Application of bacteroides sailli as biomarker in preparation of sepsis diagnosis and / or prognosis evaluation product

Abstract

The invention relates to the technical field of biomedicine, and discloses application of bacteroides sailli as a biomarker in preparation of sepsis diagnosis and / or prognosis evaluation products. The kit for the auxiliary diagnosis of the sepsis comprises a reagent for detecting the bacteroides sailli (Bacteroides sailli), and the reagent is used for detecting the bacteroides sailli (Bacteroides sailli). As a detection variable of sepsis, the single bacterium of Bacteroides sailli has high specificity and sensitivity, can be applied to diagnosis of sepsis patients as a detection marker, is high in accuracy, provides an intestinal flora detection means for the sepsis patients, provides a bacterium source basis for judging sepsis and intestinal flora disorder of the patients according to the intestinal flora detection means, and has a good application prospect. And a basis is provided for later-stage intestinal bacteria transplantation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of Bacteroides salierii as a biomarker in the preparation of sepsis diagnosis and / or prognosis evaluation products. Background Art

[0002] Sepsis is a life-threatening syndrome of organ dysfunction (SOFAScore ≥ 2) caused by a dysregulated host response to infection. With tens of millions of deaths worldwide each year, and in some countries even exceeding the death toll from cancer, sepsis has garnered widespread attention worldwide. This syndrome can strike in all age groups and has diverse triggers. With a mortality rate of 40%, and septic shock reaching a mortality rate as high as 50-60%, it poses a significant threat to human health.

[0003] The clinical manifestations of sepsis are diverse and lack distinct clinical presentations, leading many researchers to search for biomarkers for the diagnosis and treatment of sepsis. C-reactive protein (CRP) and procalcitonin (PCT) are currently the most commonly used biomarkers for the diagnosis of sepsis. Total white blood cell count, neutrophil count, and lymphocyte count are also important indicators of sepsis. However, most currently used biomarkers suffer from suboptimal sensitivity and specificity, and they are unable to accurately distinguish non-infectious conditions (such as postoperative and traumatic events) from sepsis. An ideal biomarker should be highly efficient, easily identify the cause of inflammation, be able to identify potential viral or bacterial infections, and accurately reflect the efficacy of anti-infective agents.

[0004] Therefore, new biological markers that can be used to diagnose and predict the prognosis of sepsis are in urgent need of discovery. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem that most of the markers used in the prior art for sepsis have unsatisfactory sensitivity and specificity, and to provide the use of Bacteroides salierii as a biomarker in the preparation of sepsis diagnosis and / or prognosis evaluation products. Bacteroides salierii as a biomarker has high specificity and sensitivity, and is highly accurate in diagnosing sepsis patients.

[0006] In order to achieve the above object, the present invention first provides Bacteroides salyersii ( Bacteroides salyersiae ) as a biomarker in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

[0007] The second aspect of the present invention provides a method for detecting Bacteroides salierii ( Bacteroides salyersiae) content in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

[0008] Preferably, the reagent for detecting the content of Bacteroides salyersii in a sample is used to detect the abundance of Bacteroides salyersii in the sample.

[0009] Preferably, the abundance of Bacteroides salyersii is obtained by genetic detection and / or MALDI-TOF mass spectrometry detection.

[0010] Preferably, the abundance detection process of Bacteroides salyersii comprises: isolating and extracting nucleic acids from biological samples, constructing libraries, sequencing, performing bioinformatics analysis on sequencing data, obtaining the sequence number of Bacteroides salyersii, and normalizing the sequence number to obtain the abundance.

[0011] Preferably, the sample is feces of the subject.

[0012] Preferably, the sepsis diagnosis product and the sepsis prognosis assessment product are each independently selected from at least one of a primer, a reagent, a kit, a chip, and a prediction system.

[0013] The third aspect of the present invention provides Bacteroides salierii ( Bacteroides salyersiae ) as a target in the preparation and / or screening of drugs or health foods for the treatment of sepsis.

