| PART ONEANALAYSIS OF THE BIOFILM FORMATION INCLINICAL ISOLATES OF PSEUDOMONASAERUGINOSAObjective: We analyzed biofilm formation ability of different clinicalisolates Pseudomonas aeruginosa.Method:95Pseudomonas aeruginosa were selected from the Children’sHospital of Chongqing Medical University(49)，First Affiliated Hospital ofPeking University (14) and First Affiliated Hospital of Zhejiang University(32).49strains were derived from the sputum samples,30strains werederived from the blood sample. A total of16strains were derived fromabdominal pus, urine, bile and other specimens. Using microtiter plate, weobserved the biofilm maturation and its peak period formation capacity. Atlast, we analzed the correlation between different clinical strains andbiofilm formation capability.Results: All of95Pseudomonas aeruginosa isolated from clinicalstrains could form biofilm. In49sputum’s clinical strains,45strains(91.8%) had strong biofilm formation ability. In46non sputum’s clinical strains,33strains(71.7%) had strong biofilm formation ability. To compare the strongbiofilm formation ability rate of clinical isolates from these twogroups, thedifference was statistically significant (P <0.05). Biofilm maturity ofsputum group (49) was3.63±1.20days and the non sputum group was4.27±1.23days. The biofilm maturity difference between these two groupswas statistically significant (p <0.05).Conclusion: There were big gaps in biofilm forming ability betweendifferent clinical strains. In sputum group, there was stronger biofilmforming ability and shorter biofilm maturation than those in non sputumgrouop. PART TWOEFFECT OF ANDROGRAPHOLIDE ON PSEUDOMONASAERUGINOSA INFECTION IN HUMAN BRONCHIALEPITHELIAL CELLSObjective: After establishing Pseudomonas aeruginosa infected humanbronchial epithelial cell model, we observed the intervention ofandrographolide on the bacterial infection ability and the host cellinflammatory response.Methods: Pseudomonas aeruginosa strains were selected fromChongqing, Beijing and Zhejiang. Using PCR，we detected clinical strainsT3SS virulence effector molecules encoding genes. Through analyzingT3SS virulence gene expression differences from different clinical isolatessources, we selected four clinical strains to infect BEAS-2B. These selectedstrains were Cq40（exoU+, exoS-, exoT+and exoY+）, Cq1（exoU-, exoS+,exoT+and exoY+）, Cq24（exoU-, exoS+, exoT-and exoY+）and Cq16（exoU+, exoS-, exoT-and exoY+）. Using colony counting method, wedetected Pseudomonas aeruginosa invasion force on BEAS-2B. Usingenzyme-linked immunosorbent assay(ELISA), we observed lactatedehydrogenase(LDH) releasing, inflammatory cytokines interleukin-6(IL-6)and IL-8expression. In vitro Luria-Bertani (LB) broth culture, we observedthe effect of andrographolide (concentration300μM) on bacterial growth.Using two different concentration(3μM and4.5μM) ofandrographolide, we detected its intervention effect on Pseudomonas aeruginosa infection inBEAS-2B. Colony counting was used to detect the Pseudomonas aeruginosainvasiveness on BEAS-2B after intervention of andrographolide. ELISAwas used to detect the BEAS-2B LDH releasing, IL-6and IL-8expressionafter intervention of andrographolide.Results: In95Pseudomonas aeruginosa isolated from clinical strains,the T3SS virulence gene carrier rate was as follows, the exoU21%, exoS84.2%, exoT49.5%and exoY82.1%. In Chongqing group (49), The carrierrate was as follows, exoU20%, exoS90%, exoT59%and exoY91.8%. InHangzhou group (32), exoU28%, exoS69%, exoT25%and