Effects of Tigecycline Combined with Azithromycin Against Biofilms of Multidrug-Resistant Stenotrophomonas maltophilia Isolates from a Patient in China

Abstract

Purpose: Our aim was to investigate in vitro biofilm formation by S. maltophilia and the effects of antibacterial agents used to prevent biofilm formation.

Methods: Two trimethoprim/sulfamethoxazole-resistant S. maltophilia strains were isolated from the pleural effusion of a patient with cancer. The minimum inhibitory concentrations (MICs) of amikacin, azithromycin, cefoperazone/sulbactam, and tigecycline were determined. 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. In vitro effects of azithromycin combined with tigecycline on biofilms of S. maltophilia strains were tested.

Results: 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 at 12 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 the 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± 2.31 μm, and from 32.00 μm to 13.33 ± 2.31 μm in SMA1, SMA2 and ATCC17666, respectively.

Conclusion: 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.

Keywords: Stenotrophomonas maltophilia; azithromycin; biofilm; tigecycline; trimethoprim/sulfamethoxazole.

Figure 1

Average ODs at different time points (hours) of S. maltophilia strains. Results are expressed as means ± SDs.

Figure 2

OD values of S. maltophilia strains, showing their capacity to form biofilms. Results are expressed as means ± SDs. Compared with the control group, the difference is statistically significant, ****P <0.0001. The ability of the three strains to produce biofilm was strong after 48 h of incubation.

Figure 3

Average biofilm thickness (μm) at different time points of S. maltophilia strains by confocal laser scanning microscopy. Results are expressed as means ± SDs. The growth rate of the strain biofilm was similar before culture for 12 h, however between 12 h and 48 h incubation, the growth rate of SMA1 and SMA2 became faster, which was a little faster than that of ATCC17666.

Figure 4

Confocal laser scanning microscopy images of S. maltophilia strain (ATCC17666) biofilms after 2, 6, 12, 24, 36, and 48 hours. Biofilm matrix components were stained with 0.01% AO. CLSM analysis showed bacterial biofilm beginning to form after 2 h incubation. The biological membrane structure is close, and the thickness increased over 12–24 h incubation. The three-dimensional space network structure gradually formed between 36–48 h incubation.

Figure 5

In vitro effects of azithromycin, tigecycline, amikacin, cefoperazone/sulbactam alone on S. maltophilia preformed biofilm after 12 h. Results are expressed as means ± SDs. Amikacin (all concentrations) had a far greater inhibitory effect on the biofilm of the premature biofilms (formed after 12 h incubation) than other antibiotics. Compared with the control group, the inhibitory effect of amikacin on the biofilm of the three strains was statistically significant (P <0.01). Compared with the control group, the inhibitory effect of tigecycline and cefoperazone/sulbactam on the biofilm of the two clinical isolates strains was statistically significant (P <0.01). Compared with the control group, the inhibitory effect of azithromycin S. maltophilia (SMA1, SMA2, and ATCC17666) strains that have formed biofilms had almost not reached statistical significance. *P <0.05, **P <0.01, ***P <0.001.

Figure 6

In vitro effects of tigecycline combined with azithromycin on biofilms of S. maltophilia strains after 12 h. Results are means ± SDs. ***P <0.001. Compared with tigecycline or azithromycin alone, the combination of tigecycline and azithromycin increased the inhibitory effect on S. maltophilia preformed biofilms. The best inhibitory effect was observed when 2x MIC azithromycin was combined with 1x MIC tigecycline and when 2x MIC azithromycin was combined with 1x MIC tigecycline. The biofilm inhibition rates were 64% for SMA1, 76% for SMA2, and 67% for ATCC17666. The biofilm inhibition rates of three strains were all over 60%, suggesting good results.

Figure 7

In vitro effects of tigecycline combined with azithromycin on biofilm thickness of S. maltophilia strains after 12 h. Results are expressed as means ± SDs. Compared with the control group, the difference was statistically significant in clinical isolates SMA1and SMA2. ****P <0.0001, ***P <0.001. Compared with the control group, the difference was statistically significant except 0.5x MIC tigecycline and 0.5x MIC azithromycin alone in standard strain ATCC17666. ****P <0.0001, ***P <0.001.

Figure 8

In vitro effects of 1x MIC tigecycline combined with 2x MIC azithromycin on biofilms of S. maltophilia strains after 12 h. Green fluorescent staining (AO) represents extracellular DNA. CLSM images of panel of control groups (without antibiotics) S. maltophilia biofilms have spatial biomass distribution of mature biofilms and thick coating of biofilm with compact architecture characterized by large clumps. By contrast, see panels SMA1, SMA2, and ATCC17666. CLSM images of S. maltophilia biofilm treated with 1x MIC tigecycline and 2x MIC azithromycin alone, and 1x MIC tigecycline combined with 2x MIC azithromycin. Compared with the control group (without antibiotics), the established biofilms of the antibiotic treatment group were disrupted and biofilm cells were rare. Compared with S. maltophilia biofilm treated with 1x MIC tigecycline and 2x MIC azithromycin alone, when 1x MIC tigecycline was combined with 2x MIC azithromycin, biofilms of S. maltophilia were almost disrupted. As shown in the figure, the green fluorescence is very weak and the color is very dark when observed under a laser confocal microscope. This indicates that there are few biofilm cells and the structure was destroyed. Scale bar: 50 μm.