Decrease of 'health-benefiting' microbes and increase of pathogenic bacteria (a condition termed dysbiosis) in intensive care unit patients is considered to induce or aggravate sepsis (gut-origin sepsis). Orally administered probiotics have been effective in the prevention of nosocomial infections. However, the mechanisms of probiotic-induced anti-infection and anti-sepsis remain to be explored. In the present study, 4-week-old C57BL6 mice were orally administrated with
Sepsis is one of the leading causes of mortality and morbidity in children and adults (
Probiotics are regarded as the living microorganisms, which, in adequate amounts, can bring health benefits to the host (
In the past, the colonization patterns in septic patients were investigated predominantly from culture-dependent studies. However, most of the intestinal bacteria are obligate or facultative anaerobic bacteria, which are technically challenging to culture once exposed in oxygen (
All procedures for animal care and use were approved by the Animal Care Ethics Committee of the First Affiliated Hospital, Zhejiang University (Hangzhou, China). Four-week-old male C57BL6 mice were purchased from Zhejiang University and housed in pathogen-free animal facilities under a standard 12-h light/dark cycle. Standard mouse diet and water were given throughout the study.
Four-week-old male C57BL6 mice (weight, 13.59±1.59 g) were administered daily by oral gavage with 200 µl of LGG (2×109 CFU/ml, 2.9×107 CFU/g; Culturelle, ConAgra Foods, Omaha, NE, USA; CLP+LGG group, n=23), or normal saline (control sham group, n=20; CLP+saline group, n=18) 4 weeks prior to CLP. To establish the murine septic peritonitis model, the mice were anesthetized with isoflurane and bupivacaine: 3–4% for induction and 1–3% for maintenance. A 1 cm incision was made on the middle of abdomen, and the cecum was exteriorized through the incision carefully. In order to induce mid-grade sepsis, the cecum was ligated at middle of the bottom and distal pole of the cecum, and was punctured from mesenteric toward anti-mesenteric direction using a 23-gauge needle. A droplet of feces was extruded through the holes, and the cecum was relocated into the abdominal cavity. Finally, the fascia and skin incision were closed in layers. Sham mice underwent the same operation except from the cecum ligation and puncture procedures. In all mice, 1 ml pre-warmed normal saline was injected subcutaneously following the CLP operation (
Blood samples were obtained from mice at 24 h post-CLP operation. In all instances, 1.5 ml of blood was introduced in the tube not containing any anticoagulant substance. The collected blood was left on the laboratory bench for 30 min at room temperature prior to centrifugation (3,000 × g, 5 min, 4°C), then the serum was carefully absorbed and transferred to a clean tube, and then stored at −80°C as soon as possible.
In brief, 10 µl serum sample was diluted in 40 µl sample dilution buffer per ELISA (Nuoyang Biotech, Hangzhou, China) plate well, and 100
The plate was incubated for 1 h at 37°C and washed five times. HRP-substrate goat anti-mouse IgG (cat. no. 31430; 1:100; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was then added and incubated for 15 min at 37°C in the dark. The enzyme reaction was then stopped with 50
Colon tissues were collected from mice at 24 h post-CLP operation and fixed in 10% formalin for 24 h. The fixed colon tissues were embedded in paraffin and sectioned at 4
To ensure the abundance of LGG (normal/saline, n=5; normal/LGG, n=5), DNA was extracted from fecal samples obtained from a separate set of mice prior to CLP operation. Quantification of LGG was performed by PCR using the following primers: LactoF, 5′-AGC AGT AGG GAA TCT TCC A; and LactoR, 5′-ATTYCACCGCTACACATG. The PCR reaction was performed as follows: 95°C for 5 min, followed by 35 cycles at 94°C for 15 sec, 53°C for 30 sec, and 72°C for 45 sec and a final extension at 72°C for 10 min. To analyze the microbial diversity in mice with sepsis, fecal samples were collected from mice at 24 h post-CLP operation and frozen at -80°C immediately. Microbial DNA was extracted from fecal samples using a Qiagen mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The V3-V4 region of the bacteria 16S ribosomal RNA gene was PCR-amplified using primers 341F (5′-CCT AYG GGR BGC ASC AG-3′) and 806R (5′-GGA CTA CNN GGG TAT CTA AT-3′). An eight bp unique barcode sequence was attached to each sample. A total of 50
Based on barcode and PCR primer, all raw reads were screened using the Quantitative Insights Into Microbial Ecology software (QIIME, version 1.17) (
