Application of combination composition of antibiotics and bacteria carrying DL-endopeptidase in preparation of medicine for preventing intestinal development delay of premature infants

Abstract

The invention provides an application of a combined composition of antibiotics and bacteria carrying DL-endopeptidase in preparation of a medicine for preventing intestinal development delay of premature infants, the combined composition comprises a component A and a component B which are prepared according to a specific mass ratio, the component A is selected from broad-spectrum antibiotics in an intensive care unit of newborns, and the component B is selected from bacteria carrying DL-endopeptidase and bacteria carrying DL-endopeptidase and bacteria carrying DL-endopeptidase and bacteria carrying DL-endopeptidase. And the component B is a strain which can secrete DL-endopeptidase and has endogenous drug resistance to the component A. According to the application, synergism of'clearing 'and'driving' is realized, the traditional clinical thinking of'first sterilization and then remediation 'is broken, pathogenic microorganisms (such as klebsiella and staphylococcus aureus) in a premature infant body are cleared through antibiotics, and meanwhile, bacterial components in the components are synchronously utilized to down-regulate protein expression of the intestinal mucosa intracellular cylindricity disease, so that the premature infant is prevented from suffering from the intestinal mucosa intracellular cylindricity disease, and the premature infant is prevented from suffering from the intestinal mucosa intracellular cylindricity disease. The synthesis of tight junction protein is actively realized.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of a combination of an antibiotic and bacteria carrying DL-endopeptidase in the preparation of a drug for preventing delayed intestinal development in premature infants. Background Technology

[0002] Premature infants (gestational age <37 weeks) often face a very high risk of sepsis due to their extremely immature immune systems. To prevent or treat infections, antibiotics (such as ampicillin and gentamicin) are widely and frequently used in neonatal intensive care units (NICUs). However, existing clinical observations and scientific research have revealed a serious "antibiotic paradox": an irreconcilable contradiction exists between antibiotic treatment in premature infants and intestinal development, specifically manifested as follows: The core driving force of gut development comes from specific symbiotic bacteria (such as Enterococcus faecalis). Enterococcus faecium The intestinal tract produces SagA (secretory antigen A) enzyme. This enzyme specifically processes peptidoglycan in the intestinal lumen into muramyl dipeptide (MDP), which in turn activates the host NOD2-CYLD signaling axis. This signaling pathway is a key "molecular engine" that induces the synthesis of tight junction proteins (such as ZO-1 and Occludin) in the intestinal epithelium and drives the transformation of the intestinal barrier from the embryonic to the mature state, directly determining the maturity of intestinal development. However, the clinical application of broad-spectrum antibiotics has significant side effects: while killing pathogenic bacteria, they also non-specifically eliminate beneficial symbiotic bacteria carrying the SagA gene in the intestine, causing the molecular signaling chain required for intestinal development to be completely interrupted during antibiotic treatment.

[0003] The aforementioned signal interruption directly leads to a core technological bottleneck: the gut microbiota maturity slope (MA / CA) stagnates. Even if the infection is effectively controlled, premature infants will still be at high risk of bacterial translocation and secondary late-onset sepsis (LOS) due to impaired intestinal barrier development. Currently, there is no effective technical means to overcome this dilemma. Summary of the Invention

[0004] This application aims to address the problems in the prior art, such as the exacerbation of intestinal developmental delay in preterm infants by antibiotic use, insufficient activity of probiotics under antibiotic pressure, and lack of dynamic adjustment capability of dosing regimens. It provides an application of a combination of antibiotics and bacteria carrying DL-endopeptidase in the preparation of drugs to prevent intestinal developmental delay in preterm infants, so as to achieve effective prevention of intestinal developmental delay in preterm infants, while also taking into account the needs of infection control.

[0005] To achieve the above objectives, this application provides the following technical solution: The use of a combination of an antibiotic and bacteria carrying DL-endopeptidase in the preparation of a drug for preventing delayed intestinal development in preterm infants.

[0006] Further configuration: The combination composition includes component A and component B, wherein component A is a broad-spectrum antibiotic, and component B is a bacterial strain that secretes DL-endopeptidase and has endogenous resistance to component A.

[0007] Further setting: The minimum inhibitory concentration (MIC) of component B against component A is >128 μg / mL.

[0008] Further setting: The mass ratio of component A to component B is in the range of 1:10-1:500.

[0009] Further configuration: Component A includes at least one of aminopyrine, gentamicin, and vancomycin.

[0010] Further configuration: Component B includes natural screening strains, and the screening of natural screening strains includes the following steps: screening strains that have endogenous resistance to component A and high expression of DL-endopeptidase from breast milk or the intestines of healthy full-term infants through antibiotic susceptibility testing.

