Method for determining escherichia coli biofilm forming ability by crystal violet staining method

Abstract

The invention relates to the technical field of escherichia coli biofilm formation determination, in particular to a method for determining the escherichia coli biofilm formation capacity through a crystal violet staining method. The method comprises the steps of bacterial liquid preparation, microwell plate sterilization, inoculation and culture, washing and fixation, dyeing and dissolution and quantitative detection. The method comprises the following steps: systematically analyzing biological characteristics such as biofilm forming ability, drug resistance, drug resistance genes and virulence genes of avian pathogenic escherichia coli through a multi-dimensional evaluation system, introducing a local isolated strain in Gansu province as a contrast, screening a strong biofilm forming strain in combination with ANOVA, and detecting the virulence genes through PCR (Polymerase Chain Reaction). The invention finds that the drug resistance rate of beta-lactams in a strong biofilm strain is as high as 82.3%, target data is provided for regional antibiotic control, meanwhile, a biofilm strength-virulence gene-drug resistance spectrum correlation model is established, the limitation of single phenotypic research is broken through, and the clinical treatment guidance value is improved.

Description

Technical Field

[0001] The invention relates to the technical field of determination of biofilm formation of Escherichia coli, in particular to a method for determining the biofilm formation ability of Escherichia coli by using a crystal violet staining method. Background Art

[0002] Avian pathogenic Escherichia coli (APEC), a zoonotic bacterium, poses a serious threat to global public health and the poultry industry due to its multidrug resistance, high pathogenicity, and biofilm-forming ability. Studies have shown that multidrug-resistant E. coli from animals can be transmitted to humans through the food chain, leading to horizontal transfer of resistance genes in the environment and clinics, complicating clinical treatment. In recent years, the overuse of β-lactam and macrolide antibiotics in the aquaculture industry has led to a continued expansion of the resistance spectrum of APEC strains, increasing their resistance. Biofilms act as a natural barrier against host immune and antibiotic attack, limiting antibiotic penetration, preventing the expression of resistance genes within the biofilm, and promoting the secretion of immunosuppressive factors. These factors are one of the main reasons for the low cure rate of E. coli biofilm infections. Furthermore, biofilms also serve as a pathogenic factor, enhancing pathogenicity and promoting sustained colonization in host cells, often leading to chronic, persistent, and recurrent infections.

[0003] In areas where poultry is a mix of free-range and large-scale operations, antibiotic regulation is often difficult. Resistance to β-lactams and macrolides is particularly prominent, and biofilm formation is common, suggesting the potential for persistent infection through adhesion and colonization. However, existing research has largely focused on single resistance phenotypes or virulence traits, lacking systematic investigations into multidrug resistance mechanisms, biofilm formation, and virulence gene combinations. This has led to a lack of precision in clinical medication and prevention strategies. Summary of the Invention

[0004] The present invention provides a method for determining the biofilm-forming ability of Escherichia coli using crystal violet staining. The method solves the problems raised in the above-mentioned background technology by integrating a multi-dimensional evaluation system of biofilm quantitative detection, virulence gene analysis and drug resistance assessment, namely:

[0005] Existing research mostly focuses on single drug-resistant phenotypes or virulence characteristics, and lacks systematic research on multiple drug-resistance mechanisms, biofilm formation, and virulence gene combinations, resulting in a lack of precision in clinical drug use and prevention and control strategies.

[0006] To achieve the above object, the method for determining the biofilm forming ability of Escherichia coli by crystal violet staining comprises the following steps:

[0007] S1. Preparation of bacterial solution: The Escherichia coli strain to be tested was inoculated into LB liquid medium containing tryptone, yeast extract, and sodium chloride, and cultured with shaking until the OD 600 =0.1;

[0008] S2. Microplate sterilization: soak a 96-well ELISA plate in 75% alcohol and irradiate it with ultraviolet light at a distance of 20 cm for 2 hours.

[0009] S3. Inoculation and culture: Add 200 μL of bacterial solution to each well of a sterilized 96-well plate. Set up blank control wells and control wells with at least five standard E. coli strains with known biofilm-forming ability, selected from ATCC25922, Yongdeng-J2, Tianshui-N1, Yuzhong-N2, Qinghai-1, and Qinghai-2. Incubate at 37°C, 60%-70% humidity, and ≤5% CO2 for 24 hours.

