Influence Of Iron On The Formation Of Bacterial Biofilm Of Pseudomonas Aeruginosa And Its Mechanism

Objective: To investigate the effects of iron on the drug resistance, BBF formation and toxin factors of PAO1, PAO-ΔpilHIJK and PAO-JP2, and discuss its possible function mechanism initially.Method: The colonial morphology and Gram's stain of PAO1, PAO-ΔpilHIJK and PAO-JP2 were identified by optical microscope. Biochemistry was identified by Vitek analysis; the morphology was also observed by AFM. DPD was used to establish iron-depletion environmental model in vitro. MICs of ceftazidime, tobramycin and ciprofloxacin to the 3 strains in normal or iron-depletion environment were determined by agar plate dilution method; MBEC was determined by micro-dilution method; the viable counts of bacteria in biofilms were detected after treated with the antibiotics by MTT method. The morphology of biofilms formed in different iron environment was observed by the optical microscope, SEM, CSLM and their biofilms were analyzed by computer image analysis system after being stained by AgNO₃. Twitching mobility was detected in different iron environment; the OD values were observed by enzyme-substrate reaction to compare elastase excreted by the strains; SDS-PAGE was used to compare PEA; fluorescence-quota PCR was used to compare the expression of lasR and rhlR in PAO1 and PAO-ΔpilHIJK.Results: Pyocyanin was observed in PAO1 colony, but not in PAO-ΔpilHIJK and PAO-JP2. All 3 strains wereβ-lactan enzyme negative and Gram's negative bacillus. VITEK showed slight difference between PAO1, PAO-ΔpilHIJK, PAO-JP2 and ATCC27853. AFM results showed that 3 stains are short rod-shaped, sorting according to their length, PAO1>PAO-ΔpilHIJK>PAO-JP2. 500μM DPD was confirmed as subinhibitory concentration to establishment iron-depletion environmental model. The MICs of 3 antibiotics to strains slightly increased in iron-depletion environment. The MBEC result discovered that 8MIC TOB can kill bacteria in biofilm completely; however 8MIC CAZ and CIP could not. Iron-depletion can slightly promote the bactericidal effect of antibiotics to BBR Dense black stained substance decreased significantly after PAO1, PAO-ΔpilHIJK and PAO-JP2 biofilms formed in iron-depletion environment. The SEM graphs showed that PAO1 only can form scattered cenobium in iron-depletion environment. PAO-ΔpilHIJK formed thiner biofilm than PAO1 in normal environment; situation in iron-depletion environment was similar to PAO1. PAO-JP2 even in normal environment can hardly form mature biofilm and only a small quantity of strains can adhere to the surface in iron-depletion environment. The CSLM graphs showed PAO1 and PAO-ΔpilHIJK biofilms decreased in iron-depletion environment. Iron-depletion can increase the twitching mobility of PAO1 and PAO-JP2; the elastase excretion of PAO1 and PAO-ΔpilHIJKwere also increased iron-depletion environment. Iron-depletion was necessary for the detection of PEA of PAO1 and PAO-ΔpilHIJK while no PEA was found in PAO-JP2 at any circumstances. lasR and rhlR of PAO1 were upregulated in iron-depletion environment, while little change were found in PAO-ΔpilHIJK.Conclusions: Iron-depletion can slightly decrease sensitivity of planktonic cells of PAO1, PAO-ΔpilHIJK and PAO-JP2 to the antibiotics, while increase the bactericidal effect of antibiotics to biofilm cells. Expression of elastase and PEA were increased in iron-depletion environment. Biofilm formation ability was depressed significantly in iron-depletion environment and the increased twitching mobility and the transcription of QS relative genes (lasR and rhlR) might be the possible reasons.