Application of Xylooligosaccharide in the Preparation of Attenuated Pathogenic Escherichia coli Products

Abstract

The present invention provides the use of xylo-oligosaccharides in the preparation of attenuated pathogenic Escherichia coli products. This invention, for the first time, discovers that xylo-oligosaccharides can significantly reduce the virulence of pathogenic Escherichia coli, thereby effectively preventing and / or treating colibacillosis. This provides a new raw material for attenuated pathogenic Escherichia coli products and a new application for xylo-oligosaccharides. Furthermore, the present invention also discovers that xylo-oligosaccharides do not affect the normal growth of pathogenic Escherichia coli, are unlikely to induce drug resistance in pathogenic Escherichia coli, or increase resistance to commonly used antibiotics in pathogenic Escherichia coli. In fact, they can even reduce resistance to some antibiotics (such as streptomycin).

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen attenuation, and more specifically relates to the use of xylooligosaccharides in the preparation of attenuated pathogenic Escherichia coli products. Background Art

[0002] Pathogenic Escherichia coli Escherichia Escherichia coli (PEC) infection is one of the most common bacterial diseases in poultry, severely impacting poultry performance. Previously, PEC infection was primarily controlled with antibiotics. However, with the widespread ban on antibiotics in feed and the reduction / restriction of antibiotics in livestock production, antibiotics are no longer the primary approach and their effectiveness has been limited. This is partly due to the fact that PEC can exchange virulence-related genes, including resistance genes, with commensal E. coli in the gut through gene exchange, leading to strong resistance and making it difficult to eliminate PEC with commonly used antibiotics. Furthermore, even non-antibiotic antibacterial / antimicrobial substances that share no interplay with antibiotics can contribute to cross-resistance to antibiotics in PEC. Therefore, finding strategies that are less likely to induce PEC resistance, such as mitigating PEC virulence rather than inhibiting its growth, is crucial for the prevention and control of PEC infection.

[0003] Xylooligosaccharides (XOS) are a type of oligosaccharide composed of 2 to 7 xylose molecules polymerized through β-1,4 glycosidic bonds. They are often used in the fields of medicine and nutrition, such as promoting the growth of beneficial intestinal bacteria, enhancing the body's immunity, lowering blood sugar, controlling blood lipids and regulating blood pressure. However, there are currently no reports on the toxic effects of XOS on PEC. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention aims to provide the use of xylooligosaccharides in the preparation of attenuated pathogenic Escherichia coli products, which can effectively prevent and control Escherichia coli disease caused by PEC infection without easily inducing PEC resistance.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention discovered for the first time that XOS can activate the expression of a series of genes on the PEC xylose operon, including the xylose transporter, and can enter the PEC cell. Since PEC cannot metabolize XOS, XOS can disrupt PEC's intracellular metabolism by acting as a PEC antimetabolite, thereby significantly reducing PEC's virulence. In other words, XOS can be used to prevent Escherichia coli disease caused by PEC infection. In addition, XOS can alleviate the intestinal damage and decreased growth performance of poultry caused by PEC challenge. In other words, XOS can be used to prevent and control Escherichia coli disease caused by PEC infection. Therefore, the use of xylooligosaccharides in the preparation of attenuated pathogenic Escherichia coli products and in the preparation of products for preventing and / or treating Escherichia coli disease should be within the scope of protection of the present invention.

[0007] Existing research indicates that although xylooligosaccharides (XOS) cannot be digested by animals, they can reach the back end of the animal's digestive system, selectively promoting the proliferation of beneficial bacteria in the hindgut that produce β-glucosidase, leading to the production of short-chain fatty acids. This in turn indirectly inhibits the growth of harmful bacteria, potentially leading to drug resistance in these bacteria. The present invention unexpectedly discovered that XOS can reduce the virulence of specific pathogenic bacteria (PECs) without affecting their normal growth, and is unlikely to induce drug resistance or increase resistance to commonly used antibiotics. It can even reduce resistance to some antibiotics, such as streptomycin.

[0008] Preferably, the pathogenic Escherichia coli is enterohemorrhagic Escherichia coli or avian pathogenic Escherichia coli.

[0009] Further preferably, the enterohemorrhagic Escherichia coli is enterohemorrhagic Escherichia coli O157.

[0010] Further preferably, the avian pathogenic Escherichia coli is avian pathogenic Escherichia coli O78.

[0011] Preferably, the product is a functional feed additive.

[0012] Further preferably, the functional feed additive further comprises auxiliary materials, such as one or more of starch, dextrin, zeolite powder, and corn cob powder.

[0013] Preferably, the starch is one or more of corn starch, wheat starch and potato starch.

[0014] Preferably, the dextrin is one or more of corn dextrin, maltodextrin, cyclodextrin and white dextrin.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention discovers for the first time that xylooligosaccharides can significantly reduce the virulence of pathogenic Escherichia coli, thereby effectively preventing and / or treating Escherichia coli disease. This provides a new raw material for attenuated pathogenic Escherichia coli products and a new application for xylooligosaccharides.

