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Objective To investigate the biofilm (BF)formation rule of nontypeable Haemophilus influenzae (NTHi)in vitro, and to observe the internal structure of BF by scanning electron microscope (SEM). Methods NTHi ATCC49247 was investigated in the present study,Pseudomonas aeruginosa (PA)PAO1 was cultured as positive control,at the same time blank control group was set up.The BF of the bacteria were cultured and then collected on day 1,2,3,4,5,6,and 7.The BF formation was detected by crystal violet staining and plate counting and the structure of BF formed by ATCC49247 was observed under SEM on day 3.Results The plate colony counting of biofilm BF by ATCC49247 and PAO1 raised during first 3 d, and then declined to (0.823 6±0.007 5)×107 cfu·mL-1 and (0.942 6±0.019 9)×107cfu·mL-1 respectively on day 7. The differences between two groups were statistically significant on day 3,4,5,and 6 (P<0.05).The differences between different time points in the same bacteria group were statistically significant (P<0.05).The densities of BF formed by ATCC49247 and PAO1 raised during the first 3 d.The absorbances on 570 nm wavelength (A570 )in two groups were 2.717 4±0.017 2 and 2.885 3±0.039 0 ,respectively;and then the A570 values in two groups declined to 0.151 7±0.074 5 and 1.196 9±1.108 5,respectively on day 7;the differences between bacteria groups and blank control were statistically significant (P<0.05 );the differences between two bacteria groups were statistically significant on day 3,4,5,and 6 (P<0.05);the differences between different time points in the same bacteria group were statistically significant (P<0.05).On day 3,the obvious BF formed by ATCC49247 were observed under SEM.Conclusion BF could be formed by NTHi in vitro;crystal violet staining,plate colony counting and SEM could be taken as conventional methods to detect BF.
RESUMO
<p><b>OBJECTIVE</b>To investigate the effect of Pseudomonas quinolone signal (PQS) on the virulence of Pseudomonas aeruginosa.</p><p><b>METHODS</b>Pseudomonas aeruginosa strain PAO1 was treated with PQS alone, PQS plus farnesol, or farnesol alone. The transcriptional levels of the regulator gene ExsA and virulence protein gene ExoS of type III secretion system were examined using quantitative real-time PCR, and spectrophotometry was employed to detect pyocyanin production in the bacteria. The adhesion and invasiveness of the treated PAO1 in cultured alveolar epithelial cells A549 were assessed on plate count agar, and their effects on the survival of a mouse model of peritonitis was compared.</p><p><b>RESULTS</b>The increase or decrease of PQS did not affect the growth of PAO1. Compared with the untreated bacteria, PQS-treated PAO1 showed obviously increased transcription levels of ExsA and ExoS (P<0.01) and pyocyanin production, which was significantly lowered by farnesol (P<0.01). In A549 cell cultures, farnesol-treated PAO1 exhibited significantly lowered adhesion and invasiveness, while PQS-treated PAO1 caused a significantly decreased survival time of mice with peritonitis (P<0.01). Farnesol treatment did not obviously affected ExsA transcription (P>0.05) but caused a significant reduction in the transcriptional level of Exos (P<0.05) in PAO1. PQS showed no significant effect on the adhesion and invasiveness of PAO1 (P<0.05).</p><p><b>CONCLUSION</b>PQS can maintain the adhesion and invasiveness of Pseudomonas aeruginosa, and in the hosts of the bacteria, PQS concentration is positively correlated with pyocyanin production and hence negatively with the survival time of the hosts.</p>