[0014] A fourth aspect of the present invention provides a kit for auxiliary diagnosis of sepsis, which includes a kit for detecting Bacteroides salierii ( Bacteroides salyersiae ) reagents.

[0015] Preferably, the kit further comprises a reagent for detecting at least one of procalcitonin (PCT), C-reactive protein (CRP) and Sequential Organ Failure Assessment Score (SOFA Score) in a sample.

[0016] Preferably, the sample is feces of the subject.

[0017] Through the above technical solution, the beneficial effects of the present invention are: The present invention discovered for the first time that Bacteroides salierii ( Bacteroides salyersiae ) is associated with sepsis, and the abundance of this bacterium is significantly increased in patients with sepsis; Bacteroides salilsii ( Bacteroides salyersiae ) Single bacteria have high specificity and sensitivity as a sepsis detection variable and can be used as a detection marker in the diagnosis of sepsis patients with high accuracy. This invention provides a means for detecting intestinal flora in sepsis patients, thereby providing a bacterial basis for determining sepsis and intestinal flora disturbances in patients, and providing a basis for subsequent intestinal bacterial transplantation.

[0018] Furthermore, Bacteroides salierii ( Bacteroides salyersiae ) combined with at least one of procalcitonin (PCT), C-reactive protein (CRP) and sequential organ failure assessment (SOFA Score) has higher diagnostic accuracy for sepsis and can effectively expand its application in precision medicine for sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the screening results of intestinal flora associated with sepsis in Example 1; Figure 2 This is a forest plot presentation of the ROC results of single bacteria in the sepsis-associated intestinal flora in Example 2; Figure 3 The forest plot shows the ROC results of the reference markers for clinical diagnosis of sepsis in Example 2; Figure 4 The LASSO regression results in Example 2 were used to remove the clinical indicators with strong collinearity; Figure 5 This is the ROC result of the combined diagnosis of sepsis by intestinal flora and clinical parameters in Example 2. DETAILED DESCRIPTION

[0020] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0021] In the context of the present invention, the term "abundance" refers to a measure of the number of target microorganisms in a biological sample. Quantification of the abundance of a target nucleic acid sequence within a biological sample can be absolute or relative. "Relative abundance" is typically based on one or more internal reference genes, i.e., the 16S rRNA gene from a reference strain, such as bacteria measured using universal primers and expressing the abundance of the target nucleic acid sequence as a percentage of total bacterial 16S rRNA gene copies or normalized by the 16S rRNA gene copies of Escherichia coli; "absolute abundance" gives the exact number of target molecules by comparison with a DNA standard or normalized by DNA concentration.

[0022] The first aspect of the present invention provides Bacteroides salyersii ( Bacteroides salyersiae ) as a biomarker in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

[0023] The inventors of the present invention collected stool samples from ICU patients with sepsis and non-sepsis, extracted the microbial genomic DNA from the stool samples, constructed a library, and performed metagenomic sequencing. The obtained sequencing data was processed into taxonomic abundance data. Through differential bacteria and linear discriminant analysis, the inventors found that the abundance of microorganisms significantly increased in sepsis patients: Bacteroides salierii; Bacteroides salierii was discovered for the first time. Bacteroides salyersiae ) is statistically significant in sepsis, and experiments have confirmed that Bacteroides salierii is an important biomarker for patients with sepsis. Using ROC curve analysis, Bacteroides salierii as a detection variable to distinguish ICU sepsis (sepsis) from non-sepsis (non-sepsis) patients has a specificity and sensitivity much higher than existing clinical popular markers. It can be seen that Bacteroides salierii can be used as a detection marker in the diagnosis of sepsis patients, thereby providing new tools and ideas for the early diagnosis, risk estimation, treatment and prognosis of sepsis. This is of great significance for improving the prognosis of patients with sepsis and reducing mortality, and provides a new direction for the study of the pathogenesis of sepsis and the formulation of prevention and treatment strategies.