The homogeneity of variances was verified using Bartlett's test. One-way analysis of variance (with Bonferroni as a post hoc test) was performed to compare the bacteremia, inflammatory factors and histological study variables among different groups. Survival studies were conducted via the log-rank test. Two-tailed non-parametric Kruskal-Wallis test was used to compare the differences in diversity indexes and microbial taxa. The statistical analysis was conducted using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
DNA isolated from the colon was analyzed by PCR to quantify the abundance of LGG. There was a significant increase in LGG in normal/LGG mice (9.24±0.06, log qPCR copy/fecal (g); n=5) compared with normal/saline mice (8.16±0.11, log qPCR copy/fecal (g); n=5;
A previous study demonstrated that septic mice pretreated with LGG had a markedly improved 7 day survival rate compared with the CLP+saline mice (P=0.029). Five sham mice survived (
To evaluate the effect of LGG on reducing the inflammatory response caused by sepsis, the levels of proinflammatory cytokines IL-6, TNF-α, IL-2, and of the anti-inflammatory cytokine IL-22, were detected by ELISA assay in the serum of the mice. Serum levels of IL-22, IL-2, TNF-α and IL-6 were the lowest in sham mice (
As evaluated by H&E staining and histopathological analysis, colon tissues in sham mice were histologically normal in all layers (
In the present study, colon mucosal injury was obvious in septic mice, but the injury was alleviated by LGG pretreatment. Therefore, cell proliferation marker and apoptosis of the colonic epithelium were examined next, by Ki67 staining (
The coverage index was >97% per sample, which means the 16SrRNA sequences detected in the present study were sufficient to represent the majority of the bacteria present in the samples. The OTUs, richness index (Chao 1) and diversity index (Shannon and Simpson) were decreased in control septic mice compared with sham mice (
The weighted unifrac PCoA plot was used to compare the similarities of microbiota structure among three groups. The PCoA plot revealed a significantly separate clustering in microbiota structure among the three groups. The distance between CLP+LGG and sham mice was closer compared with the distance between CLP+saline and sham mice (PC1 and PC2 were 0.242 and 0.107, respectively;
The microbial profiles of the experimental mouse groups represented 10 phyla, among which, Bacteroide, Firmicutes and Verrucomicrobia were the dominant phyla in sham mice, while Bacteroide, Proteobactria and Firmicutes composed the majority of phyla in both control CLP+saline and CLP+LGG mice (
LEfSe was used to compare the microbiota phylotypes among the three groups. The sham microbiome had a huge preponderance of Verrucomicrobia, Akkermansia, S24-7, F16 and TM_7, whereas the CLP+saline microbiome had a preponderance of Bacteroide, Enterobacteriales, Enterococcaceae, Deferribacteres, Pseudomonales and Erysipelotrichi (
Critical illness and its treatment create a hostile environment in the gastrointestinal tract by altering the microbiota. Altered mucosal oxygen gradient (
Bacteroidetes and Firmicutes are the two dominant phylum in the human and mouse microbiome. Changes in the relative abundance of Bacteroidetes and Firmicutes have been determined to affect energy balance. Firmicutes was related with energy harvest and storage, while Bacteroidetes has a capacity to consume energy (
Vollaard
Evidence suggests that several specific members of microbiota serve important roles in rebalancing dysbiosis and preventing disease (
In summary, the present study demonstrated that prophylactic LGG therapy reduced mortality through attenuating inflammatory responses, and increasing gut barrier integrity and function. LGG pretreatment increased the diversity of intestinal microbiota and expression of beneficial bacterium. Among them, Prevotellaceae, Lactobacillaceae, Staphylococcaceae, Enterococcaceae, Enterobacteriaceae, Bacteroidaceae, Deferribacteraceae can be regarded as the key bacteria in sepsis treatment. Prevotellaceae may be exploited as a potential probiotic in sepsis treatment.
The present study was supported by the Natural Science Foundation of China (grant no. 81771498) and by Diagnosis and treatment of gastrointestinal dysfunction in elderly patients with integrated traditional Chinese and Western Medicine (2017-XK-A31).
The analyzed datasets generated during the study are available from the corresponding author on reasonable request. The data that support the findings of survival studies are available from reference 10.
LC performed the histological examination of the colon, 16SrRNA analysis and was a major contributor in writing the manuscript. HL and JL performed the LGG administration and CLP model establishment. YC performed the ELISA analysis. YY was the corresponding author of this manuscript and took part in revising the manuscript. All authors read and approved the final manuscript.
