Prevention Of Pseudomonas Aeruginosa Catheter-Related Infection With Levofloxacin-Impregnated Catheter In Vitro And Vivo

Objective: To prepare levofloxacin-impregnated catheters with different drug content , assess characterization of levofloxacin-impregnated catheters ,investigate inhibition of Pseudomonas aeruginosa colonization with levofloxacin-impregnated catheters in vitro and evaluate effect of levofloxacin-impregnated catheters in catheter-related infection.Methods: (1) Both LFX and PDLLA were dissolved in acetone by solvent solution in according to ratio(weight to weight)of LFX and PDLLA, PVC cathers were placed into the above mixture solution for 30 minutes and dried at room temperature , then three kinds of levofloxacin-impregnated catheters with different drug content, i.e. LFX1:3-impregnated catheter, LFX1:6-impregnated catheter, LFX1:9-impregnated catheter were made. In the same way, PDLLA-impregnated catheters without LFX were also made. (2) Drew standard curve of LFX . Measured drug release from levofloxacin-impregnated catheters with different drug content in vitro. Calculated cumulative release of LFX from the catheter in different times. (3)Detected the MIC and MBC of LFX in PAO1. (4)Divided into LFX1:3-impregnated catheter group, LFX1:6-impregnated catheter group, LFX1:9-impregnated catheter group, PVC catheter group and PDLLA-impregnated catheter group, each catheter was immersed in 5ml 50%LB which contain 108CFU/ml Pseudomonas aeruginosa , after incubating for 6,12,24,48h at 37℃, at each time point, adhered bacteria to the catheters and bacteria in the growth culture medium were determined, the MIC and MBC of LFX in bacteria in the growth culture medium from levofloxacin-impregnated catheters with different drug content were detected, as well. Results: (1) Drug releases from LFX1:3-impregnated catheter, LFX1:6- impregnated catheter, LFX1:9-impregnated catheter were shown to be fast release, there was very small amount of drug release or no drug relase after 24h in three kinds of levofloxacin-impregnated catheters with different drug content. At each time point, accumulation release concentrations from levofloxacin- impregnated catheters with different drug content were significantly higher than MBC of LFX against PAO1.(2) The MIC and MBC of LFX in PAO1 were 0.5ug/ml, 1.0ug/ml respectively. (3) At the end of 6,12,24h incubation, LFX1:3- impregnated catheters, LFX1:6-impregnated catheters, LFX1:9- impregnated catheters were completely culture negative ,whereas PVC catheters were completely culture positive , the median numbers of CFU were 5.88log10CFU, 6.14log10CFU, 6.34log10CFU(P=0.014,P=0.014,P=0.014, respectively). At the end of 48h, LFX1:3-impregnated catheters were completely culture negative, LFX1:6-impregnated catheters and LFX1:9-impregnated catheters were completely culture positive, the median numbers of CFU were 2.66log10CFU和2.8log10CFU , whereas PVC catheters were completely culture positive,the median number of CFU was 6.11log10CFU(P=0.014,P=0.021,P=0.021, respectively).In addition, at each time point, the number of bacterial adherence to PVC catheters or PDLLA-impregnated catheters was similar (P>0.05). (4) At the end of 6,12,24,48h incubation, bacteria in the growth culture medium from LFX1:3- impregnated catheters, LFX1:6-impregnated catheters,LFX1:9- impregnated catheters,compared to PVC catheters,were significantly reduced (P<0.05).There were no evident increase in bacteria in the growth culture medium from LFX1:3- impregnated catheters, LFX1:6-impregnated catheters, LFX1:9- impregnated catheters between 6h and 48h(P=0.166, P=0.182,P=0.190), whereas, there were evident increase in bacteria in the growth culture medium from PVC catheters between 6h and 48h(P=0.02). In addition, at each time point, the number of bacteria in the growth culture medium from PVC catheters or PDLLA-impregnated catheters was similar(P> 0.05). (5) At the end of 6,12,24,48h incubation, the MICs of LFX in bacteria in the growth culture medium from levofloxacin- impregnated catheters with different drug content were 0.5ug/ml, the MBCs were 1.0ug/ml.Conclusions: Antibiotic release from LFX1:3-impregnated catheters, LFX1:6-impregnated catheters, LFX1:9-impregnated catheters were exhibited to be fast release, the higher LFX content, the more antibiotic will