[0024] The second aspect of the present invention provides a method for detecting Bacteroides salierii ( Bacteroides salyersiae ) content in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

[0025] According to the present invention, in patients with sepsis, Bacteroides salierii ( Bacteroides salyersiae ) significantly increases in abundance. Preferably, the reagent for detecting the content of Bacteroides salyersii in the sample is used to detect the abundance of Bacteroides salyersii in the sample.

[0026] According to the present invention, preferably, the abundance of Bacteroides salierii is determined by genetic testing and / or MALDI-TOF mass spectrometry. It is understood that genetic testing methods can employ metagenomic testing or PCR, and MALDI-TOF mass spectrometry can determine the unique protein composition of the bacteria themselves, using mass spectrometry to sort the measured proteins and peptides by molecular weight to form a unique proteomic fingerprint, and then identify and detect the strain based on characteristic pattern peaks.

[0027] According to the present invention, preferably, the abundance detection process for Bacteroides salyersii includes isolating and extracting nucleic acids from a biological sample, constructing a library, sequencing, and performing bioinformatics analysis on the sequencing data to determine the sequence count of Bacteroides salyersii. This sequence count is then normalized to obtain the abundance. Specifically, a normalization coefficient can be calculated based on the sequence count using MaAsLin2 (R package). The normalized relative abundance of the strain after normalization is obtained by dividing the sequence count by the normalization coefficient. Normalization by sequence count can eliminate the impact of differences in the total amount of sequencing on the results.

[0028] According to the present invention, preferably, the sample is feces of the subject.

[0029] In the present invention, the sepsis diagnosis product and the sepsis prognosis assessment product include, but are not limited to, primers, reagents, detection kits, gene chips, and sepsis prediction systems. These products utilize the abundance of a gut microbiota marker (Bacteroides salierii) in a subject's fecal sample as a detection variable to diagnose or aid in the diagnosis of sepsis. Preferably, the sepsis diagnosis product and the sepsis prognosis assessment product are each independently selected from at least one of primers, reagents, kits, chips, and prediction systems.

[0030] Further preferably, the sepsis diagnosis product and the sepsis prognosis assessment product further detect at least one of the diversity index procalcitonin (PCT), C-reactive protein (CRP) and sequential organ failure assessment score (SOFA Score or SOFA score), and combine them with the abundance of Bacteroides salierii as detection variables, thereby improving the accuracy of sepsis diagnosis or auxiliary diagnosis, risk estimation, treatment and prognosis judgment.

[0031] Exemplarily, the sepsis prediction system uses the abundance of Bacteroides salierii in a subject's stool sample as a detection variable. More preferably, Bacteroides salierii is combined with procalcitonin, C-reactive protein, and SOFA score as detection variables. Receiver-operating characteristic (ROC) curve analysis demonstrated that the prediction system and method have high sensitivity and specificity, and can serve as a new auxiliary diagnostic tool for sepsis.

[0032] As a specific embodiment, the method for establishing a sepsis prediction system based on intestinal flora in the present invention comprises the following steps: 1) Determine the inclusion criteria for patients with sepsis and healthy subjects; 2) Collect stool samples from patients with sepsis and healthy people, extract microbial genomic DNA from the stool samples, build libraries, and perform metagenomic sequencing to obtain raw data; 3) Use the same quality control and analysis methods to control the original data and eliminate unqualified data; 4) Use the same method to analyze the relative abundance of intestinal microbial flora; 5) Screen out differentially expressed bacteria and analyze their effect size using rank sum test and linear discriminant analysis; 6) The ROC curve was used to test the prediction effect of the differential bacteria.

[0033] The third aspect of the present invention provides Bacteroides salierii ( Bacteroides salyersiae ) as a target in the preparation and / or screening of drugs or health foods for treating sepsis. The health foods are health products that alleviate sepsis.

[0034] Specifically, with Bacteroides salierii as the target, substances that reduce or inhibit the abundance of Bacteroides salierii are screened out as active ingredients for preparing sepsis drugs or health foods.

