Microflora In Endotracheal Tube Biofilm Extubated Form Mechnical Ventilated Neonates And The Interplay Between Bacteria

PART Ⅰ MICROBIAL INVESTIGATION ON ENDOTRACHEAL TUBE BIOFILM AMONG INTUBATED NEONATES WITH VENTILATOR-ASSOCIATED PNEUMONIAObjective Endotracheal tube(ETT) biofilm is a significant risk factor for having ventilator-associated pneumonia(VAP). We aim to characterize the microflora in ETT biofilm among intubated neonates with and without VAP, thus investigate the significant microbial signatures in ETT biofilm among VAP neonates.Method ETT samples were achieved from neonates who were diagnosed as respiratory distress syndrome(NRDS) and pneumonia in Children’s Hospital of Chongqing Medical University from January 31,2014 to January 31, 2015. All the study patients suffered from mechanical ventilation for more than 48 hours. Microbial DNA extracted from ETT biofilm was used to sequence the 16 S r RNA gene V3-V4 region on Illumina Miseq platform. Coverage coefficients and rarefaction curve wereprocessed to evaluate the coverage of sequencing. Biodiversity index was calculated to assess the abundance and diversity of microflora. The Resulting operation taxonomy units(OTU) were analyzed with 97%similarity to characterize and compare the microbial communities in ETT biofilm between the study patients with and without VAP.Result Based on Miseq sequencing, multiple bacteria were present in ETT biofilm among intubated neonates with a relatively high diversity.Patients with different underlying diseases harbored distinct microbial signatures in ETT biofilm(P<0.05). Spearman correlation test showed a significant relationship between the presence of Streptococcus sp. in ETT biofilm and VAP(P<0.05). Although being insignificant(P>0.05), the intubation duration of patients with Streptococcus sp. present in ETT biofilm was longer and the white blood cell count was lower.Conclusion Streptococcus sp. was more easily to be identified in ETT biofilm of intubated neonates with VAP, which might suggest a role in the onset of VAP.PARTⅡ THE CONSISTENCE OF MICROFLORA IN THROAT SWAB, TRACHEAL ASPIRATES AND ENDOTRACHEAL TUBE BIOFILM AMONG NEONATES WITH VENTILATOR-ASSOCIATED PNEUMONIAObjective During intubation microbial investigations in ETT samples are unavailable due to the infeasibility of collecting ETT samples during intubation among intubated neonates. Finding alternative samples to help identify the ETT biofilm flora is urgently needed.Method ETT, throat swab and tracheal aspirate samples were collected from VAP neonates in Children’s Hospital of Chongqing Medical University from January 31, 2014 to January 31, 2015. Microbial DNA was extracted. 16 S r RNA gene V3 region PCR, denaturing gradient gel electrophoresis(DGGE), cloning and sequencing were processed.Sequences were assigned to phylogenetic species using BLAST. Microbial diversity and richness among the three types of specimens were compared based on DGGE fingerprints, and taxonomic characteristics based on the BLAST results.Result The microbial richness and diversity of ETT biofilm were similar to tracheal aspirate yet significantly different from throat swab samples(P<0.05). Compared with ETT biofilm, the overall constituent ratio of microflora was significantly different in throat swab and tracheal aspirate samples. However tracheal aspirate samples performed well in detecting Staphylococcus sp. in ETT biofilm with a sensitivity of 85.7%and a specificity of 83.3%. The sensitivity for the combination of tracheal aspirate and throat swab samples to detect Staphylococcus sp. in ETT biofilm was 100%. The detection of Pseudomonas sp. in throat swabhelped its identification in ETT biofilm especially among those with early onset VAP(sensitivity 60.0% and specificity 100%).Conclusion Our study suggests microbial investigations in throat swab and tracheal aspirate samples are beneficial to identify the ETT biofilm flora, especially on the identification of those potential pathogens such as Staphylococcus sp. and Pseudomonas sp..PART Ⅲ THE ISOLATION AND IDENTIFICATION OF STREPTOCOCCOUS SP. FROM ENDOTRACHEAL TUBE BIOFILM SPECIMEN AND ITS EFFECT ON THE BIOFIOM FORMATION OF PSEUDOMONAS AERUGINOSA IN VITROObjective To isolate and identify Streptococcus sp. in ETT biofilm,and investigate its influence on the biofilm formation of the common VAP pathogen, Pseudomonas aeruginosa.Method ETT samples were collected from VAP neonates in Children’s Hospital of Chongqing Medical University from January 31,2014 to January 31, 2015. ETT biofilm was cultured and suspected Streptococcus sp. strains were determined based on the smear test under microscope, gram stain and catalase reaction. Microbial DNA was extracted. PCR targeting 7 housekeeping genes of Streptococcus sp. was conducted. PCR products were sequenced based on which phylogenetic trees were constructed using MEGA6. Biofilm formation and the gene expression of las I, las R, rhl, rhl R and agl D were investigated and compared between biofilms of Pseudomonas aeruginosa(ATCC BAA-47)with and without the presence of the clinical Streptococcus sp. isolate.Result A Streptococcus sangunis strain was isolated and identified from the study samples. The clinical Streptococcus sangunis strain enhanced the biofilm formation as well as the growth of Pseudomonas aeruginosa. With concurrent Streptococcus sangunis strain in Pseudomonas aeruginosa biofilm, the gene expression of las I,las R,rhl,rhl R and agl D were signicantly higher than the exclusive Pseudomonas aeruginosa biofilm(P<0.05).Conclusion The clinical Streptococcus sangunis strain enhanced the biofilm formation of Pseudomonas aeruginosa, which might play a role in the pathogenicity of VAP.PART Ⅳ THE IMPACT OF MIXED BIOFILMS OF THE CLINICAL STREPTOCOCCOUS SP. ISOLATE AND PSEUDOMONAS AERUGINOSA ON HUMAN LUNG EPITHELIAR CELLSObjective To study the cell viability and cytokine secretion of the human lung epitheliar cells exposed to the clinical Streptococcus sangunis strain and Pseudomonas aeruginosa biofilm-conditioned medium(BCM).Method BCM was prepared from the clinical Streptococcus sangunis and Pseudomonas aeruginosa biofilm, Pseudomonas aeruginosa biofilm,and the clinical Streptococcus sangunis biofilm, respectively. The human lung epitheliar cells(BEAS-2B, ATCC 9609) were exposed to the the above BCM. After intubation for 24 h, cell viability was detected by CCK-8. Apoptosis and necrosis were investiaged using Hochest/PI staining.Cell supernatants were collected after intubation with BCM for 1 h, 3 h, 6 h,9 h, 12 h and 24 h, respectively, which were used for IL-8 detection.Result The clinical Streptococcus sangunis and Pseudomonas aeruginosa co-culture BCM induced less damage to BEAS-2B cells than BCM of Pseudomonas aeruginosa(P<0.05). After intubation with BCM for 3h, the level of IL-8 rose to a peak, and BCM of the Streptococcus sangunis and Pseudomonas aeruginosa induced a significant lower level of IL-8 secretion than BCM of Pseudomonas aeruginosa alone(P<0.05).Conclusion The clinical Streptococcus sangunis strain might regulate the host innate immune response induced by Pseudomonas aeruginosa biofilm.