[0017] 2. The present invention discovered for the first time that xylo-oligosaccharides do not affect the normal growth of pathogenic Escherichia coli, are not likely to induce drug resistance in pathogenic Escherichia coli, and do not increase the resistance of pathogenic Escherichia coli to existing commonly used antibiotics. In addition, they can even reduce the resistance of pathogenic Escherichia coli to some antibiotics (such as streptomycin). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the growth curve of PEC.

[0019] Figure 2 The inhibition zones of PEC after treatment with different treatment solutions.

[0020] Figure 3 This is the detection result of the expression level of PEC xylose operon gene.

[0021] Figure 4 This is a bubble diagram for KEGG pathway enrichment analysis. Figure 4 A in the figure is the KEGG pathway enrichment analysis bubble chart of differentially downregulated genes in the PEC+XOS group compared with the PEC group. Figure 4 B in the figure is the KEGG pathway enrichment analysis bubble chart of differentially upregulated genes in the PEC+XOS group compared with the PEC group.

[0022] Figure 5 The results are the detection results of the expression levels of PEC virulence factors.

[0023] Figure 6 This is an electron microscopic observation of the outer membrane morphology of PEC bacteria.

[0024] Figure 7 The adhesion of PEC to intestinal epithelial cells.

[0025] Figure 8 The results of gene expression in intestinal epithelial cells are shown in Figure 5.

[0026] Figure 9 This is the detection result of the amount of PEC colonization in the intestine.

[0027] Figure 10 The results of the detection of cytokine and tight junction protein gene expression in the intestine. Figure 10 A in the figure is the detection result of jejunum cytokine expression. Figure 10 B in the figure is the detection result of ileal cytokine expression. Figure 10C in the figure is the detection result of tight junction protein gene expression in jejunum. Figure 10 D in the figure is the detection result of ileum tight junction protein gene expression.

[0028] Figure 11 The results of the detection of PEC virulence gene expression in the cecum. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0031] PEC (serotype O157) culture medium: PEC (serotype O157) was spread on LB plate medium and cultured at 37°C for 12 h. A single colony was picked and inoculated into 5 mL of LB broth medium. The culture was cultured in a constant temperature shaker at 37°C and 180 rpm / min for 12 h. The culture medium was then inoculated into fresh LB broth medium at a volume ratio of 1:50. The culture was cultured in a shaker at 37°C and 180 rpm / min for 3 h. The culture medium was centrifuged at 9000 rpm / min for 10 min, the bacterial pellet was collected, washed with sterile PBS, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL (at this time PEC is in the logarithmic growth phase), the PEC (serotype O157) bacterial liquid is obtained.

[0032] Example 1 XOS is not likely to induce drug resistance in PEC

[0033] 1. XOS has no significant effect on the normal growth of PEC

[0034] (1) Experimental methods

[0035] Pathogenic Escherichia coli PEC (serotype O157) was spread on LB plate culture medium and cultured at 37°C for 12 h. Single colonies were picked and inoculated into LB broth medium (denoted as PEC+LB broth group), LB broth medium containing 4 mg / mL XOS (denoted as PEC+XOS+LB broth group), M9 medium with glucose (4 mg / mL) as the sole carbon source (denoted as PEC+glucose+M9 group), and M9 medium with xylooligosaccharide (XOS, 4 mg / mL) as the sole carbon source (denoted as PEC+XOS+M9 group). The culture was incubated at 37°C and 180 rpm / min for 12 h, and samples were taken every 2 h to measure the absorbance OD. 600, draw the growth curve of PEC.

[0036] (2) Experimental results

[0037] The results are as follows Figure 1 As shown. It can be seen that the addition of XOS to LB broth medium has no significant effect on the growth curve of PEC ( P >0.05), and PEC could not grow in M9 medium with XOS as the sole carbon source, indicating that XOS did not significantly affect the normal growth of PEC and could not be utilized by PEC.

[0038] 2. XOS has no inhibitory effect on PEC

[0039] (1) Experimental methods

[0040] Take 200 μL of 1×10 9 CFU / mL of PEC (serotype O157) bacteria in the logarithmic growth phase was inoculated into 200 mL of LB broth agar medium melted at 45°C and mixed thoroughly. The mixture was then poured into a sterile culture dish and cooled until solidified. A sterilized cork punch was used to punch holes in the solidified culture medium. 10 μL of different treatment solutions were dripped into the holes using a pipette. After incubation at 37°C for 12 h, the size of the inhibition zone was determined using the cross-hatch method.