[0035] A fourth aspect of the present invention provides a kit for auxiliary diagnosis of sepsis, which includes a kit for detecting Bacteroides salierii ( Bacteroides salyersiae ) reagents.

[0036] Those skilled in the art can prepare a corresponding auxiliary diagnosis kit for sepsis based on the Bacteroides salyersi biomarker disclosed in the present invention and according to known techniques in the field of kit preparation, wherein the kit contains a reagent capable of detecting the content of Bacteroides salyersi in a sample.

[0037] Preferably, the kit further comprises a reagent for detecting at least one of procalcitonin, C-reactive protein, and the SOFA score in a sample. Reagents for detecting procalcitonin, C-reactive protein, and the SOFA score can be corresponding known reagents. Specifically, the reagents for detecting Bacteroides salierii, procalcitonin (PCT), C-reactive protein (CRP), and the Sequential Organ Failure Assessment (SOFA Score) in the kit are each separately packaged.

[0038] The present invention will be described in detail below through examples.

[0039] Unless otherwise specified, the following examples were performed under conventional experimental conditions or the conditions recommended by the manufacturer's instructions. All reagents and materials used were commercially available unless otherwise specified.

[0040] Example 1 1. Study Population and Design Fecal samples were collected from 10 subjects with non-sepsis in the ICU and 13 with sepsis in the ICU. All subjects were recruited from the intensive care unit of Shanghai Zhongshan Hospital. Patients with autoimmune diseases and those taking immunomodulatory drugs were excluded. The samples were taken from patients diagnosed with sepsis or non-sepsis within 48 hours of admission to the intensive care unit. As shown in Table 1, there were no significant differences in age, gender, or BMI between the two groups of patients. Continuous variables were calculated using the Mann-Whitney Utest, and categorical variables were calculated using the Fisher's exact test.

[0041] Table 1 Basic physiological parameters of all subjects

[0042] 2. Metagenomic DNA extraction, library construction, sequencing and quality control (1) Metagenomic DNA was extracted from the fecal samples of the subjects using the TIANamp Stool DNA kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.); (2) All metagenomic DNA samples were normalized to a concentration of 1 ng / μL using the Tn5 DNA Library Prep Kit for Illumina (APExBIO) according to the instructions, and Illumina sequencing libraries were constructed. (3) Metagenomic sequencing (2 × 150 bp) was performed on the next-generation gene sequencer Illumina Novaseq 6000 platform; (4) The quality control process of metagenomic sequencing data was performed using KneadData (version 0.10.2): the trimmed non-human small fragment reads were removed using Trimmomatic (version 0.39) software (parameters set to ILLUMINACLIP: NexteraPE-PE.fa:2:30:10:8:TRUE SLIDINGWINDOW:4:20 MINLEN:75); the sequencing reads were aligned with the human reference genome (GRCh37) and the reference database SILVA 128 using Bowtie2 (version 2.4.4) software to filter and remove human and rDNA reads (parameters set to --very-sensitive --phred33); (5) The quality-controlled reads were used to determine the relative abundance of microbial taxonomy in each sample using MetaPhlan (version 3.0.13) software; (6) Species-level taxonomic units with a prevalence greater than 10% and a relative abundance greater than 0.01 of intestinal microorganisms were screened and included in subsequent differential analysis.

[0043] 3. Analysis of Intestinal Microflora Differences The data finally included in the analysis after screening in step 2 were log-transformed to avoid the influence of extreme values, and then analyzed by MaAsLin2 (version 1.14.1) software to screen out Bacteroides salicylifolia ( Bacteroides salyersiae ) was the most significant bacteria associated with sepsis, and the results were as follows Figure 1 As shown in Figure 3, its abundance was significantly increased in ICU septic patients relative to ICU non-septic patients.