[0011] Further configuration: Component B includes an engineered strain, and the construction of the engineered strain includes the following steps: using CRISPR-Cas9 gene editing technology, the DL-endopeptidase expression cassette is integrated into the chromosome of the antibiotic-resistant probiotic to obtain an engineered strain that can secrete DL-endopeptidase under antibiotic stress.

[0012] Further configuration: The combination composition is a formulation suitable for enteral administration to preterm infants.

[0013] Further configuration: The combined composition is used to promote intestinal maturation in premature infants by restoring the physiological function of the SagA-NOD2-CYLD signal axis when the slope of the gut microbiota maturity deviates from the normal range, and at least one of the following is confirmed based on the maturity slope: timing of administration, dosage, and effect of use.

[0014] Further configuration: The application relies on the preterm infant gut development and treatment collaborative management system to dynamically regulate the administration of the combined composition. The preterm infant gut development and treatment collaborative management system includes an antibiotic-strain module, a kinetic calculation server, and an automatic infusion pump linkage control module. The antibiotic-strain module is used to provide the matching relationship between antibiotics and DL-endopeptidase secretory strains resistant to the antibiotics, as well as recommendations on their compatibility and dosage ratio. The dynamics calculation server is used to process microbial community detection data, calculate the slope of microbial community maturity, identify its deviation state, and output a judgment signal. The automatic infusion pump linkage control module is used to receive the judgment signal, dynamically adjust the drug delivery rate of the combined composition, and complete the adaptation of the dosage of component B.

[0015] Further configuration: The antibiotic-strain matching module has a built-in compatibility database of antibiotics and drug-resistant DL-endopeptidase strains, which can automatically retrieve matching component B based on the selected component A.

[0016] Further configuration: The dynamics computing server is equipped with a random forest algorithm. It receives 16S rRNA gene sequencing data from fecal samples of premature infants, processes the sequencing data and calculates the microbial community maturity, and generates a microbial community maturity / actual corrected age curve and a microbial community maturity slope in real time.

[0017] Compared with existing technologies, the solution in this application has the following advantages: 1. In the application of the combination of antibiotics and bacteria carrying DL-endopeptidase in the preparation of drugs for preventing delayed intestinal development in premature infants, the combination of "clearance" and "driving" is achieved, breaking the traditional clinical thinking of "killing bacteria first and then remedial". While clearing pathogenic microorganisms (such as Klebsiella pneumoniae and Staphylococcus aureus) in premature infants through antibiotics, the bacterial components in the composition simultaneously downregulate the expression of intracellular cylindrica protein in the intestinal mucosa and actively induce the synthesis of tight junction proteins.

[0018] 2. In the application of the combination of antibiotics and bacteria carrying DL-endopeptidase in the preparation of drugs for preventing delayed intestinal development in premature infants, by means of screening or genetic engineering, the DL-endopeptidase (SagA) producing bacteria are made to have a tolerance spectrum that is compatible with the combined antibiotics, thereby solving the problem of loss of efficacy caused by antibiotic sensitivity of conventional probiotics, and improving the biorobustness of the intervention preparation in the clinical setting.

[0019] 3. In the application of the combination composition of antibiotics and bacteria carrying DL-endopeptidase in this application in the preparation of drugs to prevent delayed intestinal development in premature infants, the combination composition of this application is not a simple drug superposition. It reshapes the intestinal microecological signaling pathway during antibiotic treatment through technical means, which can effectively solve the long-standing technical contradiction between "infection control" and "development protection" in clinical practice, and provide premature infants with a robust intestinal protection solution under complex medication background.

[0020] 4. In the application of the combination composition of antibiotics and DL-endopeptidase-carrying bacteria in the preparation of drugs for preventing delayed intestinal development in preterm infants, the dynamic combined dosing method and collaborative management system can monitor changes in the intestinal flora in real time, dynamically adjust the dosage of component B based on data feedback, avoid the limitations of fixed dosing regimens, and adapt to individual differences among different preterm infants. Simultaneously, the collaborative management system achieves automated coordination of compatibility recommendations, data calculation, and dosing adjustment, reducing manual intervention. The dosing method aligns with NICU clinical diagnosis and treatment procedures and is easy to promote and apply.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a schematic diagram illustrating the mechanism of action of the combination composition of this application; Figure 2 A flowchart illustrating the dynamic combination drug delivery method based on feedback regulation for the combination compositions of this application; Figure 3 This is a hardware architecture diagram of the preterm infant gut development and treatment collaborative management system of this application; Figure 4 A diagram illustrating the mediating effect of antibiotic treatment mechanisms; Figure 5 Screening diagram of NOD2 receptor activation ability of different strains; Figure 6 This is an animal administration model diagram of the combination composition of this application; Figure 7 Survival curves showing the cumulative adverse time incidence of the combination composition of this application in septic mice; Figure 8 A bar chart showing the pathological scoring of mouse lung tissue using the combination composition of this application; Figure 9 This is a graph showing the trend of NOD2 function recovery after combined intervention in clinical preterm infants. Detailed Implementation

