| Objective:To investigate the in vitro and in vivo combination effects of fosfomycin and colistin against Carbapenem-resistant Pseudomas aeruginosa (CRPA) and to explore the intervention effect of fosfomycin on colistin-induced nephrotoxicity in rats. To preliminarily analyze the drug resistant mechanisms of clinical CRPA isolates.Method:(1) The minimum inhibitory concentration (MIC) of 11 antibiotics against 85 CRPA strains were determined by the standard agar dilution method. A checkerboard method that adhered to the recommendations of Clinical Laboratory Standards Institute (CLSI) was applied to assess the synergism effect of fosfomycin and colistin against 85 CRPA strains, and the Fraction Inhibitory Concentration Index (FICI) was calculated according to the results.The time-kill curves were applied to assess the synergism effect of fosfomycin and colistin against 5 CRPA. (2) PA-Xen5 was induced by the sub-inhibitory concentrations of meropenem with broth subculture method and then became resistant to carbapenems in experiment. The peritoneal-infection mouse model was established by intraperitoneal injection of the CRPA-Xen5 and further to investigate the in vivo combination effects of fosfomycin with high and low dosage colistin against CRPA. (3) The nephrotoxicity of rats was induced by colistin sulfate and then the intervention of fosfomycin on this renal toxicity was evaluated. (4) The Genotypes of bacterial produced Metal-beta-lactamase (MBLs) including the types of VIM?IMP? SPM?NDM-1 and GIM of were amplified by PCR for 85 CRPA isolates. The MIC of 85 CRPA isolates to meropenem and imipenem was determined by Agar dilution method with and without efflux pump inhibitor (CCCP). The MIC of meropenem or imipenem of the isolates decreased by four times or more with CCCP test and these strains was demonstrated to have efflux pump. The mRNA expression levels of four kinds of efflux pump of these isolates were detected to by real-time PCR, such as MexB, MexD, MexF and mex Y of MexAB-OprM, MexCD-OprJ, mexEF-OprN, mexXY-OprM, respectively. The outer membrane oprD2 encoding gene of 85 CRPA isolates was amplified and studied by PCR. To preliminarily analyze the drug resistant mechanisms of clinical 85 CRPA isolates. The genetic homology of 85 CRPA isolates collected from three hospitals in Beijng was determined by pulsed-field gel electrophoresis (PFGE).Result:(1) In combination, the colistin MIC values were?0.5 ?g ml-1 for 91.76% of the isolates. This result differed significantly from those obtained using a single agent treatment (The colistin MIC values were< 0.5 ?g ml-1 for only 25.88% of the isolates); Fosfomycin/colistin combination distributed in FICI?0.5,0.5<FICI<1, FICI=1, 1<FICI<2, FICI>2 were 21.18%,27.06%,5.88%,45.88%, respectively. Antagonism (FICI> 2) was not observed. The time-kill curves indicated that the combination of fosfomycin and colistin demonstrated bactericidal activity within 12 h at both 1/2× MIC and 1x MIC for PA-1, PA-2, PA-3 and PA-4, whereas the combination exhibited synergistic bactericidal activity for PA-5 only at 1 x MIC. When colistin was combined with fosfomycin, the treatment bactericidal activity was substantially enhanced, and the regrowth that occurred with colistin monotherapy was prevented.(2) In mice with peritoneal infection, the treatment effeect of fosfomycin alone or low dosage of colistin alone were inferior to their combination. The infection became serious in the treatment of low dosage of colistin at 5h. The treatment of high dosage of colistin was better than that of low dosage of colistin, which showed that antimicrobial activity of colistin was concentration-dependent. The CPS between the high-dosage colistin alone group and fosmycin-high dosage colistin combination group showed no difference (P> 0.05) however, combintion group significantly decreased the colony counts of liver, lung, kidney and blood system than that of high-dosage colistin alone group (P<0.05). The high-dosage alone group, fosmycin-low dosage colistin combination group and fosmycin-high dosage colistin combination group combination produced significant less organ lesions of liver, lung and kidney compared to untreated control group. (3) The Plasma Creatinine (Cr) and NAG level significantly increased in colistin group compared to the control group and combination group (P<0.05). No significant difference was found between the control group and combination group for Cr and NAG level (P>0.05). Histopathologic assessment showed that the colistin group was with significant kidney damage, the semiquantitative score (SQS) significantly increased in colistin group compared to other groups (P< 0.05). however, the fosfomycin group and combination group were basically normal. (4) The VIM, IMP, SPM, NDM-1 and GIM genotype of MBLs was not amplified by PCR for 85 CRPA isolates. The efflux pump inhibitor, CCCP could make MIC of 9 CRPA isolates decrease by four times or more than single drug (imipenem and meropenem, account for 10.59%) and this results indicated that they may have efflux pumps. The four kinds of efflux pump mRNA expression levels increased in 9 CRPA isolates as follow, five MexAB, five MexCD, one MexEF, seven MexXY. The loss of the large or small fragement of oprD2 was found in 81.18%(69 isolates) of 85 CRPA isolates. The 85 CRPA strains had 32 PFGE fingerprint types, The PFGE type contained the iaolates among three hospitals with high related (i.e.,?90%) was less. It demonstrated that 85 CRPA were types of dissemination and no outbreak of epidemic was found.Conclusion:(1) The fosfomycin/colistin combination displayed synergistic and partial synergistic activity against 85 isolates. Antagonism was not observed. In combination, the colistin MIC significant decreased than colistin alone. (2) The fosfomycin/ low-dosage or high-dosage colistin combination can control development of CRPA peritoneal infection in mice. (3)The fosfomycin may relieve nephrotoxicity induced by colistin. (4) The main resistant mechanism of 85 CRPA strains is the loss of OprD2, followed by efflux pump system. The VIM?IMP?SPM? NDM-1 and GIM type of MBLs were not observed. |
PDF Full Text Request