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Objective:To investigate the changes and correlation of intestinal and pulmonary microecological structures in patients with ventilator-associated pneumonia (VAP).Methods:A prospective observational study was conducted. Thirty-one patients with VAP admitted to the department of critical care medicine of General Hospital of Ningxia Medical University from May 1st 2019 to May 1st 2020 were enrolled. Feces and alveolar lavage fluid samples from patients with the same day, feces and alveolar lavage specimen flora composition and the structure of biological information analysis by 16S rRNA sequencing technologies, the comprehensive sequencing results, and clinical data of patients were analyzed.Results:① The diversity (abundance and diversity) of flora in the alveolar lavage fluid of VAP patients was higher than that of fecal flora. Among them, Ace index, Chao index and Shannon index describing the abundance of flora showed statistically significant differences [Ace index: 305.89 (214.39, 458.66) vs. 204.51 (165.15, 247.61), Chao index: 259.83 (194.20, 459.31) vs. 187.67 (153.28, 234.01), Shannon index: 3.01 (2.39, 3.54) vs. 2.55 (1.86, 2.95), all P < 0.05], but there was no significant difference in Simpson index describing diversity [0.14 (0.08, 0.27) vs. 0.19 (0.10, 0.33), P > 0.05]. ② In the sequencing results of feces and alveolar lavage fluid of VAP patients, there were some intestinal related bacteria groups with high abundance, such as Escherichia-Shigella, Faecalibacterium, Bacteroides, and Lachnospira, etc. ③ In 31 VAP patients, suspicious pathogenic bacteria was found in 20 cases (6 cases of Streptococcus viridans, 5 cases of Escherichia coli, 3 cases of Klebsiella pneumoniae, 3 cases of Acinetobacter baumannii, 2 cases of Staphylococcus aureus, 1 case of Pseudomonas aeruginosa), and the same suspected pathogens also existed in the 17 patients' alveolar lavage and waste sequencing. ④ Fourteen VAP patients combined with sepsis, 14 patients without sepsis were selected for sample size matching. The results showed that, Jaccard similarity index to describe lung-correlation of intestinal flora in VAP with sepsis group was significantly elevated, and the difference was statistically significant (0.24±0.08 vs. 0.19±0.06, P < 0.01). Conclusions:There is a certain correlation between pulmonary and intestinal flora in VAP patients. In addition to the exclusion of pulmonary infection caused by environmental and upper respiratory micro-inhalation, the lower digestive tract may also be source of infection.
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Objective:To explore the correlation between the levels of peripheral blood T lymphocyte subsets (CD3 +, CD4 +, CD8 +) and natural killer cells (NK cells) in the early stage of sepsis and intestinal injury and prognosis of patients, so as to provide the basis of immunotherapy for clinical treatment of sepsis. Methods:A prospective case-control study was conducted. Sixty-one patients with sepsis admitted to Department of Critical Care Unit (ICU) of Ningxia Medical University from September 2018 to May 2019 were selected as subjects (sepsis group). Seventeen patients with common postoperative non-sepsis were used as controls (non-sepsis group). Venous blood samples were collected from all subjects within 24 h of ICU admission, and the levels of T lymphocyte subsets and NK cells were measured. At the same time, 35 sepsis patients without chronic gastrointestinal tract and chronic renal insufficiency were selected from the sepsis group, and their serum levels of intestinal fatty acid binding protein (I-FABP) and D-lactic acid were measured. Age, sex, and underlying disease were recorded for all subjects. The acute physiology and chronic health evaluationⅡ(APACHEⅡscore) within 24 h of ICU in the sepsis group were evaluated, and the patients were followed up for 28 days. T lymphocyte subsets and NK cells, I-FABP, D-lactic acid levels were compared between the sepsis group and non-sepsis group. T lymphocyte subsets and NK cell levels were compared between the survival and death groups. Spearman correlation method was used to analyze the correlation between serum I-FABP and D-lactic acid in the early stage of sepsis and levels of CD3 +, CD4 +, CD8 + and NK cells, and multivariate logistic regression analysis was used to assess the risk factors of death in sepsis patients. Results:Compared with the non-sepsis group, serum I-FABP and D-lactic acid levels were increased in the sepsis group [I-FABP (μg/L): 18.36 (14.75, 28.34) vs 16.17 (12.12, 18.40), D- Lactic acid (mg/L): 18.70 (10.10, 40.60) vs 8.85 (7.10, 15.76), all P < 0.05]. Peripheral blood CD3 +, CD4 +, NK cells in the sepsis group were decreased [CD3 +(%): 54.30 (37.48 , 61.65) vs 60.75 (48.88, 69.95), CD4 +(%): 24.60 (17.65, 32.15) vs 31.90 (24.95, 37.10), NK cells (%): 18.20 (11.95, 31.10) vs 24.70 (19.30, 32.65), P<0.05], but there was no significant difference in peripheral blood CD8 + levels between the two groups ( P>0.05). There were no significant differences in CD3 +, CD4 +, CD8 + and NK cell levels between the survival group and death group (all P>0.05).Correlation analysis of T lymphocyte subsets, NK cells and intestinal injury markers ( n=35) showed that I-FABP was negatively correlated with CD3 + and CD8 + ( r=-0.478 and r=-0.415, respectively, both P<0.05), but not correlated with CD4 + and NK cells (both P>0.05). D-lactic acid was negatively correlated with CD3 + and CD4 + ( r=-0.344 and r=-0.423, respectively, P<0.05), and positively correlated with NK cells ( r=0.393, P<0.05), but there was no correlation between D-lactic acid and CD8 + (both P>0.05). Multivariate logistic regression analysis showed that only the APACHEⅡ score was an independent risk factor for 28-day mortality in patients with sepsis ( OR=1.222, 95% CI:1.084-1.378, P<0.01), but the early CD3 +, CD4 +, CD8 +, NK cell levels were not associated with 28-day prognosis in patients with sepsis ( P>0.05). Conclusions:Immunosuppression can occur in the early stage of sepsis, which is related to intestinal damage, but is not associated with patient prognosis.