[0041] The treatment solutions were as follows: XOS solution with a concentration of 200 mg / mL (denoted as the original group); ampicillin (AMP) solution with a concentration of 200 mg / mL (denoted as the AMP group); XOS solution with a concentration of 100 mg / mL (denoted as group A); XOS solution with a concentration of 50 mg / mL (denoted as group B); XOS solution with a concentration of 25 mg / mL (denoted as group C); XOS solution with a concentration of 12.5 mg / mL (denoted as group D); XOS solution with a concentration of 6.25 mg / mL (denoted as group E); XOS solution with a concentration of 3.125 mg / mL (denoted as group F); XOS solution with a concentration of 1.562 mg / mL (denoted as group G); and XOS solution with a concentration of 0.781 mg / mL (denoted as group H).

[0042] (2) Experimental results

[0043] The results are as follows Figure 2 As shown, no inhibition zone appeared in the original group and groups A to H, that is, XOS had no inhibitory effect on PEC, indicating that XOS is not likely to induce drug resistance in PEC.

[0044] 3. XOS does not increase the minimum inhibitory concentration (MIC) of various antibiotics against PEC

[0045] (1) Experimental methods

[0046] PEC (serotype O157) were inoculated into LB broth and cultured in a shaker at 37°C and 180 rpm / min for 12 h. A suspension with a McFarland turbidity of 0.5 was prepared, diluted 1000-fold with LB broth, and 100 μL of the suspension was added to a 96-well plate. Antibiotic solutions (including neomycin, streptomycin, gentamicin, kanamycin, tetracycline, enrofloxacin, chloramphenicol, ampicillin, amoxicillin, imipenem, meropenem, ertapenem, cefoxitin, cephalexin, and fusidic acid) were prepared by two-fold dilution and added to the 96-well plate. XOS was then added to a final XOS concentration of 4 mg / mL, representing the PEC + XOS group. A group without XOS was designated the PEC group. The two groups were incubated at 37°C for 20 h, and the bacterial growth in each well was observed, and the MIC value of each antibiotic was recorded.

[0047] (2) Experimental results

[0048] The results are shown in Table 1.

[0049] Table 1

[0050]

[0051] It can be seen that the minimum inhibitory concentrations of 14 antibiotics in the PEC+XOS group against PEC are the same as those in the PEC group, and even the minimum inhibitory concentration of one antibiotic (streptomycin) against PEC is half that of the PEC group, indicating that XOS treatment not only does not increase PEC's resistance to existing commonly used antibiotics, but can even reduce PEC's resistance to some antibiotics (such as streptomycin).

[0052] Example 2XOS can reduce the toxicity of PEC

[0053] 1. XOS can disrupt the intracellular metabolism of PECs

[0054] (1) Experimental methods

[0055] Take the concentration of 1×10 9PEC (serotype O157) cultures in the logarithmic growth phase with 0.1 CFU / mL were inoculated into M9 medium without any carbon or nitrogen sources (starvation group), M9 medium containing 4 mg / mL XOS (PEC+XOS group), and M9 medium with glucose (4 mg / mL) as the sole carbon source (PEC+glucose group). Cultures were performed at 37°C and 180 rpm for 6 h. One mL of each culture medium was collected and centrifuged at 4°C and 12,000 rpm for 5 min. The supernatant was discarded and the bacterial pellet was collected. According to the method in the literature (Dong Tao. Mechanism of intestinal inflammation induced by heat shock protein HtpG of Salmonella typhimurium. Master's thesis. South China Agricultural University. 2021.), the total RNA of the bacteria was extracted using a bacterial RNA extraction kit, and its concentration and purity were detected using a micro-UV spectrophotometer NanoDrop 2000. Its integrity was then detected by agarose gel electrophoresis. The RNA concentration was then diluted to 500 ng / μL with ultrapure water, and 2 μL of the RNA dilution was taken for reverse transcription using the reverse transcription kit HiScript II Q RTSuperMix (Novagen Biotech) (the reverse transcription system was: 4 μL 5×HiScript II qRTSuperMix, 2 μL RNA, and 14 μL double-distilled water. The reaction conditions were: 25 ℃, 5 min; 50 ℃, 15 min; 85 ℃, 2 min). The cDNA sample obtained by reverse transcription was placed in a fluorescent quantitative PCR instrument and the RNA polymerase subunit of PEC ( rpoA ) was used as the internal reference gene, and the expression level of PEC xylose operon gene was used as the internal reference gene. xylA 、 xylB 、 xylE 、 xylF 、 xylG 、 xylH For the target gene, real-time fluorescence quantitative PCR was performed (the qRT-PCR reaction system was: 2 μL cDNA, 0.6 μL Forword Primer (10 μM), 0.6 μL Reverse Primer (10 μM), 10 μL 2×ChamQ Universal SYBR qPCR Master Mix and 6.8 μL dd H2O. The reaction conditions were: pre-denaturation (95 ℃, 10 min); denaturation (95 ℃, 15 s), annealing (52-63 ℃, 15 s), extension (72 ℃, 40 s), for a total of 40 cycles; denaturation (95 ℃, 60 s); annealing (52-63 ℃, 30 s); extension (95 ℃, 30 s). The primer sequences are shown in Table 2 and were synthesized by Shanghai Sangon Biotechnology Co., Ltd.). -ΔΔCtThe relative expression of mRNA of each target gene was determined by one-way analysis of variance. The overall data were analyzed by one-way analysis of variance, and the Duncan's multiple comparison method was used for pairwise comparison if the difference was significant (significance was defined as P <0.05).