[0023] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0024] It should be understood that the steps described in the method embodiments of this application may be performed in different orders and / or in parallel. Furthermore, the method embodiments may include additional steps and / or omit the steps shown. The scope of this application is not limited in this respect.

[0025] The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to". The term "connection" can refer to a direct connection or an indirect connection via intermediate components (elements). The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". Definitions of other terms will be given in the following description.

[0026] It should be noted that the concepts of "first" and "second" mentioned in this application are used only to distinguish between devices, modules or units, and are not intended to limit these devices, modules or units to necessarily be different devices, modules or units, nor are they intended to limit the order or interdependence of the functions performed by these devices, modules or units.

[0027] Please see Figures 1 to 9 This application relates to the use of a combination of an antibiotic and DL-endopeptidase-carrying bacteria in the preparation of a drug for preventing delayed intestinal development in preterm infants. By screening or constructing SagA (secreted antigen A) producing bacteria with specific resistance to antibiotics, it ensures that during the window period when antibiotics clear pathogens, the NOD2-CYLD (a signaling pathway formed by nucleotide-binding oligomerization domain protein 2 and cylindromatosis protein) signaling axis in the host intestine can still obtain continuous molecular drive, thereby maintaining the synthesis of proteins with tight junctions in the intestine (such as ZO-1 and Occludin) and ensuring the normalization of the intestinal development trajectory of preterm infants.

[0028] It should be noted that DL-endopeptidase, also known as SagA (secretory antigen A), is a specific hydrolase. These two names refer to the same substance. DL-endopeptidase is named based on its function (DL-type peptide bond hydrolysis activity), while SagA is the antigenic name given when it was first discovered in the bacterial strain. Essentially, both are peptidoglycan hydrolases that regulate intestinal peptide metabolism. This peptidoglycan hydrolase can help improve intestinal flora structure and barrier function by regulating intestinal peptide metabolism and promoting intestinal mucosal cell proliferation. Its mechanism of action involves processing peptidoglycan in the intestinal lumen into muramyl dipeptide (MDP), thereby activating the host's NOD2-CYLD signaling axis. This process induces the synthesis of intestinal epithelial tight junction proteins (ZO-1, Occludin), driving the transformation of the intestinal barrier from the embryonic to the mature state.

[0029] The composition comprises component A and component B formulated in a specific mass ratio. Component A is selected from broad-spectrum antibiotics commonly used in neonatal intensive care units, specifically including at least one of ampicillin, gentamicin, and vancomycin. Component B is a bacterial strain that can secrete DL-endopeptidase (SagA) and has endogenous resistance to component A. The minimum inhibitory activity (MIC) of component B against component A is >128 μg / mL, ensuring that component B can stably survive and secrete DL-endopeptidase (SagA) under the administration pressure of component A, avoiding inhibition by antibiotics. DL-endopeptidase (SagA) can generate ligands by hydrolyzing bacterial peptidoglycan, activating the NOD2-CYLD signaling pathway, synergistically regulating intestinal immune balance and mucosal barrier repair, thereby improving intestinal flora maturity.

[0030] In one specific embodiment, component B can be a natural screening strain. Specifically, through antibiotic susceptibility testing, strains exhibiting endogenous resistance to the antibiotics in component A and high expression of DL-endopeptidase (SagA) are screened from breast milk or the intestines of healthy full-term infants, with Enterococcus faecalis strains being the preferred candidate. Breast milk and the intestinal samples of healthy full-term infants are safe sources, and the screened strains stably secrete DL-endopeptidase (SagA), exhibit good biocompatibility, and can adapt to and regulate the intestinal microenvironment of preterm infants.

[0031] In another embodiment, component B may be an engineered strain that uses CRISPR-Cas9 gene editing technology to integrate the DL-endopeptidase (SagA) expression cassette into the chromosome of drug-resistant Bifidobacterium or Lactobacillus, thereby constructing an engineered strain that can stably secrete DL-endopeptidase (SagA) under antibiotic stress.

