| Bacterial biofilm (BF) was first proposed by the American professorCosterton in1987, it is a way of life that bacterial form multicellularcomplexes on some inert solid surface, it is a bacterial population withcertain space configuration being covered by bacterial, bacteriallipopolysaccharide, fiber protein, DNA, RNA and other ingredients. It iswidely believed that more than80%of clinical infection have a relationwith BF. Haemophilus influenzae is a Gram-negative bacterial. It has beenconfirmed Haemophilus influenzae BF relate to a variety of respiratorydiseases such as chronic obstructive pulmonary disease, pulmonary fibrosis,chronic nasal-sinusitis, secretory otitis media, adenoidal hypertrophy, etc.After bacterial BF formation, antibiotics resistance can increase10-1000times. As a common expectorant, Ambroxol (AMB) is used in clinicalrespiratory mucus dilution to improve ventilation conditions. Recentlystudies reported that AMB can destroyed BF of Pseudomonas aeruginosa,Staphylococcus aureus and bactericidal within BF. It is not reportedwhether AMB could destroy BF of Haemophilus influenzae andbactericidal within BF. The study isolated Haemophilus influenzae fromthe adenoids, established BF model in vitro, measured the antibioticssusceptibility comparison of planktonic bacterial. After selecting2strainsof Haemophilus influenzas with strongest BF, we observed destroyfunction of the AMB against Haemophilus influenzae BF and bactericidalwithin BF. Finally the effect of combination AMB and antibioticbactericidal effect on bacterial BF is observed.Firstly adenoids specimens are collected from the children withadenoidal surgery, then after being isolated, cultured and identified, Haemophilus influenzae is preserved. The BF formation capacity is testedby Crystal violet staining in96well plate and the stucture of the BF isobserved by scanning electron microscope (SEM). Then its susceptibility toantibiotic ampicillin (AMPC), ceftriaxone (CRO), levofloxacin (LVFX),and azithromycin (AZM) is compared after BF formation. Then2strainsHaemophilus influenzaes with strongest BF are selcetced. The effect ofAMB against BF is observed through Crystal violet and SEM. AMBbactericidal effect on bacterial within BF is observed by colony counts.Finally the bactericidal effect of combined AMB with AMPC on BF wasassayed with MTT.30strains Haemophilus influenzae were collected from36adenoidsspecimens, all of the Haemophilus influenzaes can form various size BFafter72hours culture, and HI12, HI23formed strongest BF. The structureof BF of Haemophilus influenzae was very clear by scanning electronmicroscope (SEM). Compared to the planktonic bacterial, the minimalbiofilm eradication concentration (MBEC) for AMPC againstbiofilm-forming bacterial was more than100times higher than the minimalinhibitory concentration (MIC) and minimal bactericidal concentration(MIC). The MBEC of CRO was dozens of times than MBC and MIC. TheMBEC of LVFX was several times than MBC and MIC. And the MBEC ofAZM is very close to MBC and MIC. Through Crystal violet assay, whenAMB reached at0.24mg ml-1and0.47mg ml-1, respectively, to HI23BFand HI12BF has a clear effect (P<0.05) with the control group. The BFwas destroyed by SEM. AMB had the effect on bactericidal by plate culturecount, and the effect in a dose dependent. Conclusion: AMB destroyed theBF of Haemophilus influenzae, and had bactericidal within BF. Statisticsshow that AMB combined AMPC have a synergistic bactericidal effect onHaemophilus influenzae bacterial within BF (P<0.05).Comprehensive experimental results: Haemophilus influenzae clinicalisolates can form BF. After the BF formations, sensitivity to AMPC, CRO, LVFX and AZM decline. AMB have a destroy and bactericidal effect onHaemophilus influenzae BF. AMB combined with AMPC have asynergistic bactericidal effect on Haemophilus influenzae BF. The studyprovides a theoretical foundation for AMB in clinical treatment, andprovide a useful medication guide to treatment of infectious diseases by BFof Haemophilus influenzae. |
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