All procedures for animal care and use were approved by the Animal Care Ethics Committee of the First Affiliated Hospital, Zhejiang University (Hangzhou, China).
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
LGG pretreatment decreases systemic inflammatory response in sepsis. The serum levels of inflammatory factors were examined in mice with different treatment. All four inflammatory factors were the lowest in sham mice. Serum levels of IL-22 and IL-2 were decreased with LGG pretreatment, while LGG had no effect on TNF-α and IL-6 levels in septic mice. Data are expressed as mean ± standard deviation (n=10 per group). *P<0.05, **P<0.01 and ***P<0.001 with comparisons indicated by lines. LGG,
LGG pretreatment attenuates the injury of colon mucosa. Representative images (magnification, ×100) from (A) H&E. (B) Occludin (brown signal). (C) Ki67 (positive cells indicated by black arrows). (D) TUNEL (positive cells indicated by black arrows) staining of ileum sections from the sham, CLP+saline and CLP+LGG mouse groups and (E) Quantification of the immunohistochemistry results. Data are expressed as mean ± standard deviation (n=10 per group). ***P<0.001 with comparisons indicated by lines. LGG,
Principal coordinates analysis plots based on weighted unifrac metrics in mice with different treatment. Stars represent the sham group (n=5), triangles represent the control septic CLP+saline group (n=8), and dots represent the CLP+LGG group (n=8). LGG,
Comparison of the major microbiomes at the (A–D) phyla, (E and F) family and (G and H) genus levels between sham mice (red), CLP+saline (blue) and CLP+LGG mice (green) respectively. (A) Comparison of the abundances of bacterial phyla of each sample. (B) Comparison of the average abundance of each bacterial phylum in the sham, CLP+saline and CLP+LGG mice, respectively. (C and D) Significant differences among the abundances of discriminatory phyla among three groups. (E and G) Enrichment in sham mice; (F and H) Enrichment in CLP+saline mice; the upper and lower ranges of the box represent the 75 and 25% quartiles, respectively. Sham mice, n=5; CLP+saline mice, n=8; CLP+LGG mice, n=8. *P<0.05, **P<0.01 and ***P<0.001 with comparisons indicated by lines. LGG,
LEfSe and LDA analysis based on OTUs characterize the microbiomes between the sham and CLP+saline mice. (A) Cladogram using LEfSe method indicating the phylogenetic distribution of fecal microbes associated with sham (green) and CLP+saline mice (red). (B) LDA scores showed the significant bacterial difference between the sham and CLP+saline mice. Sham mice, n=5; CLP+saline mice, n=8. LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis; OUT, operational taxonomic unit; CLP, cecal ligation and puncture.
LEfSe and LDA analysis based on OTUs characterize the microbiomes between the sham and CLP+LGG mice. (A) Cladogram using LEfSe method indicating the phylogenetic distribution of fecal microbes associated with sham (green) and CLP+LGG mice (red). (B) LDA scores showed the significant bacterial difference between the sham and CLP+LGG mice. Sham mice, n=5; CLP+LGG mice, n=8. LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis; OUT, operational taxonomic unit; CLP, cecal ligation and puncture; LGG,
LEfSe and LDA analysis based on OTUs characterize the microbiomes between the CLP+saline mice and CLP+LGG mice. Cladogram using LEfSe method indicating the phylogenetic distribution of fecal microbes associated with CLP+LGG (green) and CLP+saline mice (red). LDA scores showed the significant bacterial difference between the CLP+LGG and CLP+saline mice. CLP+saline mice, n=8; CLP+LGG mice, n=8. LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis; OUT, operational taxonomic unit; CLP, cecal ligation and puncture; LGG,
Normal/LGG (n=5) | Normal/saline (n=5) | P-value | |
---|---|---|---|
9.24±0.06 | 8.16±0.11 | <0.001 |
Quantification data from PCR analysis are expressed as log qPCR copy/fecal (g). Data are expressed as mean ± standard deviation. LGG,
Summary of alpha analysis data.
Sham | CLP+saline | CLP+LGG | |
---|---|---|---|
OTUs | 1,299.60±166.56 | 1,058.75±199.53 | 1,184.63±343.76 |
Coverage | 99% | 99% | 99% |
Chao1 | 2,151.26±230.87 | 1,800.81±325.82 | 1,953.06±569.96 |
Shannon | 6.27±0.39 | 5.29±0.72 | 5.84±1.12 |
Simpson | 0.97±0.02 | 0.92±0.06 | 0.94±0.07 |
Data are expressed as mean ± standard deviation. CLP, cecal ligation and puncture; LGG,