[0032] The specific mass ratio of component A to component B in this application is in the range of 1:10 to 1:500. Within this range, the anti-infective effect of antibiotics and the intestinal regulatory effect of the strain can be balanced, avoiding excessive inhibition of strain activity by antibiotics, while ensuring that the strain can effectively alleviate the interference of antibiotics on the intestinal flora.

[0033] Furthermore, the combination composition of this application adopts a dosage form suitable for enteral administration to preterm infants, including microcapsules, powders, suspensions, or enteric-coated granules. After aseptic processing, it can achieve the simultaneous or sequential release of component A and component B in the intestine of preterm infants, adapting to the physiological characteristics of the preterm infant's intestine and improving bioavailability. The combination composition of this application achieves protection of the intestines of preterm infants through three functional levels: (1) Infection control and signal protection functions.

[0034] Component A, containing antibiotics, recognizes and destroys the cell walls of pathogenic Klebsiella or Staphylococcus aureus, leading to bacterial death and infection control. Simultaneously, component B, due to its endogenous resistance to component A, stably colonizes the intestine and continuously secretes DL-endopeptidase (SagA), providing the material basis for subsequent signal regulation. The key mechanism of this function is that DL-endopeptidase (SagA) specifically cleaves free peptidoglycan (PGN) in the intestinal lumen into muramyl dipeptide (MDP), thereby activating the intestinal protective signaling pathway.

[0035] (2) Molecular signal axis repair function.

[0036] The MDP generated in the above process enters intestinal epithelial cells via solute carrier family transport proteins, thereby activating the intracellular nucleotide-binding oligomerization domain 2 receptor (NOD2). The key mechanism of this function is that the activated NOD2 signal downregulates the transcriptional level of cylindromatosis protein (CYLD), relieving the "brake" inhibitory effect of CYLD on intestinal development pathways and promoting the positive regulation of intestinal development-related signals.

[0037] (3) Physical barrier blocking function.

[0038] As CYLD expression levels decrease, it can induce efficient assembly of tight junction protein-1 (ZO-1) and occludens-1 (Occludin) at the apical junction complex of intestinal epithelial cells, thereby strengthening the integrity of the intestinal physical barrier and reducing the impact of intestinal flora imbalance and external stimuli on intestinal development.

[0039] In addition, it can be specifically combined Figure 1 , Figure 1 This study demonstrates the synergistic effect of antibiotics and DL-endopeptidase (SagA) bacteria at the intestinal lumen and cellular levels from a spatial perspective. Figure 1 The upper middle area is labeled "sterilization and removal," which shows that component A powder is destroying the cell wall structure of pathogenic bacteria (such as Klebsiella pneumoniae), thereby causing the pathogen to lyse and die, thus realizing the infection control function. Figure 1 The central region is the "signal retention region," which shows that component B, which has endogenous resistance to component A, maintains its structural integrity in an antibiotic-compatible environment and continuously secretes DL-endopeptidase (SagA) into the intestinal lumen, providing a stable material basis for subsequent signal regulation. Figure 1The area below shows a cross-section of intestinal epithelial cells, depicting the cell wall acyl dipeptide particles produced after DL-endopeptidase specifically cleaves free peptidoglycan in the intestinal lumen. These particles enter the cell via solute carrier family transport proteins on the intestinal epithelial cell membrane, thereby activating the intracellular nucleotide-binding oligomerization domain 2 receptor (NOD2). The activated NOD2 points an inhibitory arrow toward cylindromatosis protein (CYLD), downregulating its transcription level, and ultimately inducing tight junction protein-1 (ZO-1) to assemble at cell junctions to form a thickened protein band, strengthening the intestinal physical barrier.

[0040] therefore, Figure 1 Through the layered display of spatial dimensions, it is clearly confirmed that in the combination composition of this application, the "bactericidal" function of component A and the "intestinal development-promoting" function of component B are non-interfering parallel relationships in the same space and time. The two work synergistically and do not affect each other, jointly achieving precise protection of the intestines of premature infants.

[0041] Through the progressive and synergistic effects of the above three functional levels, a significant improvement in the MA / CA (microbiota age / actual corrected age, i.e., maturity slope K) index is ultimately achieved, providing core efficacy support for preventing delayed intestinal development in premature infants.

