The Effect Of Chlorogenic Acid On The Mucoid Pseudomonas Aeruginosa Biofilm And Its Synergistic Effect With Levofloxacin

Pseudomonas aeruginosa is an important opportunistic pathogen in Clinical medicine. It is also the main pathogenic bacterium of iatrogenic infection. It is common in clinical biomedical materials associated infections and certain chronic intractable infectious diseases such as chronic panbronchiolitis, cystic fibrosis, osteomyelitis, etc. The extensive use of antibiotics as well as its formation of resistance biofilm (biofilm, BF) leads to the extremely refractory infection of Pseudomonas aeruginosa. The out of control of mucoid Pseudomonas aeruginosa in alginate production, making BF formation easily and with more complicate structure, then making bacterial escape from immune clearance and increase resistance to antimicrobial agents.MucA gene mutation, the main mechanism for phenotypic change, hold high rate in mucoid Pseudomonas aeruginosa from both experimental strains and clinical isolates strains[1,2]. However, the research on BF about MucA gene mutation strains was few, an efficient widely used method in clinical treatment is urgent needed.Chlorogenic acid is one of the main active ingredient of many herbs such as honeysuckle, Eucommia, capillaries, etc. It has a broad antimicrobial effect on gram-negative and positive bacteria. Previous findings by our group showed that chlorogenic acid can inhibit the formation of the wild type PAO1 BF, and can destruct the mature BF[3]. So, this study we chose classical mucoid Pseudomonas aeruginosa PDO300 (MucA gene mutation) as the experimental strain, to study the intervention of chlorogenic acid on the BF formation ability of mucoid Pseudomonas aeruginosa. Then, establish the BF model in vitro to investigate the effect of chlorogenic acid alone and in combination with levofloxacin on mucoid Pseudomonas aeruginosa biofilm in vitro. OBJECTIVE Establish the BF model of mucoid Pseudomonas aeruginosa in vitro, to observe the inhibitory effect of chlorogenic acid on the biofilm formation of mucoid Pseudomonas aeruginosa.METHODS (1) Mucoid strains of pseudomonas aeruginosa PDO300 was used for biofilm formation in vitro, Medical materials (PVC) as the carriers, set up the static biofilm model of PDO300 in vitro by using 50% LB medium as liquid mediums. After cultivation for 3 days and 7 days, determine the biofilm on the surface of carriers by silver staining. (2) Minimum inhibitory concentration (MIC) of chlorogenic acid was measured by double dilution. The control group and chlorogenic acid group were set up, and then add chlorogenic acid (512μg/ml) at the beginning of construction of biofilm models. On the 3rd and 7th day, observe the structure of biofilm by SEM, detect viable bacteria counts in the biofilm by serial dilution method,semi-quantity biofilm by crystal violet staining.RESULTS (1) On the 3rd day, young and thin biofilm on the surface of carriers can be seen by silver staining, and the 7th day, thick mature biofilm on the surface of carriers can be seen (2) The MIC of chlorogenic acid to PDO300 is 2048μg/ml. After cultivating for 3 days, the young biofilm was thin but full covered on the surface of the carriers in the control group by SEM and grew into thick, large, three-dimensional and Mushroom-like mature biofilm on the 7th day.Compared with the control group, the scale of the biofilm in the chlorogenic acid group are smaller than the control group. Consistently, the viable bacteria counts in the biofilm of chlorogenic acid group is less than control group (P<0.01) and the biofilm by crystal violet staining in chlorogenic acid group is less than the control group after incubated for 3d or 7d (P<0.01).CONCLUSIONS Chlorogenic acid can restrain mucoid Pseudomonas aeruginosa from forming biofilm on the surface of carriers. OBJECTIVE: To investigate the destruction role of chlorogenic acid on alone mucoid Pseudomonas aeruginosa biofilm and its synergistic bactericidal action with LFX on mucoid Pseudomonas aeruginosa BF bacterial.METHODS MIC of LFX and minimum bactericidal concentration (MBC) of LFX was measured by double dilution. Construction the biofilm model in vitro, then dividing into control group, chlorogenic acid group, LFX group (2μg/ml), LFX group (4μg/ml) chlorogenic acid + LFX group (2μg/ml), and chlorogenic acid + LFX group (4μg/ml). After incubating for 24h, BF was observed by scanning electron microscope (SEM), viable bacterial counts were determined by serial dilution, crystal violet staining method was used for biofilm quantitation.RESULTS The MIC of chlorogenic acid to PDO300 is 0.5μg/ml, and the MBC is 2μg/ml. 24 hours after the effect of chlorogenic acid alone or combined with different concentrations of LFX, when compared with the control group, the biofilm on the carriers were destroyed and reduced by SEM, and with significant less viable bacterial counts (P<0.05) and production of biofilm (P<0.05), and the effect were in a dose-dependent of LFX. Further more, the combination of LFX in different concentrations with chlorogenic acid making the destruction effect more obvious with less viable bacterial counts (P<0.05) and production of biofilm (P<0.05) than the chlorogenic acid or LFX alone, the high concentration of LFX (4μg/ml) group had the significant synergistic bactericidal effect than the low concentration of LFX (2μg/ml) group (P<0.05).CONCLUSIONS Chlorogenic acid can destroy the BF of PDO300 in vitro. Combined with LFX, it can also destroy the biofilm of PDO300 and can enhance the bactericidal effect of LFX on PDO300 within the biofilm.