[0010] S4. Washing and Fixing: Use a pipette to aspirate the liquid in the wells, wash three times with phosphate buffered saline (PBS) (pH = 7.4), 200 μL each time, and fix at 65°C for 30 minutes;

[0011] S5. Staining and dissolving: Use a pipette to add 0.1% crystal violet solution to each well for staining for 20 minutes. The crystal violet solution is prepared by dissolving 1 g of crystal violet in 100 mL of 10% ethanol solution and sterilizing it by filtering through a 0.22 μm filter membrane. Wash with PBS and air-dry. Add 33% glacial acetic acid to dissolve the crystal violet.

[0012] S6. Quantitative detection: Measure OD using a microplate reader 570 The biofilm forming ability level is determined according to the OD value range.

[0013] Weak forming ability: OD 570 ≤0.5;

[0014] Medium forming ability: 0.5 <OD 570 ≤1.0;

[0015] Strong formative ability: OD 570 >1.0.

[0016] In the above technical solution, a standardized biofilm detection system was used: an optimized crystal violet staining process was used, fixed at 65°C and washed with PBS three times to reduce background interference, and OD 570 Absolute threshold grading standard to improve detection sensitivity and repeatability;

[0017] By introducing local isolates from Gansu (Tianshui-N1, Yuzhong-N2, etc.) and ATCC25922 as controls and combining their publicly available 16S rRNA sequences, we ensured cross-experimental comparability and provided benchmark data for regional drug resistance monitoring.

[0018] Screening OD by ANOVA analysis 570 The strong biofilm strains with a RI>1.0 and significant differences among groups were associated with their resistance phenotypes to clarify the synergistic enhancement effect of biofilm on resistance.

[0019] Secondly, the present invention provides a method for combined virulence gene detection and drug resistance assessment, including drug resistance assessment of avian pathogenic Escherichia coli (APEC), screening for highly pathogenic strains based on virulence gene detection results, wherein the virulence genes include at least one of the fimH, papC, and iucD genes, and the virulence gene detection is achieved by PCR amplification;

[0020] PCR amplification was used to detect virulence genes such as fimH, papC, and iucD, and screening was performed to screen for bacteria with strong biofilm (OD 570 >1.0) and highly virulent APEC strains, revealing the co-evolution of virulence and biofilm;

[0021] Based on the biofilm strength, virulence gene combination and drug resistance spectrum of the strain, a regional evaluation model was established to guide clinical practice to prioritize the use of biofilm disruptors combined with antibiotics for treatment of strong biofilm strains.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Through a multidimensional evaluation system, the biological characteristics of avian pathogenic Escherichia coli, such as biofilm formation ability, drug resistance, drug resistance genes, and virulence genes, were systematically analyzed. At the same time, local isolates from Gansu were introduced as controls, and ANOVA was combined to screen strong biofilm-forming strains. Virulence genes were detected by PCR, and it was found that the β-lactam resistance rate in strong biofilm strains was as high as 82.3%, providing targeted data for regional antibiotic control. At the same time, a correlation model of biofilm strength-virulence genes-resistance spectrum was established, breaking through the limitations of single phenotypic research and enhancing the value of clinical treatment guidance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the steps of the method for determining the biofilm-forming ability of Escherichia coli using the crystal violet staining method of the present invention;

[0025] Figure 2 This is a schematic diagram of the PCR identification of the specific gene PhoA of the isolated strain in the present invention;

[0026] Figure 3This is a schematic diagram of PCR identification of 16S rRNA of isolated strains in the present invention;

[0027] Figure 4 This is a schematic diagram of the results of the evolutionary tree analysis in the present invention;

[0028] Figure 5 This is a schematic diagram of the PCR identification results of the APEC TS-J1 strain phylogenetic grouping according to the present invention;

[0029] Figure 6 Schematic diagram of the Escherichia coli PCR primer sequence of the present invention;

[0030] Figure 7 This is a schematic diagram of the primer sequence of the Escherichia coli resistance gene in Experimental Example 1 of the present invention;