[0042] Furthermore, the combination composition of this application is suitable for scenarios where the intestinal flora maturity slope of preterm infants deviates from the normal range. It can restore the normal physiological function of the SagA-NOD2-CYLD signaling axis, that is, DL-endopeptidase (SagA) first specifically cleaves free peptidoglycan in the intestinal lumen into muramyl dipeptides (MDPs), thereby activating intracellular nucleotide-binding oligomerization domain 2 receptor (NOD2). The activated NOD2 can downregulate the transcription level of deubiquitinase (cylindromaeosis protein CYLD), and through the negative regulation of the NF-κB signaling pathway by CYLD, balance the intestinal inflammatory response and promote the positive transduction of intestinal development-related signals, providing a key molecular driver for the intestinal maturation of preterm infants, and thus promoting the orderly transformation of the intestinal barrier from the embryonic state to the mature state. Therefore, in order to achieve precise prevention of delayed intestinal development in preterm infants, the combination composition of this application adopts a dynamic combination dosing method based on feedback regulation. It uses flora maturity as the criterion for determining at least one of the following: timing of administration, dosage, and effect, so as to achieve precise adaptation of the composition to the intestinal maturation status of preterm infants.

[0043] Please combine Figure 2 , Figure 2 The entire clinical intervention process of the combination composition of this application using a feedback-based dynamic combination dosing method is illustrated in time-logic. This dynamic combination dosing method specifically includes the following steps: S001. Initial Status Assessment. The baseline microbial maturity (MA) of preterm infants before drug administration was determined using 16S rRNA gene sequencing and metabolomics analysis to provide baseline data for subsequent drug administration adjustments.

[0044] S002. Composition Administration. Based on the premature infant's weight, age, and clinical infection risk, adjust and administer the pre-prepared ratio of component A and component B to meet the needs of infection control and intestinal protection, respectively.

[0045] S003. Implement slope detection. Collect fecal samples from premature infants every 48 hours, and calculate the gut microbiota maturity slope K=ΔMA / ΔCA through detection and analysis, where ΔMA is the change in gut microbiota maturity and ΔCA is the actual change in corrected age, thereby reflecting in real time the degree of interference of antibiotics on gut microbiota and the regulatory effect of strains.

[0046] S004. Logical Judgment. If the calculated slope K ≥ 0.8, it indicates that the maturity of the intestinal flora has improved stably and the interference of antibiotics is small, so the original dosing regimen should be maintained; if the slope K < 0.5, it is determined that the interference of antibiotics is significant and the development of intestinal flora is inhibited, so proceed to the next step S005.

[0047] S005. Dynamic dose compensation: To ensure the anti-infective effect, the dosage of component A is kept constant. The dosage of component B is increased by 50%-100%. After each concentration increase, the monitoring operation in step S003 is performed again after 48 hours until the slope K rises back to ≥0.5. The current dosing regimen is maintained to achieve individualized dynamic adjustment.

[0048] The entire clinical intervention process of the above-mentioned dynamic combined drug administration method takes the successful closure of the intestinal barrier and the successful prevention of sepsis as the core endpoints. Intestinal barrier closure can be confirmed by the stable maintenance of intestinal physical barrier-related indicators (such as the expression levels of tight junction protein-1 and atresia protein) and the ratio of microbial maturity / actual corrected age (MA / CA) within the normal range; successful prevention of sepsis can be confirmed by clinical infection-related signs and pathogen detection results. Once both are achieved, the clinical intervention process of this dynamic combined drug administration can be terminated.

[0049] In addition, to efficiently implement the above-mentioned dynamic drug delivery method, this application also provides a preterm infant intestinal development and treatment co-management system. This application relies on the preterm infant intestinal development and treatment co-management system to dynamically regulate the drug delivery of the combined composition. For details, please refer to... Figure 3 , Figure 3The physical device connection method for implementing a collaborative management system for the intestinal development and treatment of preterm infants is illustrated. This system includes an antibiotic-strain matching module, a kinetic calculation server, and an automatic infusion pump linkage control module. These modules work together to achieve dynamic drug administration regulation. Specifically, the antibiotic-strain matching module has a built-in compatibility database of antibiotics and drug-resistant DL-endopeptidase strains. It can automatically retrieve matching component B based on a selected component A and generate pre-mixing ratios and compatibility suggestions for components A and B within a specific mass ratio range. The kinetic calculation server includes a microprocessor unit equipped with a random forest algorithm. It rapidly processes 16S rRNA gene sequencing data from preterm infant fecal samples, calculates microbial maturity (MA), and generates MA / CA curves and microbial maturity slope K in real time, thus providing accurate and efficient data support for the logical determination of S004. The automatic infusion pump linkage control module is electrically connected to the kinetic calculation server. It includes two high-precision micro-injection pumps corresponding to components A and B, specifically an antibiotic pump and a bacterial preparation pump. By receiving a logical judgment command of the maturity slope K, it can adjust the inflow rate of the bacterial preparation pump for component B to achieve dynamic adjustment of the drug concentration while keeping the drug dosage of component A constant.