exoY63%. InBeijing group (14), exoU7%, exoS100%, exoT71%, exoY93%. To confirmevery strain virulence effector molecules encoding gene carrying, we found11types. There were9strains encoded as exoU-,exoS-,exoT-and exoY-,4strains encoded as exoU-,exoS+,exoT-and exoY-，4strains encoded asexoU-,exoS+,exoT+and exoY-，28strains encoded as exoU-,exoS+,exoTandexoY+，2strains encoded as exoU-,exoS-,exoT+and exoY+，29strainsencoded as exoU-,exoS+,exoT+and exoY+，2strains encoded asexoU+,exoS-,exoT-and exoY+，2strains encoded as exoU+,exoS-,exoT+and exoY+，2strains encoded as exoU+,exoS+,exoT+and exoY-，5strainsencoded as exoU+,exoS+,exoT-and exoY+，and8strains encoded asexoU+,exoS+,exoT+and exoY+. exoU-, exoS+, exoT-, exoY+andexoU-,exoS+, exoT+, exoY+are two main phenotypes in95clinical strainsvirulence genes carrying. exoU-、exoS+、exoT+、exoY+genotypecarrying rate in sputum group is33%, and30%in non-sputum group.exoU-、exoS+、exoT-、exoY+genotype carrying rate in sputum group is37%,and22%in non sputum group. Two genotypes carrier rate is no statisticallysignificant between the sputum group and non sputum group (p>0.05).Cq40、 Cq1、 Cq24and Cq16could invade into BEAS-2B.3μMandrographolide could significantly reduce the number of Pseudomonasaeruginosa invasive intracellular colony number, which dropped from1840±121CFU/ml to1090±101CFU/ml in Cq1,2900±100CFU/ml to1060±196CFU/ml in Cq40,3200±264CFU/ml to1090±125CFU/ml inCq16and1740±121CFU/ml to980±190CFU/ml in Cq24. There wasstronger inhibition effect of4.5μM andrographolide. The invasiveintracellular colony number dropped to360±45CFU/ml in Cq1,500±36CFU/ml in Cq40430±30CFU/ml in Cq16and360±26CFU/ml in Cq24.With the corresponding，3μM andrographolide could significantly affect theLDH releasing， from3128.3±456.939μmol/ml to1875.8±16.061μmol/ml in Cq1,4082.45±133.307μmol/ml to3175.266±298.927μmol/mlin Cq40,3518.467±264.113μmol/ml to2131.6±178.922μmol/ml in Cq16and2382.4±63.161μmol/ml to1636.466±55.930μmol/ml in Cq24. IL-6level was affected by3μM andrographolide, from25.133±0.204pg/ml to17.557±0.318pg/ml in Cq1,12.495±0.691pg/ml to8.576±0.657pg/mlin Cq40,15.130±0.449pg/ml to10.780±0.982pg/ml in Cq16and24.106 ±1.107pg/ml to18.360±0.221pg/ml in Cq24. IL-8production was alsodropped by3μM andrographolide, from593.866±48.981pg/ml to372.466±3.2653pg/ml in Cq1,472.5±48.350pg/ml to339.6±5.556pg/ml inCq40,488.833±39.480pg/ml to251.93±2.9004pg/ml in Cq16, and569.833±26.385pg/ml to345.4±8.006pg/ml in Cq24. Using4.5μMandrographolide, the LDH level dropped to1061.366±165.731μmol/ml inCq1,1813±92.528μmol/ml in Cq40,1459.7±137.157μmol/ml in Cq16and1016.133±77.309μmol/ml in Cq24. The IL-6level dropped to8.865±0.175pg/ml in Cq1,3.761±0.209pg/ml in Cq40,3.348±0.341pg/ml in Cq16and8.833±0.574pg/ml in Cq24. IL-8production dropped to215.266±8.558pg/ml in Cq1,187.566±3.677pg/ml in Cq40,89.76±4.677pg/ml inCq16and300.466±13.9004pg/ml in Cq24. The difference before and afterandrographolide intervention was statistically significant (p <0.05). Thedifference between two different concentration was also statisticallysignificant (p <0.05).Conclusion: Clinical Pseudomonas aeruginosa could invade intoBEAS-2B, which promoted the cells upregulation in LDH releasing andstimulated cells up expression of inflammatory cytokine IL-6and IL-8.Andrographolide could inhibit Pseudomonas aeruginosa clinical isolatesstrains invasion force on human bronchial epithelial cell, and reduce the celldamage degree and improve cell inflammatory response. |
PDF Full Text Request