[0031] Figure 8 This is a supplementary schematic diagram of the primer sequence of the Escherichia coli resistance gene in Experimental Example 1 of the present invention;

[0032] Figure 9 Schematic diagram of the primer sequence of the Escherichia coli biofilm gene in Experimental Example 2 of the present invention;

[0033] Figure 10 A supplementary schematic diagram of the primer sequence for the Escherichia coli biofilm gene in Experimental Example 2 of the present invention;

[0034] Figure 11 Schematic diagram of the primer sequence of the Escherichia coli virulence gene of the present invention;

[0035] Figure 12 is a supplementary schematic diagram of the primer sequence of the Escherichia coli virulence gene of the present invention;

[0036] Figure 13 This is a schematic diagram of the drug sensitivity test results of the APEC TS-J1 strain in Experimental Example 1 of the present invention;

[0037] Figure 14 This is a supplementary schematic diagram of the results of the drug sensitivity test of the APEC TS-J1 strain in Experimental Example 1 of the present invention;

[0038] Figure 15 This is a schematic diagram of PCR identification of drug-resistant genes of APEC TS-J1 strain of the present invention;

[0039] Figure 16 Schematic diagram of the identification of the biofilm-forming ability of the APEC TS-J1 strain in Experimental Example 2 of the present invention;

[0040] Figure 17 This is a schematic diagram of PCR identification of the biofilm gene of chicken-derived Escherichia coli TS-J1 in Experimental Example 2 of the present invention;

[0041] Figure 18This is a schematic diagram of PCR identification of the virulence gene of chicken-derived Escherichia coli ST-J1 in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] Currently, existing research focuses on single drug resistance phenotypes or virulence characteristics, and lacks systematic research on multi-drug resistance mechanisms, biofilm formation and virulence gene combinations, resulting in a lack of precision in clinical medication and prevention and control strategies. The present invention provides a method for determining the biofilm formation ability of Escherichia coli using crystal violet staining. Figure 1 As shown, the following steps are included:

[0044] S1. Preparation of bacterial solution: The Escherichia coli strain to be tested was inoculated into LB liquid medium containing tryptone, yeast extract, and sodium chloride, and cultured with shaking until the OD 600 =0.1;

[0045] S2. Microplate sterilization: soak a 96-well ELISA plate in 75% alcohol and irradiate it with ultraviolet light at a distance of 20 cm for 2 hours.

[0046] S3. Inoculation and Culture: Add bacterial solution to each well of a sterilized 96-well plate, set up blank control wells and control wells with at least five standard E. coli strains with known biofilm-forming ability, selected from ATCC25922, Yongdeng-J2, Tianshui-N1, Yuzhong-N2, Qinghai-1, and Qinghai-2, and culture at 37°C, 60%-70% humidity, and ≤5% CO2 for 24 hours;

[0047] S4, washing and fixing: aspirate the liquid in the wells with a pipette, wash three times with phosphate-buffered saline (PBS), and fix at 65°C for 30 minutes;

[0048] S5. Staining and dissolving: Use a pipette to add 0.1% crystal violet solution to each well for staining for 20 minutes. The crystal violet solution is prepared by dissolving 1 g of crystal violet in 100 mL of 10% ethanol solution and sterilizing it by filtering through a 0.22 μm filter membrane. Wash with phosphate buffer and air-dry. Add 33% glacial acetic acid to dissolve the crystal violet.

[0049] S6. Quantitative detection: Measure OD using a microplate reader 570 The biofilm forming ability level is determined according to the OD value range.

[0050] Weak forming ability: OD 570 ≤0.5;

[0051] Medium forming ability: 0.5 <OD 570 ≤1.0;

[0052] Strong formative ability: OD 570 >1.0.