[0050] In addition, to facilitate the dynamic adjustment of component B, the preterm infant gut development and treatment collaborative management system of this application is also equipped with a host display. The host display is connected to the kinetic calculation server and can display two comparison curves in real time. One curve is the standard development trajectory curve of the preterm infant gut microbiota, and the other curve is the real-time development trajectory curve of the host (preterm infant) gut microbiota. By comparing the overlap of the two curves, the development status of the gut microbiota can be determined, which serves as a supplementary basis for the automatic infusion pump linkage control module to adjust the infusion rate of component B, further improving the accuracy of dynamic adjustment of component B and facilitating medical staff to intuitively monitor and intervene in the drug administration process.

[0051] When the maturity slope K is less than 0.5, the pumping rate of component B is automatically increased (corresponding to a concentration increase of 50%-100%) until the maturity slope K rises back to ≥0.5, at which point the current flow rate is maintained, so as to achieve automated and precise control of the drug delivery process and reduce human error.

[0052] Therefore, this application adopts a dynamic intervention method based on the feedback of "maturity slope K". By implementing the detection of the ratio of the preterm infant's gut microbiota age (MA) to the actual corrected age (CA), when the slope deviates, the concentration of component B in the combination composition is adjusted to achieve precise site treatment. This not only provides an individualized level of intervention, but also effectively prevents bacterial translocation and late-onset sepsis caused by a weak intestinal barrier. It also allows the developmental trajectory of preterm infants to approach the Jinkang standard line even under complex medication backgrounds, providing core technical support for the clinical precision medicine of preterm infants.

[0053] This application also validated the technical feasibility and significant effects through rigorous controlled experiments and clinical data simulations.

[0054] To verify the necessity of combined use of antibiotics and DL-endopeptidase, statistical analysis of clinical samples was conducted to examine the statistical relationship between antibiotic exposure, gut microbiota maturity, and late-onset sepsis (LOS). The results are shown below. Figure 4 As shown, the data analysis results indicate that although antibiotic exposure can achieve the clinical goals of clearing pathogens and controlling infection, it also has the side effect of non-specifically clearing beneficial commensal bacteria carrying the SagA gene in the gut, which directly hinders the maturation process of the gut microbiota. This side effect is a key mediating factor in the increased risk of late-onset sepsis in premature infants (P=0.04). That is, antibiotics do not directly induce late-onset sepsis, but rather indirectly increase the risk of late-onset sepsis in premature infants by disrupting the molecular signaling chain of intestinal development and weakening the integrity of the intestinal barrier through the above-mentioned side effects.

[0055] The above results directly confirm the indirect negative impact of antibiotics alone on the gut microbiota and development of premature infants. Furthermore, from a clinical data perspective, they clearly establish the technical concept of this application, which requires the combined use of DL-endopeptidase-secreting strains that effectively promote gut microbiota maturation during and after antibiotic use. In other words, through this combined regimen, the core anti-infective efficacy of antibiotics can be guaranteed while precisely blocking their key side effect of hindering gut microbiota maturation, thus mitigating the risk of subsequent infections caused by antibiotic use at its source.

[0056] Secondly, please combine Figure 5 , Figure 5 A screening diagram of the NOD2 receptor activation capacity of different strains was created, clearly defining the key biological criteria for screening the core strains in this application, providing direct experimental evidence for the precise selection of functional strains in the combination composition. The experiment systematically compared multiple strains of Enterococcus faecalis (Gastroenterococcus faecium) using an in vitro detection system. E. faecium SagA gene positive, SagA + ) and Enterococcus faecalis ( E. faecalis SagA gene negative, SagA - The activation capacity of NOD2 receptor (quantified by OD value).

[0057] The test results showed that Enterococcus faecalis strains carrying only the SagA gene and stably expressing and secreting DL-endopeptidase (SagA) (such as strains Ef1 and Ef3) could produce significant immune activation activity against the NOD2 receptor, while Enterococcus faecalis strains without the SagA gene and unable to express this enzyme did not have this activation effect. This verifies that not all common probiotic strains can achieve the technical effects of this application; only specific Enterococcus faecalis strains producing DL-endopeptidase (SagA) are the key effective components for promoting intestinal flora maturation and activating the NOD2-CYLD signaling axis. This confirms the necessity and uniqueness of selecting this type of specific strain as a combination component in this application, excluding the possibility of using common non-SagA-producing bacteria, and laying a crucial biological foundation for the screening, identification, and subsequent preparation of the combination composition of the core strain.

