| Objective Our aim was to investigate in vitro biofilm formation by Stenotrophomonas maltophilia and the effects of antibacterial agents used to prevent biofilm formation.MethodsTwo multi-drug resistant strains were screened from clinical isolates of S.maltophilia.These two strains were isolated from pleural effusion of a cancer patient.Two strains of S.maltophilia were resistant to trimethoprim / sulfamethoxazole.The separation interval was 20 days.Two trimethoprim/sulfamethoxazole-resistant S.maltophilia strains were isolated from the pleural effusion of a patient with cancer.The minimum inhibitory concentration(MIC)of antibacterial drugs such as amikacin,azithromycin,cefoperazone/sulbactam,tigecycline,etc.were determined by the micro broth dilution method.The checkerboard method was used to determine the fractional inhibitory concentration indices(FICIs).A crystal violet biofilm assay and confocal laser scanning microscopy(CLSM)were used to observe biofilm formation.The adhesion and morphology of biofilm and the structural changes under the action of antimicrobials were observed by scanning electron microscope.In vitro effects of azithromycin combined with tigecycline on biofilms of S.maltophilia strains were tested.ResultsThe combination of azithromycin and tegacycline had synergistic antibacterial effect on two clinical isolates and ATCC17666,and the FICI value ranged from 0.1875 to 0.375.The two S.maltophilia isolates were confirmed to produce strong biofilms.Crystal violet biofilm assay and CLSM analysis of S.maltophilia biofilm were in the initial adhesive stage after 2 h incubation.Biofilm was in the exponential phase of growth at12 h,and reached maximal growth at 36–48 h.Compared with tigecycline or azithromycin alone,the combination of tigecycline and azithromycin increased the inhibiting effect S.maltophilia biofilm biomass after incubation for 12 h.Compared with the control group,in almost all strains treated with tigecycline and azithromycin,the biofilm was significantly suppressed significance(P<0.001).We found that 2x MIC azithromycin combined with 1x MIC tigecycline had best inhibiting effect against the biofilm,the biofilm inhibition rates of three strains were all over 60%,the biofilm thickness was inhibited from 36.00 ± 4.00 μm to 8.00 μm,from 40.00 μm to 6.67± 2.31μm,and from 32.00 μm to 13.33 ± 2.31 μm in SMA1,SMA2 and ATCC17666,respectively.The standard strain ATCC17666 was selected to observe the effect of 1x MIC tegacycline and 2x MIC azithromycin on the biofilm of Stenotrophomonas maltophilia in vitro by scanning electron microscope.The results of scanning electron microscope image analysis showed that the bacteria on the coverslips conglomerated in thick,heterogeneous and multiple layers with columnar clusters in the control group(without antibiotics).Contrary to the results of the control group,some aggregated microcolonies could be observed in the SEM images of the bacteria treated with 1x MIC tegacycline and 2x MIC azithromycin alone and the combination of 1x MIC tegacycline and 2x MIC azithromycin.The results showed that the morphology of bacterial cells in the antimicrobial treatment group was damaged,and the cellular content and leakage were inconsistent.The formation of wrinkles and the loss of membrane integrity were also observed in the surface morphology of Stenotrophomonas maltophilia treated with tegacycline and azithromycin,which was also obvious in CLSM analysis.The combination of 1x MIC tegacycline and 2x MIC azithromycin caused more significant damage to the structural integrity of the biofilm than that of 1x MIC tegacycline and 2x MIC azithromycin alone.ConclusionThe combination of azithromycin and tegacycline had synergistic antibacterial effect on two clinical isolates and ATCC17666,and the FICI value ranged from 0.1875 to 0.375.Azithromycin combined with tigecycline inhibited biofilm formation by S.maltophilia.Our study provides an experimental basis for a possible optimal treatment strategy for S.maltophilia biofilm-related infections. |
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