[0044] Example 2 1. ROC analysis of sepsis-associated bacteria The pROC R package was used to calculate the specificity and sensitivity of the test indicators and plot ROC curves. Receiver Operation Characteristic (ROC) analysis combines sensitivity and specificity to comprehensively evaluate diagnostic accuracy or discriminant efficacy. The logistic regression model in R was used to calculate the predictive value of sepsis-associated bacteria for sepsis and non-sepsis classification. This predictive value was used to plot ROC (Receiver Operating Characteristic) curves and calculate the AUC value.

[0045] Specifically, the threshold of the actual measurement value is first calculated within the system, and then the number of true positive cases (TP), false positive cases (FP), true negative cases (TN), and false negative cases (FN) corresponding to the threshold are calculated. The specificity and sensitivity are calculated according to the following formula: Specificity (true negative rate) = TN / (TN+FP), Sensitivity (true positive rate) = TP / (TP+FN), The ROC curve can be constructed through specificity and sensitivity, and the integral of the ROC curve is the AUC.

[0046] The results are as follows Figure 2 As shown, Bacteroides salierii ( Bacteroides salyersiae ) had the best effect in distinguishing sepsis from non-sepsis, with an AUC of 0.842.

[0047] 2. ROC analysis of reference markers for clinical diagnosis of sepsis The reference markers for clinical diagnosis of sepsis are obtained as follows: Immune cell count (WBC / lymphocyte count / neutrophil count): Use a blood routine tester to detect the number of major immune cell subsets in the patient's peripheral venous blood; NLR: The ratio of neutrophils to lymphocytes based on blood count results is called granulocyte-lymphocyte ratio. CRP: Peripheral venous blood was drawn from the patient, and an enzyme-linked immunosorbent assay (ELISA) kit (purchased from Wuhan Saipei Biotechnology Co., Ltd., catalog number: SP11257) was used in combination with a Bio-Tek automatic microplate reader to quantitatively detect CRP in the patient's blood, indicating the patient's inflammation level; PCT: Peripheral venous blood was drawn from the patient, and the enzyme-linked immunosorbent assay (ELISA) kit (purchased from Wuhan Saipei Biotechnology Co., Ltd., catalog number: SP10720) was used in combination with a Bio-Tek automatic microplate reader to quantitatively detect the PCT in the patient's blood, indicating the patient's bacterial infection level; SOFA Score: The score is accumulated based on the real-time status of the patient's respiratory system (whether respiratory support is needed, the patient's oxygenation status measured by a bedside arterial blood gas analyzer), circulatory system (mean arterial pressure calculated from blood pressure data recorded by an automatic sphygmomanometer, and the dosage of hormonal drugs), liver (serum bilirubin level measured by a fully automatic biochemical analyzer), kidney (serum creatinine level measured by a fully automatic biochemical analyzer, and the patient's 24-hour urine output), central nervous system (Glasgow score obtained by the doctor based on the patient's eye opening response, verbal response, and limb movement), and coagulation system (platelet count measured by a routine blood tester). For specific scoring criteria, please refer to "Mervyn Singer, et al . The ThirdInternational Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), JAMA . 2016;315(8):801-810”; SOFA Score ≥2 is one of the necessary criteria for the clinical diagnosis of sepsis.

[0048] The results of the intergroup comparison of clinical diagnostic reference markers for sepsis are shown in Table 2. The intergroup differences were obtained using the Mann-Whitney U test. It was found that the differences in commonly used clinical markers among ICU patients were small. Only CRP showed a significant difference between sepsis and non-sepsis (P = 0.02), which further suggests the need for more accurate reference markers for the diagnosis of sepsis.

[0049] Table 2 Common clinical indicators related to sepsis diagnosis in all subjects

[0050] ROC analysis results of reference markers for clinical diagnosis of sepsis Figure 3 As shown in the figure, consistent with the results of the significance test, CRP showed the highest potential for distinguishing sepsis from non-sepsis, with an AUC of 0.792.