[0056] Table 2

[0057]

[0058] Another concentration of 1×10 9 PEC (serotype O157) bacteria in the logarithmic growth phase were inoculated at a volume ratio of 1:50 into LB broth (PEC group) or LB broth containing 4 mg / mL XOS (PEC+XOS group). After incubation at 37°C with an air shaker at 180 rpm / min for 3 hours, the cells were harvested and snap-frozen in liquid nitrogen for 15 seconds. RNA samples were extracted and tested for concentration, purity, and integrity. The samples were then used for library construction and quality control, sequencing, and bioinformatics analysis. Bioinformatics analysis included data quality control, reference sequence alignment, novel transcript prediction, gene structure analysis, gene expression quantification, differential expression analysis, GO and KEGG analysis of differentially expressed genes, and gene set enrichment analysis.

[0059] (2) Experimental results

[0060] The detection results of PEC xylose operon gene expression are as follows Figure 3 As shown. It can be seen that the PEC+XOS group xylA 、 xylB 、 xylE 、 xylF 、 xylG 、 xylH The expression levels of xylose operon genes in PEC were significantly higher than those in PEC+glucose group, indicating that XOS can significantly activate xylA 、 xylB 、 xylE 、 xylF 、 xylG 、 xylH The expression of PEC xylose operon genes indicated that XOS could enter PEC cells through xylose transporters, and because PEC could not utilize XOS, XOS could act as an antimetabolite in PEC cells, disrupting PEC metabolism and thus reducing the toxicity of PEC.

[0061] KEGG pathway enrichment analysis bubble diagram Figure 4As shown in the figure, compared with the PEC group, metabolic pathways such as carbon metabolism, citrate cycle, glycolysis / gluconeogenesis, and oxidative phosphorylation were significantly downregulated in the PEC+XOS group (no metabolic pathways were significantly upregulated in the PEC+XOS group), further confirming that XOS can disrupt the intracellular metabolism of PEC and thus reduce the toxicity of PEC.

[0062] 2. XOS can reduce the expression of PEC outer membrane porins

[0063] Outer membrane porins are a class of membrane proteins with complex structures embedded in the outer membrane layer of the cell wall of Gram-negative bacteria such as PEC. They include ompC and ompF, and are important virulence factors regulated by the virulence regulatory factor ompR. They are closely related to the integrity of the bacterial outer membrane, bacterial adhesion and invasion, and can cause host cell damage.

[0064] (1) Experimental methods

[0065] PEC (serotype O157) was spread on LB plates and cultured at 37°C for 12 h. A single colony was picked and inoculated into 5 mL of LB broth and cultured at 37°C at 180 rpm / min for 12 h. The culture was then inoculated into fresh LB broth (denoted as the PEC group) and LB broth containing 4 mg / mL XOS (denoted as the PEC+XOS group) at a volume ratio of 1:50. After culture at 37°C at 180 rpm / min for 3 h, the cells were collected. The RNA of the cells was extracted, tested for concentration, purity, and integrity, and then reverse transcribed and expressed as rpoA As the internal reference gene, ompR 、 ompC 、 ompF Real-time fluorescence quantitative PCR was performed for the target gene using 2 -ΔΔCt The relative expression of mRNA of each target gene was determined by the t-test method (primer sequences are shown in Table 3 and synthesized by Shanghai Sangon Biotechnology Co., Ltd.). P <0.05).

[0066] Table 3

[0067]

[0068] (2) Experimental results

[0069] The results are as follows Figure 5 As shown, the PEC+XOS group ompR and outer membrane porin genes ompC 、 ompF The expression levels of the virulence factors in PEC were significantly lower than those in the PEC group, indicating that XOS treatment can significantly reduce the expression levels of PEC virulence factors, thereby alleviating the virulence of PEC.

[0070] 3. XOS does not affect the overall structure of the PEC cell wall

[0071] (1) Experimental methods

[0072] PEC (serotype O157) was plated on LB plates and incubated at 37°C for 12 h. A single colony was then picked and inoculated into 5 mL of LB broth and incubated at 37°C at 180 rpm / min for 12 h. The culture was then inoculated into fresh LB broth (PEC group) and LB broth containing 4 mg / mL XOS (PEC+XOS group) at a volume ratio of 1:50. After incubation at 37°C at 180 rpm / min for 3 h, the cells were collected. After gentle rinsing with phosphate-buffered saline (PBS), the pellet was centrifuged and fixed with electron microscopy fixative at 25°C for 2 h. The fixed cell samples were rinsed three times with 0.1 mol / L PBS (15 min each time) and then fixed in 30% ( v / v ) → 50% ( v / v ) → 70% ( v / v ) → 80% ( v / v ) → 90% ( v / v )→95%( v / v ) → 100% ( v / v ) in ethanol solution for 15 min, the bacterial sample was placed in a critical point dryer (Quorum K850) for drying, then adhered to a conductive carbon film double-sided tape and placed on the sample stage of an ion sputtering instrument (Hitachi MC1000) for gold spraying for 30 s. Finally, it was observed and photographed under a scanning electron microscope (Hitachi SU8100).