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Objective@#To investigate the characteristics of gut microbiota dysbosis in patients with severe pneumonia using 16SrDNA sequencing.@*Methods@#A prospective observational research was conducted. The stool samples retained by natural defecation or enema within 2 days after hospital were collected from 16 patients with severe pneumonia admitted to department of intensive care unit (ICU) of General Hospital of Ningxia Medical University from June to December in 2018 and 10 persons for physical exam were enrolled as the healthy control group. The 16SrDNA sequencing technology was used to detect fecal flora and analyze biological information.@*Results@#① 1 015 475 effective sequences were obtained from the stool samples from the severe pneumonia group and the healthy control group. Using 16SrDNA method, it was found that the average effective length of the sample sequence was 458.35 bp and the average sequence number of the total samples was 39 056.73. ② Analysis of α diversity of gut microbiota showed that, compared with the healthy control group, the Ace index, Chao index and the Shannon index of gut microbiota diversity in the severe pneumonia group were significantly decreased [Ace index: 167.23 (143.14, 211.26) vs. 227.71 (214.53, 247.05), Chao index: 152.38 (138.09, 182.54) vs. 228.25 (215.49, 248.95), Shannon index: 2.37 (1.68, 2.89) vs. 3.39 (3.03, 3.63), all P < 0.01], and the Simpson index was significantly increased [0.21 (0.11, 0.33) vs. 0.07 (0.06, 0.12), P < 0.01], which indicated the gut microbiota diversity of the severe pneumonia group was decreased. ③ Analysis of β diversity of gut microbiota, principal coordinate analysis (PCoA) showed that gut microbiota structural with the healthy control group was similar, while that in the severe pneumonia group was different. Adonis analysis showed that the structural of the gut microflora revealing significant differences between the severe pneumonia group and the healthy control group (R2 = 0.061, P = 0.05). ④ Analysis of phylum difference gut microflora showed that, compared with the healthy control group, the proportion of Firmicutes in severe pneumonia group was decreased [27.36 (18.12, 39.28)% vs. 52.25 (38.36, 63.82)%, P = 0.02], the proportions of Actinobacterias, Synergistetes and Fusobacterias were increased [2.30 (0.30, 4.80)% vs. 0.02 (0.00, 0.06)%, 0.36 (< 0.01, 0.57)% vs. < 0.01 (< 0.01, < 0.01)%, 0.01 (< 0.01, 0.08)% vs. < 0.01 (< 0.01, < 0.01)%, all P < 0.05]. ⑤ Analysis of genus difference gut microflora showed that, the proportions of Bifidobacterium, Ruminococcus, Pseudobutyrivibrio, Coprococcus, Lachnospira and Prevotella in the severe pneumonia group were significantly lower than those in healthy control group [0.18 (0.01, 0.25)% vs. 3.40 (0.46, 5.78)%, 0.01 (< 0.01, 0.29)% vs. 2.26 (0.84, 4.86)%, 0.01 (< 0.01, 0.02)% vs. 2.73 (1.87, 5.74)%, 0.02 (< 0.01, 0.07)% vs. 0.80 (0.50, 2.32)%, < 0.01 (< 0.01, < 0.01)% vs. 0.88 (0.33, 2.08)%, 0.02 (< 0.01, 0.31)% vs. 7.74 (0.07, 36.27)%, all P < 0.05]; the proportions of Escherichia and Enterococcus in the severe pneumonia group were higher than those in healthy control group, but there was no difference between the two groups [2.00 (0.57, 10.23)% vs. 1.16 (0.23, 2.68)%, 0.02 (< 0.01, 0.42)% vs. < 0.01 (< 0.01, 0.04)%, both P > 0.05]; the proportions of Fusobacterium and Staphylococcus in severe pneumonia group were significantly higher than those in healthy control group [0.01 (< 0.01, 0.08)% vs. < 0.01 (< 0.01, < 0.01)%, 0.01 (< 0.01, 0.02)% vs. < 0.01 (< 0.01, < 0.01)%, both P < 0.05].@*Conclusion@#Gut microbiota dysbiosis in patients with severe pneumonia shows that the abundance and diversity decrease, structure of intestinal flora changes, and beneficial symbiotic bacteria decrease and pathogenic bacteria increase, which may be associated with the occurrence and development of severe pneumonia.