be released. Three kinds of levofloxacin-impregnated catheters with different drug content can inhibit Pseudomonas aeruginosa adherence to the catheters and kill Pseudomonas aeruginosa in peri-catheter medium and restrain Pseudomonas aeruginosa to grow, the LFX1:3-impregnated catheters which contained the most drug content and had the most drug release were the most effective formulation to inhibit Pseudomonas aeruginosa adherence to the catheters, which indicated that levofloxacin-impregnated catheters were able to prevent Pseudomonas aeruginosa catheter-related infection . In addition, at 2h, drug release concentrations from three kinds of levofloxacin-impregnated catheters with different drug content remained higher than MBC of LFX against PAO1, the MBCs of LFX in bacteria in the growth culture medium from levofloxacin-impregnated catheters with different drug content were the same as that of LFX in bacteria before incubation, which suggested the low risk of resistance from levofloxacin-impregnated catheters with different drug content. Objectives: To mimic clinical catheter-related infection, establish an murine model of"tissue cage"infection, investigate inhibition of Pseudomonas aeruginosa adherence and biofilm formation with LFX1:3-impregnated catheters in vivo, evaluate preventive effects of LFX1:3-impregnated catheters in Pseudomonas aeruginosa catheter-related infection.Methods:LFX1:3-impregnated catheters or PVC catheters were implanted subcutaneously in mice, 50ul(107CFU) of PAO1 was injected along subcutaneous implanted catheters as well. 1,5 days after challenging, mice were sacrificed, samples were collected, bacterial counts on implanted catheters and in peri- implanted catheters tissues were determined. Bacterial colonization and biofilm formation on implanted catheters were assessed by SEM.Results: (1) 1 day after challenging, for the LFX1:3-impregnated catheter group , 3 of 8 catheters were culture positive for Pseudomonas aeruginosa, whereas, for the PVC catheter group, 8 catheters were completely culture positive, the median number of CFU was 4.11log10CFU (P=0.001). 5 day after challenging, for the LFX1:3-impregnated catheter group, 8 catheters were completely culture negative,whereas, for the PVC catheter group, the numbers of CFU adherent to the catheters were still very high, the median number of CFU was 4.99log10CFU (P=0.000). (2)1,5days after challenging, for the LFX1:3-impregnated catheter group and the PVC catheter group, peri-implanted catheters tissues were completely culture positive, the median numbers of CFU were 3.96log10CFU , 5.44log10CFU (P=0.001);4.33log10CFU,5.42log10CFU (P=0.001). Compared to the PVC implanted catheters, the LFX1:3-impregnated implanted catheters significantly reduced numbers of CFU in peri-implanted catheters tissues. (3)SEM images: 1 day after challenging, significantly less bacteria, single bacteria or even no bacteria adhering to the LFX1:3-impregnated implanted catheters, whereas, a large amount of dispersal bacteria and small numbers of microcolonies adhering to the PVC implanted catheters. 5 day after challenging, no bacteria on the LFX1:3-impregnated implanted catheters, bacteria colonized to the PVC implanted catheters were in clusters surrounding shaggy, deep mucus-like substance, coral-shaped biofilms formation in which scattered PMN can be seen. (4) Macroscopic observation: 1 day after challenging, secretion can be seen both inside the LFX1:3-impregnated implanted catheters and the PVC implanted catheters, peri-implanted catheters tissues exhibited mild hyperemia, swollen, 5 days after challenging, very small secretion can be seen inside the LFX1:3-impregnated implanted catheters, peri-implanted catheters tissues showed no inflammation or abscess formation, whereas, purulent secretion can be seen inside the PVC implanted catheters, peri-implanted catheters tissues indicated abscess formationConclsions:LFX1:3-impregnated implanted catheters strongly inhibited Pseudomonas aeruginosa adherence and biofilm formation to the catheters, reduced the number of CFU of Pseudomonas aeruginosa from peri-implant tissue, demonstrating the potent bactericidal activity in vivo. Therefore, levofloxacin-impregnated catheter was a promising new strategy for prevetion of Pseudomonas aeruginosa catheter-related infection.