[0073] (2) Experimental results

[0074] Electron microscopic observation of the outer membrane morphology of PEC bacteria Figure 6 As shown in the figure, compared with the PEC group, although the outer membrane layer of the cell wall in the PEC+XOS group was partially damaged (in the yellow circle), its overall structure was not affected (it would not destroy the overall structure of bacteria like antibiotics). This shows that the mechanism by which XOS reduces the virulence of PEC is only through disrupting the intracellular metabolism of PEC and reducing the expression of virulence factors such as its outer membrane porins, and will not affect the normal growth of bacteria.

[0075] Example 3XOS can alleviate PEC-induced intestinal epithelial cell damage

[0076] 1. Adhesion rate of PEC to intestinal epithelial cells

[0077] (1) Experimental methods

[0078] Chicken intestinal epithelial cells were isolated according to the method in the literature (Zhang Beibei. Study on the alleviating effect and mechanism of arginine on intestinal mucosal damage in broiler chickens. Doctoral dissertation. China Agricultural University. 2018) and inoculated into 12-well plates in DMFM / f12 medium (containing 5% fetal bovine serum). The cells were cultured at 37°C until the cell density reached 80%. The culture medium was discarded, the residual culture medium was washed with PBS, and fresh DMFM / f12 medium containing 5% fetal bovine serum was added. The cells were then divided into PEC group (100 μL of 1×10 9 CFU / mL of PEC (serotype O157) in the logarithmic growth phase, mixed) and PEC+XOS group (add 100 μL of 1×10 9 A PEC (serotype O157) bacterial suspension with 100 CFU / mL and in the logarithmic growth phase was prepared, and XOS was added to a final XOS concentration of 4 mg / mL and mixed thoroughly. The 12-well plate was then placed in a biochemical incubator and incubated at 37°C for 1 hour. The cell culture supernatant was collected and placed in a 1.5 mL centrifuge tube. The cells were washed three times with sterile PBS (to remove unadhered bacteria). 1 mL of PBS containing 1% Triton-100 was then added to the cells for 30 minutes and thoroughly pipetted to disrupt the cells. The collected liquid was then diluted 10,000-fold with PBS, and 5 μL of the diluted liquid was dripped onto LB broth agar medium. After incubation in a 37°C constant temperature incubator for 12 hours, the single colonies in each drop were counted, and single colonies were picked for Giemsa staining. The average number of single colonies in all drop points corresponding to each sample is the number of single colonies of the sample, and the adhesion rate is calculated according to "adhesion rate (%) = (number of single colonies × 200 × dilution factor) / number of added bacteria".

[0079] (2) Experimental results

[0080] The results are as follows Figure 7 As shown in the data, the adhesion rate of PEC to chicken intestinal epithelial cells in the PEC+XOS group was significantly lower than that in the PEC group, indicating that XOS can significantly inhibit the adhesion of PEC to chicken intestinal epithelial cells, thereby alleviating PEC-induced intestinal epithelial cell damage.

[0081] 2. Gene Expression in Intestinal Epithelial Cells

[0082] (1) Experimental methods

[0083] Chicken intestinal epithelial cells were isolated according to the method in the literature (Zhang Beibei. Study on the alleviating effect and mechanism of arginine on intestinal mucosal damage in broiler chickens. Doctoral dissertation. China Agricultural University. 2018) and inoculated into 12-well plates in DMFM / f12 medium (containing 5% fetal bovine serum). The cells were cultured at 37°C until the cell density reached 80%. The culture medium was discarded, the residual culture medium was washed with PBS, and fresh DMFM / f12 medium containing 5% fetal bovine serum was added. The cells were then divided into PEC group (100 μL of 1×10 9 CFU / mL of PEC (serotype O157) in the logarithmic growth phase, mixed well), PEC+XOS group (add 100 μL of 1×10 9 CFU / mL of PEC (serotype O157) in the logarithmic growth phase, and XOS was added to a final XOS concentration of 4 mg / mL and mixed thoroughly) and a CON group (i.e., no PEC culture solution or XOS was added). The 12-well plate was then placed in a biochemical incubator and incubated at 37°C for 1 hour. The cell culture supernatant was collected in a 1.5 mL centrifuge tube and the cells were washed three times with sterile PBS (to remove unadhered bacteria). 1 mL of PBS containing 1% Triton-100 was then added to permeabilize the cells for 30 minutes and thoroughly pipetted to disrupt the cells. The collected liquid was then diluted 10,000-fold with PBS, and 5 μL of the diluted liquid was dripped onto LB broth agar medium. After incubation in a constant temperature incubator at 37°C for 12 hours, single colonies were picked to extract cellular RNA. After concentration, purity, and integrity testing, reverse transcription was performed and 3-phosphoglycerate dehydrogenase ( GAPDH ) as the internal reference gene, and inflammatory factors ( IL-1β 、 TNF-α 、 IL-6 、 IFN-γ 、 IL-8 ), cell proliferation-related genes PCNA and apoptosis-related genes Caspase 3 Real-time fluorescence quantitative PCR was performed for the target gene using 2 -ΔΔCt The relative mRNA expression of each target gene was determined by the method (primer sequences are shown in Table 4 and were synthesized by Shanghai Sangon Biotechnology Co., Ltd.). The overall data results were analyzed by one-way analysis of variance. If the differences were significant, pairwise comparisons were performed using the Duncan's multiple comparison method. The significance was defined as P <0.05.