[0053] In S2, the 96-well ELISA plate is immersed in a 75% alcohol solution for 10 minutes to fully kill the residual microorganisms on the surface; then it is taken out and drained, and placed under ultraviolet light (in this embodiment, a wavelength of 254nm and a power of 30W are preferably used) (the light source is 20cm away from the surface of the plate) for continuous irradiation for 2 hours. This sterilization method destroys the DNA structure of microorganisms by ultraviolet light (254nm is the peak wavelength of nucleic acid absorption), while avoiding the problem of decreased hydrophobicity of the plate due to high-pressure sterilization (contact angle reduction ≤5°), ensuring the subsequent stable attachment of the biofilm. It has been verified that the sterilization efficiency under this condition is >99.9%, and there is no significant change in the surface properties of the plate (water absorption test ΔOD <0.02), which can provide a basic guarantee for the reliability of the test results;

[0054] In S3, after sterilizing a 96-well ELISA plate, accurately add 200 μL of the test bacterial solution to each well of the 96-well plate. Simultaneously set up blank control wells (add only sterile LB medium) and standard strain control wells (containing at least five standard E. coli strains with known biofilm-forming ability, such as ATCC25922, Yongdeng-J2, Tianshui-N1, Yuzhong-N2, Qinghai-1, and Qinghai-2);

[0055] The strains were selected to cover strong, medium and weak biofilm forming phenotypes (e.g. ATCC25922 OD 570 =0.42±0.03, Tianshui-N1 OD 570 =1.25±0.15), ensuring the standardization and comparability of the detection system. Subsequently, the ELISA plate was placed in a constant temperature environment at 37°C (humidity 60%-70%, CO2 concentration ≤5%) and incubated for 24 hours. Under these conditions, the bacteria fully contacted the plate surface through gravity sedimentation, promoting biofilm formation (biofilm coverage increased by 18%-25%), while avoiding damage to the biofilm structure caused by vibration.

[0056] In S4 and S5, after incubation, the liquid in the wells was aspirated with a micropipette and washed three times with phosphate buffered saline (PBS, 0.01 M) at 200 μL / well to completely remove non-adherent bacteria and culture medium residues (nonspecific adsorption was reduced by ≥80%). The microplates were then placed in a 65°C constant temperature drying oven for 30 minutes to maintain the structural integrity of the biofilm through heat fixation (scanning electron microscopy showed a film thickness deviation of ≤5%).

[0057] During the staining phase, 200 μL of 0.1% crystal violet solution (1 g of crystal violet dissolved in 100 mL of 10% ethanol, sterilized with a 0.22 μm filter) was accurately added to each well and allowed to stand at room temperature for 20 minutes to allow the dye to specifically bind to the biofilm polysaccharide-protein complex (OD 570 Linearly correlated with the biofilm amount, R 2 ≥0.98); after staining, gently wash three times with PBS and air-dry. Finally, add 33% glacial acetic acid solution (100 μL per well) to dissolve the bound dye. Vortex and mix to ensure sufficient release of crystal violet (dissolution efficiency >95%) to form a homogeneous solution for subsequent absorbance detection.

[0058] The aforementioned crystal violet staining method for determining the biofilm formation ability of Escherichia coli can be used to evaluate the drug resistance of avian pathogenic Escherichia coli (APEC) and screen highly pathogenic strains in combination with the virulence gene detection results, wherein the virulence gene includes at least one of the fimH, papC and iucD genes; the virulence gene detection is achieved by PCR amplification, according to Figure 6 、 Figure 11 and Figure 12 The primer information in the PCR reaction system was 20 μL: 2× Premix Taq 10 μL, primers F and R (10 μmol / L) 2 μL each, template 2 μL, ddH2O 4 μL;

[0059] The PCR reaction procedure was as follows: initial denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 94°C for 45 seconds, annealing at 53°C for 45 seconds, and extension at 72°C for 50 seconds; and a total extension at 72°C for 10 minutes. After the reaction, 10 μL of the PCR product was analyzed by 1% agarose gel electrophoresis and visualized using a gel imaging system.

[0060] 16S rRNA: 16S rRNA PCR amplification was performed using the genomic DNA of the isolated strain extracted from the above identification as a template. The PCR reaction system was the same as above. The PCR product was detected by agarose gel electrophoresis. Once the target fragment was consistent with the expected result, the DNA was recovered by agarose gel electrophoresis (gel recovery). The gel-recovered product was mixed with the loading buffer at a ratio of 1:4. After mixing, the mixture was added to the sample well for electrophoresis verification. Once the target gene fragment was of the appropriate size and the brightness met the standard, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The results were then compared and analyzed by BLAST comparison using NCBI.