ABSTRACT
Objective To investigate the characteristics of gut microbiota dysbosis in patients with severe pneumonia using 16SrDNA sequencing. Methods A prospective observational research was conducted. The stool samples retained by natural defecation or enema within 2 days after hospital were collected from 16 patients with severe pneumonia admitted to department of intensive care unit (ICU) of General Hospital of Ningxia Medical University from June to December in 2018 and 10 persons for physical exam were enrolled as the healthy control group. The 16SrDNA sequencing technology was used to detect fecal flora and analyze biological information. Results ① 1015475 effective sequences were obtained from the stool samples from the severe pneumonia group and the healthy control group. Using 16SrDNA method, it was found that the average effective length of the sample sequence was 458.35 bp and the average sequence number of the total samples was 39056.73. ② Analysis of α diversity of gut microbiota showed that, compared with the healthy control group, the Ace index, Chao index and the Shannon index of gut microbiota diversity in the severe pneumonia group were significantly decreased [Ace index: 167.23 (143.14, 211.26) vs. 227.71 (214.53, 247.05), Chao index: 152.38 (138.09, 182.54) vs. 228.25 (215.49, 248.95), Shannon index:2.37 (1.68, 2.89) vs. 3.39 (3.03, 3.63), all P < 0.01], and the Simpson index was significantly increased [0.21 (0.11, 0.33) vs. 0.07 (0.06, 0.12), P < 0.01], which indicated the gut microbiota diversity of the severe pneumonia group was decreased. ③ Analysis of β diversity of gut microbiota, principal coordinate analysis (PCoA) showed that gut microbiota structural with the healthy control group was similar, while that in the severe pneumonia group was different. Adonis analysis showed that the structural of the gut microflora revealing significant differences between the severe pneumonia group and the healthy control group (R2 = 0.061, P = 0.05). ④ Analysis of phylum difference gut microflora showed that, compared with the healthy control group, the proportion of Firmicutes in severe pneumonia group was decreased [27.36 (18.12, 39.28)％ vs. 52.25 (38.36, 63.82)％, P = 0.02], the proportions of Actinobacterias, Synergistetes and Fusobacterias were increased [2.30 (0.30, 4.80)％ vs. 0.02 (0.00, 0.06)％, 0.36 (< 0.01, 0.57)％ vs. < 0.01 (< 0.01, < 0.01)％, 0.01 (< 0.01, 0.08)％ vs. < 0.01 (< 0.01, < 0.01)％, all P < 0.05]. ⑤ Analysis of genus difference gut microflora showed that, the proportions of Bifidobacterium, Ruminococcus, Pseudobutyrivibrio, Coprococcus, Lachnospira and Prevotella in the severe pneumonia group were significantly lower than those in healthy control group [0.18 (0.01, 0.25)％ vs. 3.40 (0.46, 5.78)％, 0.01 (< 0.01, 0.29)％ vs. 2.26 (0.84, 4.86)％, 0.01 (< 0.01, 0.02)％ vs. 2.73 (1.87, 5.74)％, 0.02 (< 0.01, 0.07)％vs. 0.80 (0.50, 2.32)％, < 0.01 (< 0.01, < 0.01)％ vs. 0.88 (0.33, 2.08)％, 0.02 (< 0.01, 0.31)％ vs. 7.74 (0.07, 36.27)％, all P < 0.05]; the proportions of Escherichia and Enterococcus in the severe pneumonia group were higher than those in healthy control group, but there was no difference between the two groups [2.00 (0.57, 10.23)％ vs. 1.16 (0.23, 2.68)％, 0.02 (< 0.01, 0.42)％ vs. < 0.01 (< 0.01, 0.04)％, both P > 0.05]; the proportions of Fusobacterium and Staphylococcus in severe pneumonia group were significantly higher than those in healthy control group [0.01 (< 0.01, 0.08)％ vs. < 0.01 (< 0.01, < 0.01)％, 0.01 (< 0.01, 0.02)％ vs. < 0.01 (< 0.01, < 0.01)％, both P < 0.05]. Conclusion Gut microbiota dysbiosis in patients with severe pneumonia shows that the abundance and diversity decrease, structure of intestinal flora changes, and beneficial symbiotic bacteria decrease and pathogenic bacteria increase, which may be associated with the occurrence and development of severe pneumonia.