[0084] Table 4

[0085]

[0086] (2) Experimental results

[0087] The results are as follows Figure 8 As shown, the inflammatory factors of chicken intestinal epithelial cells in the PEC+XOS group IL-1β 、 TNF-α and cell proliferation-related genes PCNA The expression of inflammatory factors IFN-γ and apoptosis-related genes Caspase-3 The expression of also recovered to a level with no significant difference from that in the CON group, indicating that XOS can not only significantly reduce the inflammatory response induced by PEC, but also alleviate the enhancement of PEC-induced cell apoptosis, thereby alleviating the abnormal cell proliferation caused by the feedback of enhanced cell apoptosis. Therefore, XOS can alleviate PEC-induced intestinal epithelial cell damage.

[0088] Example 4XOS can alleviate PEC-induced intestinal damage

[0089] To facilitate the detection of PECs colonizing the intestine, this example used the method described in the literature (Liu Fengzhi. Aerosol Generation and Transmission Patterns of Escherichia coli in Chicken Houses. Master's Thesis. Shandong Agricultural University. 2009). The spectinomycin-resistant plasmid pMB1-spect-PthrC3_8-eGFP (purchased from Adegene) was transformed into PECs (serotype O78) and used in the following animal experiments.

[0090] 144 one-day-old male Lingnan yellow-feathered broilers were selected and divided into three groups based on weight balance: the CON group, the PEC group, and the PEC+XOS group. Each group consisted of six replicates, with eight broilers per replicate. The initial weight of each group was measured, and the following 12-day experiment was conducted:

[0091] (1) CON group: From day 1 to day 12, the rats were fed a basic diet (nutritional composition shown in Table 5, nutritional level shown in Table 6), and were allowed to eat freely. From day 7 to day 12, 2 mL of PBS was administered orally once daily, and the rats were fasted for 6 h before and after each administration.

[0092] (2) PEC group: The animals were fed with a basic diet and free access to food from day 1 to day 12; 2 mL of PEC bacterial solution (2 × 10 9 CFU / mL), and fasted for 6 h before and after each gavage.

[0093] (3) PEC+XOS group: The rats were fed with a basic diet (mixed with 1600 mg / kg XOS) from day 1 to day 12, and were allowed to eat freely. From day 7 to day 12, 2 mL of PEC solution (2×10 9 CFU / mL), and fasted for 6 h before and after each gavage.

[0094] The body weight of each group of broilers at the end of the experiment and the weight of the feed consumed during the experiment were weighed. From each replicate of each group, a broiler with the closest body weight to the average was selected and its live weight was recorded. After fasting for 12 h (without food or water), the following procedures were performed: ① A 1 cm sample of tissue near the jejunum and mid-ileum was cut with sterile scissors and forceps. The chyme was rinsed with PBS and divided into two parts. One part was fixed with 4% (w / v) paraformaldehyde solution (for intestinal morphological analysis) and the other part was quickly frozen in liquid nitrogen and stored in a refrigerator at −80°C (for intestinal tissue gene expression determination); ② Duodenal and jejunal mucosal samples were scraped with a glass slide (for determination of PEC colonization); ③ Cecal chyme was collected, quickly frozen in liquid nitrogen, and stored in a refrigerator at −80°C.

[0095] Table 5

[0096]

[0097] The premix in Table 5 uses zeolite powder as a carrier and provides the following per kilogram of feed: vitamin A 12,000 IU, vitamin D 3,600 IU, vitamin E 45 IU, vitamin K 32.5 mg, vitamin B12.2 mg, vitamin B2 8 mg, pantothenic acid 40 mg, niacin 4 mg, biotin 0.4 mg, folic acid 1.0 mg, vitamin B12 0.013 mg, iron 80 mg, copper 8 mg, zinc 60 mg, manganese 110 mg, selenium 0.3 mg, and iodine 1.1 mg.