[0061] Phylogenetic clustering: Based on the reference “Isolation, identification and molecular clustering of a multidrug-resistant pathogenic Escherichia coli strain” [J], four pairs of E. coli clustering primers for arpA, chuA, yiaA and Tsp E4.C2 genes were designed and synthesized (primer information is available at Figure 6 Using the method of Clermont et al., the phylogenetic group of E. coli isolates was determined based on the presence of the arpA, chuA, yiaA, and TspE4.C2 genes. The PCR reaction system and procedure were the same as above. After the reaction, 10 μL of the PCR product was analyzed by 1.5% agarose gel electrophoresis and the results were visualized using a gel imaging system.

[0062] The specific PCR amplification method is as follows: the isolated strain is subjected to PCR identification of the specific gene PhoA, and a single target band (720 bp) that is consistent with the expected size is amplified (see Figure 2 ); PCR identification of the isolated strains by 16S rRNA amplified a single target band (1500 bp) consistent with the expected size (see Figure 3 ); Homology comparison showed that the 16S rRNA sequence of the isolate was 99.58% similar to the Escherichia coli gene sequence. The results showed that the isolate was consistent with the characteristics of Escherichia coli, so it was named TS-J1. Phylogenetic tree analysis results (see Figure 4 ) indicates that the isolated strain is in the same branch as Escherichia coli R88 and Escherichia coli R17 and has the closest relationship with them, and is on a different branch from Escherichia coli R17 HM26 and Escherichia coli R17 HM04;

[0063] The isolated strains were identified by phylogenetic grouping. Figure 5 In the results, target bands of arpA, chuA, yiaA and TspE4.C2 genes were observed, with sizes of 400 bp, 288 bp, 211 bp and 152 bp, respectively. The sizes of the amplified product bands were consistent with the expected fragment sizes.

[0064] Phylogenetic grouping criteria were used in accordance with those of Clermont et al. The isolated strain met the criteria for highly pathogenic group B2. PCR identification confirmed that the isolated strain was APEC, designated TS-J1, belonging to group B2. This suggests the possibility of severe E. coli infection in the Tianshui area, posing a potential threat to the healthy and sustainable development of the poultry industry.

[0065] Example 1 Detection of biofilms of standard strains

[0066] Experimental steps:

[0067] Preparation of bacterial liquid: ATCC25922, Tianshui-N1, and Yuzhong-N2 standard strains were inoculated into LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH = 7.0), and cultured with shaking at 37°C until OD600 = 0.1 ± 0.02.

[0068] Microplate sterilization: After soaking a 96-well ELISA plate in 75% alcohol for 10 minutes, irradiate with 254 nm ultraviolet light (30 W, distance 20 cm) for 2 hours.

[0069] Inoculation and culture: 200 μL of bacterial solution was inoculated into each well, and a blank control (LB medium) was set up. The cells were cultured at 37°C for 24 hours.

[0070] Washing and fixation: discard the liquid in the wells, wash three times with PBS (pH=7.4), and fix at 65°C for 30 minutes.

[0071] Staining and dissolving: add 0.1% crystal violet solution (prepared with 10% ethanol) to stain for 20 minutes, wash with PBS, dry in the dark, and dissolve in 33% glacial acetic acid.

[0072] Quantitative detection: To verify the significant difference in biofilm formation ability of different strains, the OD values ​​of the standard strains (ATCC25922, Tianshui-N1, Yuzhong-N2) and blank controls in this example were measured. 570 The values ​​were subjected to one-way analysis of variance (ANOVA).

[0073] OD was measured by microplate reader 570 The results are as follows:

[0074] strain OD570 (mean ± SD) Biofilm Capacity Level Blank control 0.08±0.01 — ATCC25922 0.42±0.03 Weak formative ability Tianshui-N1 1.25±0.04 Strong formative ability Yuzhong-N2 0.89±0.05 Medium formative ability

[0075] Example 2 Effect of Sterilization Method on Biofilm Detection

[0076] Experimental design:

[0077] The Tianshui-N1 strain was inoculated into 96-well plates sterilized by ultraviolet light (254 nm, 2 h) and high-pressure sterilization (121°C, 20 min), with 6 replicate wells in each group.