[0051] 3. LASSO regression to deal with the collinearity problem of clinical indicators For the five clinical parameters obtained from blood tests, LASSO (Least Absolute Shrinkage and Selection Operator) regression was used to eliminate indicators with strong collinearity to reduce the impact of internal correlations on subsequent modeling. LASSO regression is a linear regression analysis method that addresses collinearity and other issues by introducing an L1 regularization term. This method is implemented using the "glmnet" R package.

[0052] The results are as follows Figure 4 As shown in Figure 2, after 1000 LASSO regression screenings, PCT and CRP were selected for subsequent modeling more than 800 times, indicating that PCT and CRP had weak collinearity. Therefore, these two clinical indicators were subsequently compared with Bacteroides salyersiae Joint modeling.

[0053] 4. Combining microbiome with clinical indicators can improve the accuracy of sepsis diagnosis In addition to the above PCT / CRP / Bacteroides salyersiae , also included one of the gold standards for clinical diagnosis of sepsis: SOFA Score ≥ 2, and Bacteroides salyersiae Joint modeling.

[0054] The results are as follows Figure 5 As shown in the figure, the highest AUC of the combined diagnosis of clinical indicators is the combination of PCT, CRP and SOFA Score ≥ 2 (AUC = 0.800), which is higher than that of the single PCT / CRP / SOFA Score ≥ 2, but still lower than that of the single Bacteroides salyersiae Effect.

[0055] Bacteroides salyersiae The effect of combined diagnosis with any clinical indicator is better than that of a single clinical indicator or a combination of clinical indicators. Bacteroides salyersiae, When PCT, CRP, and SOFA Score ≥ 2 were combined to diagnose sepsis, the accuracy was further improved, with an AUC of 0.885.

[0056] From the above results, it can be seen that Bacteroides salyersii ( Bacteroides salyersiae) has a good effect in diagnosing or assisting in the diagnosis of sepsis, and its performance is better than the currently recognized clinical indicators. When used in combination with clinical indicators, its accuracy will be further improved.

[0057] In summary, the present invention discovered for the first time the proto-saliersii Bacteroides ( Bacteroides salyersiae ) is associated with patients with sepsis, specifically showing that its abundance is significantly higher than that in non-septic people. When this microbiome is combined with clinical indicators as a predictor of sepsis, the accuracy rate is high. The present invention provides a new approach for the diagnosis and treatment of patients with sepsis.

[0058] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. Bacteroides salierii ( Bacteroides salyersiae ) as a biomarker in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

2. Used to detect Bacteroides salicyloides in samples ( Bacteroides salyersiae ) content in the preparation of sepsis diagnosis products and / or sepsis prognosis evaluation products.

3. The use according to claim 2, wherein: The reagent for detecting the content of Bacteroides salyersii in a sample is used to detect the abundance of Bacteroides salyersii in the sample.

4. The use according to claim 3, wherein: The abundance of the Bacteroides salyersii is obtained by genetic detection and / or MALDI-TOF mass spectrometry detection.

5. The use according to claim 4, wherein: The abundance detection process of Bacteroides salyersii includes: isolating and extracting nucleic acids from biological samples, constructing libraries, sequencing, performing bioinformatics analysis on sequencing data, obtaining the sequence number of Bacteroides salyersii, and standardizing the sequence number to obtain the abundance.

6. The use according to claim 2, wherein: The sample is feces of the subject to be tested.

7. The use according to any one of claims 1 to 6, wherein The sepsis diagnosis product and the sepsis prognosis assessment product are each independently selected from at least one of a primer, a reagent, a kit, a chip, and a prediction system.

8. Bacteroides salierii ( Bacteroides salyersiae ) as a target in the preparation and / or screening of drugs or health foods for the treatment of sepsis.

9. A kit for auxiliary diagnosis of sepsis, wherein: The kit includes a kit for detecting Bacteroides salierii ( Bacteroides salyersiae ) reagents.

10. The kit according to claim 9, wherein The kit further comprises a reagent for detecting at least one of procalcitonin, C-reactive protein and sequential organ failure score in the sample; Preferably, the sample is feces of the subject.

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