[0058] In addition, this application demonstrates the timeline of the overall experimental design for the combination dosing regimen by constructing standardized in vivo validation in preterm mice. Please refer to [link / reference needed] for details. Figure 6 . Figure 6 The timeline shows that newborn mice were first given continuous meropenem antibiotic treatment from day 3 to day 7 after birth (P3-P7) to accurately simulate the clinical antibiotic exposure status of preterm infants in the neonatal intensive care unit. Then, from day 7 to day 9 after birth (P7-P9), they were given the SagA-positive Enterococcus faecalis (see application). E. faecium The control group used SagA-negative Enterococcus faecalis ( E. faecalis The mice underwent gavage intervention to achieve parallel experiments between the combined treatment regimen and the control regimen. Finally, on day 9 after birth (P9), pathogenic bacteria challenge modeling was carried out to provide a unified experimental judgment criterion for subsequent evaluation of the effects of different intervention regimens on the protection of the intestinal barrier and anti-infection ability of mice, and also to provide direct in vivo experimental evidence for the scientificity, feasibility and protective scope of the combined drug administration method.

[0059] This application uses Kaplan-Meier survival curves (cumulative adverse event occurrence curves) to evaluate the efficacy of the combination composition. For specific validation results, please refer to [link to relevant documentation]. Figure 7 , Figure 7 The dashed line represents bacteria treated with antibiotics alone, without DL-endopeptidase combination therapy. E. faecalis The control group was treated with antibiotics and those treated with DL-endopeptidase bacteria ( ). The solid line represents the control group treated with antibiotics and those treated with DL-endopeptidase bacteria ( ). E. faecalisSurvival analysis of the experimental group showed that, compared with the control group, the cumulative incidence of adverse events in the experimental group using the antibiotic and DL-endopeptidase in combination was significantly lower. Relevant statistical indicators verified the significance and reliability of this difference (HR=3.55, P=0.045). HR=3.55 indicates that the cumulative risk of adverse events in the control group was 3.55 times that of the experimental group, directly quantifying the protective effect of the bacteria using DL-endopeptidase. P=0.045<0.05 confirms that the difference between the two groups was not due to random error and has clear statistical significance.

[0060] The above results demonstrate that the pharmaceutical composition of this application can significantly reduce the risk of adverse events and exert a clear protective effect in the context of antibiotic exposure, providing quantitative and rigorous in vivo experimental evidence for the efficacy of the pharmaceutical composition.

[0061] To further verify the efficacy of this application, an in vivo functional verification experiment was also conducted in animals. This involved constructing a mouse model of antibiotic exposure and using the histoathology score (a measure of inflammatory infiltration and tissue damage) as the evaluation index. The results were combined with intergroup comparisons (control group vs. combination therapy intervention group) and statistical tests. For detailed results, please refer to [link to relevant documentation]. Figure 8 The bar chart shows the pathological scores of lung tissue in each group of mice. The results show that, compared with the control group, mice treated with the combination of this application had significantly lower pathological scores for lung inflammatory infiltration and tissue damage. (P<0.001) confirms that the combination composition of this application can effectively reduce the damage of secondary lung infection caused by antibiotic exposure through the systemic regulation of the gut-lung axis, fully verifying the in vivo efficacy and extended value of the technical solution of this application in the prevention and control of anti-infective complications.

[0062] The histopathology scores of lung tissue in each group of mice are presented. The bar chart shows that, compared to the control group, mice treated with the combination composition of this invention had significantly lower scores for lung inflammatory infiltration and tissue damage. (P<0.001), demonstrating that the composition can systematically reduce secondary infection damage following antibiotic treatment via the gut-lung axis.

[0063] Please see Figure 9 This application also conducted human clinical trials, clarifying the human suitability and clinical translation potential of the pharmaceutical composition of this application. Figure 9The results of the validation of the pharmaceutical composition of this application in humans are presented. The experiment involved subjects receiving SagA-containing probiotics (Lactobacillus reuteri). L. reuteri Preterm infants receiving the combined intervention were designated as the experimental group (purple line), while preterm infants who did not receive the combined intervention after antibiotic exposure served as the blank control group (gray line). The ability of fecal filtrate to activate NOD2 receptors was used as the core detection indicator, and the trend of this indicator over time was dynamically monitored and compared between the two groups of preterm infants.

