder, proteinase K, restriction endonuclease Xba I , low - melt agarose gel, lambda DNA, PCR primer, ethidium bromide, DNAfragment nurification Wit. f!C.f!P nnwder rf?s*?rnin*? nnwrWExperiment methods2.1 DNA pulsed field gel electrophoresisA total of 92 clinical S. maltophilia isolates were incubated with LB broth and purified with proteinase K. Xba I was used for restriction endonuclease digestion. PFGE was performed with the CHEF Mapper XA system. The gels were stained with ethidium bromide and photographed under UA light.2.2 Detection of minimal inhibitory concentrations (MIC)MICs were determined by standard agar dilution method on MH medium after a 24h incutation at 35T).2. 3 Detection of MPCThe MPC was defined as the lowest drug concentration that prevented recovery of lolonies when more than 1010 cells. High - density cultrues were prepared from overnight cultures grown in MH agar medium followed by 6h of incubation with shaking in MH broth at 37 T! o Inoculated fluoroquinolone - impregnated plates were incubated for 72 h, at which time the MPC was recorded as the lowest fluoroquinolone concentration completely inhibiting bacterial growth.2.4 Amplification and sequence analysis of QRDRs of gyrA, gyrB, parC and parEDNA template was prepared from a loop of a fresh overnight culture suspended in water, followed by boiling for lOmin and centrifugation. Amplification was performed in a final volume of 50ul containing 5ul of DNA template, lOmM Tris -HCL, 50mM KCL, 2.5mM MgCl2, each primer at a concentration of 2. 5pM, each primer at a concentration of 2. 5pM, each deoxynucleoside triphos-phate at a concentration of 200uM, and 5. OU of Taq polymerase. The reactions were performed on a DNA thermal cycler. The samples were denatured at 941 for 5 min, followed by 30 amplification cycles with the following parameters;941 for 1 min, 54^ at 1 min for annealing, and 72^ at 1 min for polymerization. A final cycle of T2X. for 5 min was used to fully extend the amplicons. The PCR products were purified with a PCR purification kit. Sequencing of both strands of the gyrA, gyrB, parC, and parE fragments was done by Shanghai Sengon Biological Energying Technology Corporation.2.5 Effect of MIC by pump inhibitorThe MICs of 5 kinds of fluoroquinolone were determined in combination with 5mg of CCCP per ml and 20 ug of reserpine per ml. An efflux mechanism was inferred to be present when the quinolone MIC in the presence of CCCP and /or reserpine was at least fourfold less than the corresponding MIC in the absence of these compounds.Results1. Molecular epidemiological characteristicsThe PFGE conditions under Xba I restriction resolved DNA fragment sizes ranging from 30 kbp to 420 kbp, including 8 to 16 bands. There were 63 clones in the 92 clinical isolates. A total of 39 isolates were detected clone transmission , involved in clone A to clone J. Clone A had ten isolates, except one, nine of which were isolated from the same hospital, 5 from ICU ward and 3 from respiratory ward. The MIC of ciprofloxacin increased from 1 mg/L to 8 mg/L in clone E after four times transmission.2. MIC of fluoroquinolones for S. maltophiliaThe susceptibility of moxifloxacin, levofloxacin, lomefloxacin and ciprofloxacin were 100%, 90. 3%, 71% and 29% , respectively. The susceptibility of ciprofloxacin by disk diffusion methods was higher than agar dilution methods, which had statistic significance.3. MPC of fluoroquinolones for S. maltophiliaThe MPCs were determined increasingly by the order moxifloxacin < levofloxacin < ciprofloxacin < lomefloxacin. MPCs of moxifloxacin and levofloxacin were mainly 32 mg/L, and that of ciprofloxacin and lomefloxacin were mainly 128 mg/L.4. PCR and sequence analysis of QRDRs of gyrA, gyrB, parC and parE With respect to the corresponding proteins described in Genbank for S. maltophilia ATCC 13637, no amino acid changes were detected in gyrA, gyrB and parC. 18 isolates were identified amino acid substitution in parE accouting for 64.3%. The sequences of ten laboratory resistant isolates were identical with their parents isolates. Compared with ciprofloxacin sensitive group, the positiverate of amino acid substition had no statistic significance in ciprofloxacin resistant group.5. Phenotypic characterization of efflux mechanismCCCP and reserpine reduced the resistance of partial S. maltophilia to flu-roquinolones. 27 isolates showed positive efflux pump which not only in resistant strains but also in suceptible strains. Compared with pump negative group, the susceptibility of levofloxacin, lomefloxacin and ciprofloxacin decreased while resistance rate of lomefloxacin and ciprofloxacin increased in pump positive group, which had statistic significance.Conclusions1. Enviromental contamination contributes to disseminate epidemic S. maltophilia in the same medical unit. Medical transmission is the main route of dessemination among different units. Clone transmission is one of the reasons of resistance.2. Fluoroquinolones can induce resistnance while routine used alone. Disk diffusion method shouldnt replace agar dilution method. Ciprofloxacin shouldnt replace other fluoroquinolones while evaluationg suceptibility.3. GyrA, gyrB and parC of S. maltophilia have no amino acid substitution. A high frequency of amino acid replacements was present in parE, but that has no related to fluoroquinolone resistance in S. maltophilia.4. CCCP and reserpine can enhance the activities of fluoroquinolones. Efflux pump is related to resistance.