Remodeling gut microbiota by Streptococcus thermophilus 19 attenuates inflammation in septic mice

Background Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and is the leading cause of death in burn patients. S. thermophilus 19 is highly effective probiotic strains with well-studied health benefits, but its role in protecting viscera against injury caused by sepsis and the underlying mechanism is poorly understood. Methodology/Principal Findings We tested the utility of S. thermophilus 19 in attenuating inflammation in vitro and vivo of a Lps-induced sepsis mouse model. We also evaluated the influence of sepsis and S. thermophilus on microbial community. In vitro, S. thermophilus 19 can decrease the expression of inflammatory factors. Additionally, in a lipopolysaccharide (LPS)-induced septic mouse model, mice administered the probiotics 19 was highly resistant to LPS and exhibited decreased expression of inflammatory factors compared to LPS-treated control mice. A MiSeq-based sequence analysis revealed that gut microbiota alterations in mice intraperitoneally injected with 1 mg/ml LPS were mitigated by the administration of oral probiotics 19. Furthermore, changes in the levels of inflammatory factors in different organs related to different gut microbiota alterations were observed. Conclusions/Significance Together these findings indicate that S. thermophilus 19 may be a new avenue for interventions against inflammation caused by sepsis and other systemic inflammatory diseases. Author summary Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and is the leading cause of death in burn patients, responsible for up to 50 to 60% of burn injury deaths. Although our understanding of sepsis has increased substantially in recent years, it is still reported to be the leading cause of death in seriously ill patients, and the incidence of sepsis has increased annually. Therefore, new insights into the causes of sepsis are urgently needed. Here, we use Miseq method to determine the composition of gut microbiota. We also use a probiotic (Streptococcus thermophilus 19) to investigate the therapeutic effect on sepsis. We found that the exceptional efficacy of probiotic Streptococcus thermophilus 19 in attenuating the inflammation in septic mice by modifying the structure and function of gut microbiota. Our study proposed oraling Streptococcus thermophilus 19 might be a new avenue for interventions against inflammation caused by sepsis and other systemic inflammatory diseases.

Author summary 36 Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection 37 and is the leading cause of death in burn patients, responsible for up to 50 to 60% of burn injury 38 deaths. Although our understanding of sepsis has increased substantially in recent years, it is still 39 reported to be the leading cause of death in seriously ill patients, and the incidence of sepsis has 40 increased annually. Therefore, new insights into the causes of sepsis are urgently needed.Here, 41 we use Miseq method to determine the composition of gut microbiota.We also use a probiotic 4 64 Introduction 65 Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection 66 and is the leading cause of death in burn patients, responsible for up to 50 to 60% of burn injury 67 deaths [1,2]. Although our understanding of sepsis has increased substantially in recent years, it 68 is still reported to be the leading cause of death in seriously ill patients, and the incidence of 69 sepsis has increased annually. Therefore, new insights into the causes of sepsis are urgently 70 needed.

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The gut microbiota is a complex ecosystem consisting of trillions of bacteria that live in 72 the digestive tracts of humans and other animals [3]. Growing evidence supports the key role of a 73 healthy gut microbiota in promoting and maintaining a balanced immune response and in the 74 establishment of the gut barrier immediately after birth [4,5]. Moreover, a dysbiotic state of the 75 gut microbiota can lead to dysregulation of various processes, which can in turn contribute to the 76 development of autoimmune conditions [6]. For instance, the presence or overabundance of 77 specific types of bacteria may contribute to inflammatory disorders such as IBD [6]. Additionally, 78 metabolites from certain members of the gut flora may influence host signaling pathways, 79 contributing to disorders such as colon cancer and obesity. Sepsis is an extreme response to 80 inflammation that has profound effects on all parts of the body. For decades, the gut has been 81 regarded as the motor of sepsis [7], and it has recently been shown that a healthy gut microbiota 82 has a protective role during systemic inflammation. Thus, we hypothesized that intestinal 83 bacteria play an important role in sepsis since the gut microbiota is associated with many 84 diseases.

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Probiotics are live microbes that have beneficial effects on human and animal health 86 when ingested in sufficient amounts [8]. Probiotics play an important role in maintaining the 87 normal microbiota composition and have been used to treat or prevent a number of gut health 88 disorders, such as irritable bowel syndrome, hypercholesterolemia, gastritis, gut infection, 89 parasitic infestation, hypersensitivity (including food allergies), and even certain types of cancers 90 (e.g., colorectal cancer) [9,10]. The use of microbes as probiotics also hold potential for oral 91 health in preventing and treating oral infections, dental plaque-related diseases, periodontal 92 diseases and halitosis. Furthermore, probiotics can alleviate inflammation associated with some 93 human diseases by promoting changes in the gut microbiota composition [11,12]. Streptococcus 94 thermophilus is a highly effective probiotic that has well studied health benefits, including the 95 production of antibiotics that prevent infections from pneumonia-causing microbes and C.

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In this study, we used a coculture system (probiotics and RAW264.7 cells) to assess the 98 ability of probiotics to decrease the expression of inflammatory factors. We showed that 99 Streptococcus thermophilus 19 can decrease the inflammation induced by LPS in RAW264.7 100 cells. Furthermore, we investigated the ability of Streptococcus thermophilus 19 to protect mice 101 against LPS-induced inflammation and gut microbiota alterations when administered as a 102 probiotic. We observed that the administration of S. thermophilus 19 as probiotics could alter the 103 gut microbiota composition of untreated mice or mice with LPS-induced sepsis, with the 104 symptoms of sepsis mitigated in the latter group. Moreover, the levels of several inflammatory 105 factors in various organs were correlated to a diverse gut microbiota composition. We 6 106 hypothesize that the supplementation of diets with probiotics protects visceral organs by 107 reducing inflammation through alterations in the gut microbiota after sepsis. 110 To assess the influence of the assayed probiotics on the expression of inflammatory factors, we 111 developed a coculture system (probiotics and RAW264.7 cells). After incubating for 6 hours,       137 To test whether the assayed probiotics could alleviate inflammation in vivo, we induced sepsis in   demonstrating that the probiotics has no influence on the host in the absence of sepsis. 146 Sepsis is an extreme response to inflammation, which has profound effects on all parts of the     200 We showed that probiotic intervention can attenuate the inflammation in septic mice ( Figure.    Overall, these data showed that LPS and probiotics significantly impacted the microbiota 239 composition of mice.

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The function of gut microbiota was specifically altered after the administration oral probiotics 241 Next, we used a Kruskal-Wallis/Wilcoxon rank-sum test to determine how the altered 242 community structure of the gut microbiota affects its function. One KEGG database biochemical    354 The body weight, water and food intake, and stool appearance were documented for all groups of 355 mice every other day throughout the experiment. After 1 week, the liver, kidneys, lungs, heart 356 and small intestines were collected from each mouse and were divided into triplicate samples, 357 with one stored in liquid nitrogen, a second stored in RNAiso Plus for RNA extraction, and the 18 358 third was fixed in 4% (w/v) paraformaldehyde at 4°C for later histological analysis. 360 After the animals were sacrificed, different tissue samples were collected. After fixation in 4% paired-end sequenced (2 × 300) on an Illumina MiSeq platform according to standard protocols.

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The taxonomy of each 16S rRNA gene sequence was analyzed using RDP classifier    Table 2.  Table1   Group Treatment Groups(n=8) Gavaging