| Objective:The changes of pulmonary bacterial community structures and function between prebacillary and infected samples from Hospital Acquired Pneumonia( HAP) patients infected by Pseudomonas aeruginosa in Neurosurgical Intensive Care Unit(NICU) were analyzed by 16S rRNA gene sequencing technology to explore a new way to diagnose and cure HAP.Method:Patients that admitted to NICU from 2013.12 to 2015.12 were strictly selected according to the following conditions: 1) The moderately severe craniocerebral injury individuals that combined no lung injury and pneumonia on admission; 2) Tracheotomy was performed within 48 hours after admission; 3) The speciemens were collected by fiber bronchoscope through the trachea incision under no HAP status at 48 hours after admission, and the cultivation results indicated no pathogenic bacteria; 4) The speciemens were collected when HAP occurred for the first time, the cultivation results only indicated Pseudomonas aeruginosa, and subsequently two or more consecutive cultivations also only indicated Pseudomonas aeruginosa. The individuals were removed if contained one of the following conditions: antibiotic usage history resently, not suitable for fiber bronchoscope, the absence of the second specimens. The specimens were divided into prebacillary and infected groups based on whether HAP occurred when specimens were collected. Simultaneously the specimens were divided into two parts: one was delivered for bacterial cultivation, the other was cryopreservated for further sequencing. The information of participators including epidemiological data, clinical symptoms and signs, laboratory and imageological examination results.etc were collected. The variations of bacterial community structures especially predominant bacteria and functional gene were compared between prebacillary group and infected group.Results:A total number of 20 self-control specimens from 10 patients were enrolled as per patient provided two specimens at prebacillary and infected time points respectively. We analysis the results from five aspects: classification levels?? diversity?principal component analysis?? diversity and functional genes. 1) A total of 459 species were obtained on genus level. Haemophilus, Prevotella, Streptococcus and Neisseria were the dominant bacteria for prebacillary group and the percentages between prebacillary and infected groups were 25.0% vs 0.8%, 21.0% vs 0.8%, 12.7% vs 5.1%, 9.0% vs 12.9%. Prevotella, Pseudomonas, Neisseria and Stenotrophomonas were the dominant bacteria for prebacillary group and the percentages between infected and prebacillary groups were 24.4% vs 21.0%, 20.2% vs 0.3%, 12.9% vs 9.0%, 6.3% vs 0.02%. The rates of shared flora between prebacillary and infected specimens from the same person were below 60%. 25 of 459 species existed statistical differences, including Pseudomonas and Haemophilus. 2) A total of 289 species were obtained on familly level. Pasteurellaceae,Streptococcaceae and Prevotellaceae were the dominant bacteria for prebacillary group and the percentages between prebacillary and infected groups were27.95% vs1.34%, 12.65% vs 5.11%, 11.39% vs 11.82%. Pseudomonadaceae, Neisseriaceae and Paraprevotellaceae were the dominant bacteria for prebacillary group and the percentages between infected and prebacillary groups were 20.24% vs 0.31%, 13.78% vs 9.28%, 12.58% vs 9.62%. 15 of 289 species existed statistical differences, including Pasteurellaceae and Pseudomonadaceae. 3) A total of 172 species were obtained on order level. Pasteurellales, Bacteroidales and Lactobacillales were the dominant bacteria for prebacillary group and the percentages between prebacillary and infected groups were 28.66% vs 24.33%, 23.28% vs 0.58%, 13.78% vs 9.28%. Bacteroidales, Pseudomonadales and Neisseriales were the dominant bacteria for prebacillary group and the percentages between infected and prebacillary groups were 20.24% vs 0.31%, 13.78% vs 9.28%, 12.58% vs 9.62%. 8 of 172 species existed statistical differences, including Pasteurellales and Pseudomonadales. 4) A total of 87 species were obtained on class level. Gammaproteobacteria, Bacteroidia and Bacilli were the dominant bacteria for prebacillary group and the percentages between prebacillary and infected groups were 33.39% vs 31.34%, 24.33% vs 28.66%, 14.87% vs 5.67%. Gammaproteobacterias, Bacteroidia and Betaproteobacteria were the dominant bacteria for prebacillary group and the percentages between infected and prebacillary groups were 31.34% vs 33.39%, 28.66% vs 24.33%, 14.48% vs 10.03%. 3 of 87 species existed statistical differences, including no dominant bacteria.5) A total of 37 species were obtained on phylum level. Proteobacteria, Bacteroidetes and Firmicutes were the common dominant bacteria for prebacillary and infected groups and the percentages between prebacillary and infected groups were 44.8% vs 46.5%, 26.4% vs 30.6%, 18.7% vs 12.1%. 2 of 37 species existed statistical differences, including no dominant bacteria. The rates of shared flora between prebacillary and infected specimens from the same person ranged from 39.1% to 88.9%. Only one of the matched-paired ratio was 39.1%, the others were above 60%. 6) ? diversity and principal component analysis on genus level The Unifrac analysis including PCo A and NMDS were performed based on the distance matrix information obtained by calculating evolution and abundance information of species in specimens. Principal component analysis were conducted by R software based on classification and bacteria abundance information on genus level. The community differences were reflected by distance between two points and results show that prebacillary and infected specimens from the same individual, between and within prebacillary group and infected groups speciemens existed obvious differences. 7) ? diversity indexes Chao index, ACE index, Shannon index and Simpson index reflected the community diversity and richness existed no statistical difference between two groups. 8) functional gene The functional genes were divided into 7 categories and 41 subcategories. Membrane Transport belonged to Environmental Information Processing, Replication and Repair belonged to Genetic Information Processing, Carbohydrate Metabolism and Amino Acid Metabolism belonged to Metabolism were dominant genes with percentages of 11.2% vs 10.1%, 9.7% vs 8.5%, 9.4% vs 8.9%, 9.1% vs 10.1% between prebacterial and infected groups. 8 of 41 subcategories including Amino Acid Metabolism existed statistical difference.Conclusion:1.The discovery between prebacillary and infected groups: 1) The compositions on genus, family and order levels existed obvious difference especially for the dominant bacteria, but on the class and phylum levels existed minor differences; 2) The evolution information and species abundance information existed obvious differences indicated the bacterial composition varied greatly; 3) The distribution of functional genes varied little.2. The discovery between prebacillary and infected specimens from the same individual: 1) The compositions on genus, family, order and class levels existed obvious difference especially for the dominant bacteria, but on phylum level existed minor differences; 2) The evolution information and species abundance information existed obvious differences indicated the bacterial composition varied greatly; 3) The distribution of functional genes varied little.3. The discovery of specimens within prebacillary and infected groups indicated the bacterial composition varied greatly but distribution of functional genes varied little.4. The research results showed that the bacterial composition varied greatly when HAP occurred for moderately severe craniocerebral injury patients during the treatment in NICU. |
PDF Full Text Request