The Relation Between Infections By Nontypeable Haemophilus Influenzae And Biofilm Formation

PART1Biofilm Formation by nontypeable Haemophilus influenzae in vitroObjective To investigate biofilm formation by nontypeable haemophilus influenzae (NTHi) in vitro, so as to pave the way for infection pathogenesis study of biofilm formed by NTHi.Methods Nontypeable haemophilus influenzae (NTHi) ATCC49247was investigated in the present study, Pseudomonas aeruginosa (PA) ATCC27853as positive one. Biofilms were single-cultured on cover glasses and micro-cell plate, and then collected on day2,3,4and7after incubation, and the condition of biofilm formation was determined by crystal violet staining and plate counting. Scanning electron microscope was also observed under on day4.Results The absorbances of biofilm in the first4days were from 0.0591±0.0013to0.269±0.0028for NTHi vs.0.0634±0.0016to0.332±0.0028for PA, followed by an decrease on day7to0.1135±0.0021for NTHi vs.0.1265±0.0050for PA, when using crystal violet staining. The blank control was0.0365±0.0028. The viable count of NTHi in the first4days were from (0.530±0.028)×107CFU/ml to (2.275±0.0212)×107CFU/ml, followed by an decrease to (1.036±0.0198)×107CFU/ml on day7, while the count of PA in the corresponding points were from (0.55±0.014)×107CFU/ml to (3.705±0.021)×107CFU/ml, followed by an decrease to (1.405±0.035)×107CFU/ml on day7. There were significant differences in time points and group of bacteria and blank control (P<0.05). However, except for day4(P<0.05), there were no statistical differences in comparisons between the two bacteria in the other days (P>0.05). On day4, obvious biofilms were seen using scanning electron microscope.Conclusions Biofilm could be formed by NTHi in vitro. Crystal violet staining, plate counting, and SEM could be taken as Conventional detection. PART IIThe relation between common diseases infected by NTHi and biofilm formation among childrenObjective To study the variation of biofilm formation in common diseases infected by NTHi among children, and to lay a foundation for biofilm effect in different diseases.Methods80strains of NTHi were isolated from respiratory tract from sinusitis, otitis media, bronchitis and pneumonia and ATCC49247were investigated in the study. Consecutive5days were cultured, the density of biofilm were determined by crystal violet staining, and confirmed by scanning electron microscope on day2.Results The average absorbances for biofilm formation by clinical strains the time to form biofilm by clinical strains were0.3056±0.021838on day1, went highest to0.5516±0.04653on day2, and came down to0.2554±0.02229on day5, while the absorbance of ATCC49247were only0.0305±0.0007on day1, and went highest to0.255±0.0339on day4, then came down to0.1945±0.0057on day5. The four diseases’ groups owned different absorbances, shown as0.67477±0.064936for otitis media,0.62190±0.057424for penumonia,0.44705±0.015081for bronchitis0.43817±0.017277for sinusitis on day2, and0.32559±0.031621,0.27371±0.024501,0.22098±0.013289,0.17689±0.028316in the corresponding disease on day5. Except for diseases between bronchitis and sinusitis (P>0.05), there were significant differences between other diseases’ group (P<0.05).Conclusion Variations of biofilm formation could be seen in different diseases infected by NTHi among children. Among the common diseases infected by NTHi in pediatrics, biofilm functions biggest in otitis media, followed by pneumonia, and smallest in sinusitis and bronchitis. PART ⅢThe relation between proinflammatory protein E and biofilm formation caused by NTHiObjective To study the relation between proinflammatory protein E and biofilm formation caused by NTHi among children, and provide evidences for biofilm prevention of biofilmMethods A total of59clinical NTHI isolates from300pediatric patients with otitis media and/or sinusitis,36isolates with sinusitis and23with otitis media were obtained. White blood cell were measured by bloodanalyser. RNAs were extracted from the specimens in state of biofilm and free-floating, and performed on day2,3,4and5, with reverse transcription-polymerase chain reaction (RT-PCR) for pe and luxS genes mRNA.Results In inflammation response, the expression of PE mRNA were correlated with the inflammation(P<0.05, r=0.5253). When pe mRNA were positively transcripted, luxS presented as negative, which indicated no biofilm formation, while pe mRNA were negatively transcripted, luxS presented as positive, which indicated biofilm formation.Conclusion Proinflammatory protein E could influence biofilm formation.