[0098] Table 6

[0099]

[0100] 1. Growth performance

[0101] (1) Experimental methods

[0102] According to the body weight of broilers in each group at the beginning and end of the experiment and the weight of feed consumed during the experiment, the average final weight, average daily weight gain, average daily feed intake and feed-to-weight ratio of broilers were calculated.

[0103] (2) Experimental results

[0104] The results are shown in Table 7. It can be seen that compared with the CON group, the average final weight and average daily weight gain of the PEC group were significantly reduced, and the feed-to-weight ratio was significantly improved. The PEC+XOS group could reverse the changes in the above indicators caused by PEC and significantly increase the average daily feed intake, indicating that XOS can offset the decline in broiler growth performance caused by PEC challenge. This is because XOS can alleviate the intestinal damage induced by PEC.

[0105] Table 7

[0106]

[0107] 2. Intestinal morphology and structure

[0108] (1) Experimental methods

[0109] Jejunal and mid-ileal tissue samples fixed with 4% (w / v) paraformaldehyde were paraffin-embedded and stained with hematoxylin-eosin. 4-μm sections were obtained. Complete and representative villus-crypt units were selected from each section (at least eight per section). Villus height and crypt depth were measured using Image J software (villus height was the linear distance from the villus tip to the crypt opening, and crypt depth was the linear distance from the crypt opening to the base). The ratio of villus height to crypt depth (i.e., villus-crypt ratio) was then calculated.

[0110] (2) Experimental results

[0111] The experimental results are shown in Table 8. It can be seen that compared with the CON group, the jejunal crypt depth of the PEC group was significantly reduced, while the PEC+XOS group could reverse the changes in indicators caused by PEC and significantly increase the height of ileal villi, indicating that XOS can alleviate the PEC-induced destruction of the intestinal morphology and structure of broiler chickens, thereby alleviating the PEC-induced intestinal damage.

[0112] Table 8

[0113]

[0114] 3. PEC colonization in the intestine

[0115] (1) Experimental methods

[0116] Scraped duodenal and jejunal mucosal samples were dissected longitudinally and rinsed with sterile PBS containing 0.25 mg / mL spectinomycin to remove other intestinal microorganisms. The samples were then transferred to sterile EP tubes containing 1 mL of sterile PBS containing 0.25 mg / mL spectinomycin. The tissues were homogenized using a rapid sample grinder at 4°C (2 cycles of 30 s, with a 30 s pause in between). The homogenate was then diluted 10-fold with sterile PBS containing 0.05 mg / mL spectinomycin. Five μL of the dilution was then dripped onto LB broth agar medium containing 0.05 mg / mL spectinomycin. After incubation at 37°C for 9 h, the number of single colonies per drop was counted. The average number of single colonies per drop for each sample was calculated as the single colony count for that sample, which was used to characterize the intestinal colonization of PECs.

[0117] (2) Experimental results

[0118] The results are as follows Figure 9As shown in the data, compared with the CON group, the colonization of PEC in the intestine (duodenum and jejunum) in the PEC group was significantly increased, while the PEC+XOS group could reverse the changes in indicators caused by PEC, indicating that XOS can inhibit the colonization of PEC in the intestine and thereby alleviate PEC-induced intestinal damage.

[0119] 4. Intestinal Tissue Gene Expression

[0120] (1) Experimental methods

[0121] According to the instructions of the RNA extraction kit FastPure® Cell / Tissue Total RNA Isolation Kit V2 (Novagen Biotech), total RNA was isolated from tissue samples of the jejunum and mid-ileum stored at −80°C. After its concentration and purity were determined using a NanoDrop 2000 micro-volume UV spectrophotometer, 1 μL of RNA was reverse transcribed using the reverse transcription kit HiScript II Q RT SuperMix (Novagen Biotech). The reverse transcription system consisted of 4 μL of 5× HiScript II qRT SuperMix, 2 μL of RNA, and 14 μL of double-distilled water. The reaction conditions were: 25°C for 5 min; 50°C for 15 min; and 85°C for 2 min. The cDNA sample obtained by reverse transcription was placed in a fluorescence quantitative PCR instrument and PCR was performed using reduced glyceraldehyde-phosphate dehydrogenase ( GAPDH ) as the internal reference gene, and inflammatory cytokines ( IL-1β 、 TNF-α 、 IL-6 、 IL-8 ), anti-inflammatory cytokines IL-10 and tight junction proteins ( ZO-1 、 Occludin 、 Claudin-1 ) was used as the target gene for real-time fluorescence quantitative PCR (qRT-PCR reaction system: 2 μL cDNA, 0.6 μL Forword Primer (10 μM), 0.6 μL Reverse Primer (10 μM), 10 μL 2×ChamQ Universal SYBR qPCR Master Mix and 6.8 μL dd H2O. Reaction conditions were: pre-denaturation (95°C, 10 min); denaturation (95°C, 15 s), annealing (52-63°C, 15 s), extension (72°C, 40 s), for a total of 40 cycles; denaturation (95°C, 60 s); annealing (52-63°C, 30 s); extension (95°C, 30 s). Primer sequences are shown in Table 9 and were synthesized by Shanghai Sangon Biotechnology Co., Ltd.) with 2 -ΔΔCtThe relative mRNA expression of each target gene was determined by one-way analysis of variance. The overall data were analyzed, and the Duncan's multiple comparison method was used to compare the two groups after the difference was significant. The significance was defined as P <0.05.