[0064] The results showed that the ability of fecal filtrate from premature infants in the experimental group to activate NOD2 receptors increased significantly with the progress of intervention time, and the detection results at each time point were significantly higher than those of the control group without intervention. This fully demonstrates that the combined intervention regimen of this application can effectively activate the NOD2 signaling pathway in human premature infants, achieving the same mechanism of action as in animal experiments. This directly verifies the practical application effect of the regimen in humans and clarifies its good clinical translation potential and feasibility.

[0065] In summary, the combination of antibiotics and bacteria carrying DL-endopeptidase provided in this application achieves a synergistic effect of "elimination" and "driving," breaking the traditional clinical thinking of "killing bacteria first and then remedial treatment." While eliminating pathogenic microorganisms (such as Klebsiella pneumoniae and Staphylococcus aureus) in premature infants through antibiotics, it simultaneously utilizes the bacterial components in the composition to downregulate the expression of intracellular cylindromatosis protein in the intestinal mucosa and actively induce the synthesis of tight junction proteins.

[0066] By using screening or genetic engineering, DL-endopeptidase (SagA) producing bacteria can be made to have a tolerance spectrum compatible with combined antibiotics, thereby solving the problem of loss of efficacy caused by antibiotic sensitivity in conventional probiotics and improving the biorobustness of interventional preparations in clinical settings.

[0067] The combination composition of this application is not a simple drug additive; it reshapes the intestinal microecological signaling pathway during antibiotic treatment through technical means, which can effectively solve the long-standing technical contradiction between "infection control" and "development protection" in clinical practice, and provide a robust intestinal protection solution for premature infants under complex medication backgrounds.

[0068] The above description is only a partial embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. The use of a combination of an antibiotic and bacteria carrying DL-endopeptidase in the preparation of a drug for preventing delayed intestinal development in preterm infants.

2. The application according to claim 1, characterized in that, The combination composition includes component A and component B, wherein component A is a broad-spectrum antibiotic and component B is a bacterial strain that secretes DL-endopeptidase and has endogenous resistance to component A.

3. The application according to claim 2, characterized in that, The minimum inhibitory concentration (MIC) of component B against component A is >128 μg / mL.

4. The application according to claim 2, characterized in that, The mass ratio of component A to component B is in the range of 1:10 to 1:

500.

5. The application according to claim 2, characterized in that, Component A includes at least one of aminopyrine, gentamicin, and vancomycin.

6. The application according to claim 2, characterized in that, Component B includes natural selection strains, and the screening of natural selection strains includes the following steps: screening strains that are endogenously resistant to component A and have high expression of DL-endopeptidase from breast milk or the intestines of healthy full-term infants by means of antibiotic susceptibility testing.

7. The application according to claim 2, characterized in that, Component B includes an engineered strain, the construction of which includes the following steps: using CRISPR-Cas9 gene editing technology, the DL-endopeptidase expression cassette is integrated into the chromosome of the antibiotic-resistant probiotic to obtain an engineered strain that can secrete DL-endopeptidase under antibiotic stress.

8. The application according to claim 2, characterized in that, The combination composition is a formulation suitable for enteral administration to preterm infants.

9. The application according to claim 2, characterized in that, The combination composition is used to promote intestinal maturation in premature infants by restoring the physiological function of the SagA-NOD2-CYLD signal axis when the slope of the gut microbiota maturity deviates from the normal range. At least one of the following is determined based on the slope of the maturity: timing of administration, dosage, and effect of use.

10. The application according to claim 9, characterized in that, The application relies on the preterm infant gut development and treatment co-management system to dynamically regulate the administration of the combined composition. The preterm infant gut development and treatment co-management system includes an antibiotic-strain module, a kinetic calculation server, and an automatic infusion pump linkage control module. The antibiotic-strain module is used to provide the matching relationship between antibiotics and DL-endopeptidase secretory strains resistant to the antibiotics, as well as recommendations on their compatibility and dosage ratio. The dynamics calculation server is used to process microbial community detection data, calculate the slope of microbial community maturity, identify its deviation state, and output a judgment signal. The automatic infusion pump linkage control module is used to receive the judgment signal, dynamically adjust the drug delivery rate of the combined composition, and complete the adaptation of the dosage of component B.

11. The application according to claim 10, characterized in that, The antibiotic-strain matching module has a built-in compatibility database of antibiotics and drug-resistant DL-endopeptidase strains, and can automatically retrieve matching component B based on selected component A.

12. The application according to claim 10, characterized in that, The dynamic computing server is equipped with a random forest algorithm. It receives 16S rRNA gene sequencing data from fecal samples of premature infants, processes the sequencing data and calculates the microbial community maturity, and generates a microbial community maturity / actual corrected age curve and microbial community maturity slope in real time.

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