[0122] Table 9

[0123]

[0124] (2) Experimental results

[0125] The results are as follows Figure 10 As shown, Figure 10 A in the figure is the detection result of jejunum cytokine expression. Figure 10 B in the figure is the detection result of ileal cytokine expression. Figure 10 C in the figure is the detection result of tight junction protein gene expression in jejunum. Figure 10 D in the figure is the detection result of ileum tight junction protein gene expression.

[0126] It can be seen that compared with the CON group, the jejunal inflammatory factors in the PEC group TNF-α The expression of ileal inflammatory factors was significantly downregulated. IL-1β The expression of jejunal anti-inflammatory factors was significantly upregulated; the PEC+XOS group could reverse the changes of the above indicators caused by PEC and IL-10 There is also a certain upregulation effect on the expression of jejunal tight junction proteins in the PEC group compared with the CON group. Occludin The expression of jejunal tight junction protein was downregulated, while the PEC+XOS group could reverse the change of this index caused by PEC. Moreover, compared with the CON group, the PEC+XOS group could significantly upregulate the expression of jejunal tight junction protein. ZO-1 The expression of ZO-1 The expression trend indicated that XOS could regulate PEC-induced intestinal inflammation and reduce intestinal barrier damage, thereby alleviating PEC-induced intestinal damage.

[0127] 5. Expression of cecal virulence genes

[0128] (1) Experimental methods

[0129] Total RNA from cecal digesta was extracted using the PowerFecal Pro kit (QIAGEN, Germany). After its concentration and purity were determined using a NanoDrop 2000 micro-volume UV spectrophotometer, 1 μL of RNA was reverse transcribed using the HiScript II QRT SuperMix reverse transcription kit (Novagen Biotech). The reverse transcription system consisted of 4 μL of 5× HiScript II qRTSuperMix, 2 μL of RNA, and 14 μL of double-distilled water. The reaction conditions were: 25°C for 5 min; 50°C for 15 min; and 85°C for 2 min. The cDNA sample obtained by reverse transcription was placed in a fluorescence quantitative PCR instrument and PCR was performed using reduced glyceraldehyde-phosphate dehydrogenase ( GAPDH ) as the internal reference gene, and PEC virulence gene fimH 、 csgA 、 hycA 、 luxS 、 relA and ompR Real-time fluorescence quantitative PCR was performed for the target gene (the qRT-PCR reaction system was: 2 μL cDNA, 0.6 μL Forword Primer (10 μM), 0.6 μL Reverse Primer (10 μM), 10 μL 2×ChamQ Universal SYBR qPCR Master Mix and 6.8 μL dd H2O. The reaction conditions were: pre-denaturation (95°C, 10 min); denaturation (95°C, 15 s), annealing (52-63°C, 15 s), extension (72°C, 40 s), for a total of 40 cycles; denaturation (95°C, 60 s); annealing (52-63°C, 30 s); extension (95°C, 30 s). The primer sequences are shown in Table 10 and were synthesized by Sangon Biotechnology (Guangzhou)). -ΔΔCt The relative mRNA expression of each target gene was determined by one-way analysis of variance. The overall data were analyzed, and the Duncan's multiple comparison method was used to compare the two groups after the difference was significant. The significance was defined as P <0.05.

[0130] Table 10

[0131]

[0132] (2) Experimental results

[0133] The results are as follows Figure 11As shown in the data, compared with the CON group, the expression levels of PEC virulence genes in the PEC group were significantly increased, while the PEC+XOS group could reverse the changes in indicators caused by PEC, indicating that XOS can inhibit the expression of PEC virulence genes and thereby alleviate PEC-induced intestinal damage.

[0134] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of xylo-oligosaccharide in the preparation of a product for reducing the resistance of pathogenic Escherichia coli to streptomycin, characterized in that: The pathogenic Escherichia coli is enterohemorrhagic Escherichia coli O157 or avian pathogenic Escherichia coli O78.

2. The application according to claim 1, characterized in that The product is a functional feed additive.

3. The application according to claim 2, characterized in that: The functional feed additive further comprises auxiliary materials.

4. The application according to claim 3, characterized in that The auxiliary material is one or more of starch, dextrin, zeolite powder and corn cob powder.

5. The application according to claim 4, characterized in that: The starch is one or more of corn starch, wheat starch and potato starch.

6. The application according to claim 5, characterized in that The dextrin is one or more of corn dextrin, maltodextrin, cyclodextrin and white dextrin.

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