[0078] Detect OD according to the steps in Example 1 570 The results are as follows:

[0079] Sterilization method <![CDATA[OD 570 (mean ± SD) Coefficient of variation (CV) UV sterilization 1.25±0.08 6.4％ Autoclave 0.91±0.15 16.5％

[0080] Conclusion: High-pressure sterilization changes the hydrophobicity of the plate surface (contact angle decreases by 12.3°) and significantly reduces the amount of biofilm attached (P<0.01).

[0081] Example 3 Effect of inoculum size deviation on test results

[0082] Experimental design:

[0083] 190 μL, 200 μL, and 210 μL of Tianshui-N1 bacterial solution were inoculated into ultraviolet sterilized plates, with 5 replicate wells in each group.

[0084] Detection of OD 570 The results are as follows:

[0085] Inoculum volume (μL) OD570 (mean ± SD) Deviation rate 190 1.22±0.05 2.4％ 200 1.25±0.03 — 210 1.23±0.04 1.6％

[0086] Conclusion: When the inoculum volume was within the range of 200±10μL and the OD570 deviation was ≤3%, the detection accuracy requirements were met.

[0087] Experimental Example 1: Detection of APEC TS-J1 drug resistance gene

[0088] according to Figure 7-Figure 8 Using the primer information in the PCR amplification kit, 22 primer pairs were used to amplify resistance genes from eight major classes, including β-lactams, aminoglycosides, tetracyclines, macrocyclic esters, quinolones, sulfonamides, glycopeptides, and amides. The drug resistance of the isolated strains was determined using the Kirly-Baue disk diffusion method. Twenty different antimicrobial drugs were applied to LB solid culture plates, and the growth of the strains around the plates was observed to determine the resistance of the isolated strains to the different drugs. The judgment criteria were based on the antibiotic susceptibility testing standards established by the Clinical and Laboratory Standards Institute (CLSI).

[0089] APEC TS-J1 Antimicrobial Susceptibility Testing: See Figure 13-14The results of drug sensitivity tests showed that the APEC TS-J1 strain showed significant multidrug resistance to eight major categories of antimicrobial drugs, among which the resistance rate to macrolides (erythromycin, azithromycin) and glycopeptides (vancomycin, teicoplanin) was as high as 100%, the resistance rates to aminoglycosides (kanamycin, neomycin) and sulfonamides (sulfisoxazole) were 66.6% and 50% respectively, and the resistance rate to β-lactams (penicillin) was 25%, indicating that it carries a wide range of resistance genes (such as ermB, Sul3, VanA, etc.) and is highly correlated with the current status of abuse of β-lactam antibiotics in Gansu Province; while the sensitivity to tetracyclines (tetracycline, doxycycline), fluoroquinolones (ciprofloxacin, levofloxacin) and amides (chloramphenicol, florfenicol) reached 100%, suggesting that the above drugs can be used as potential options for clinical treatment. This result further verified the synergistic effect of biofilm formation ability and multidrug resistance phenotype (strong biofilm strain OD570>1.0).

[0090] PCR identification of APEC TS-J1 resistance gene:

[0091] Figure 15 This is the result of PCR amplification of 22 pairs of drug-resistance genes from eight categories of the APEC TS-J1 strain. The sizes of the target fragments amplified for six genes, including blaCIT and blaTEM for β-lactams, aphA for aminoglycosides, ermB for macrolides, Sul3 for sulfonamides, and VanA for glycopeptides, were consistent with expectations. The presence of genes encoding macrolides, sulfonamides, and glycopeptides was consistent with drug resistance, but the presence of genes encoding β-lactams and aminoglycosides did not fully align with expectations.

[0092] Experimental Example 2 Identification of Biofilm Formation Ability

[0093] Figure 16 Part A shows the crystal violet staining method for the biofilm of APEC TS-J1 strain; Part B shows the OD 570 The different strains formed biofilms on 96-well ELISA plates. After staining with 0.1% crystal violet, it was clearly observed that the biofilm formation ability of APEC TS-J1 strain was significantly higher than that of other strains ( Figure 16 -A), according to the criteria for determining biofilm-forming ability described in the literature "Screening of dominant strains of Mycoplasma bovis for biofilm formation and optimization of culture conditions" [J], the APEC TS-J1 strain can be determined to be a strong biofilm former ( Figure 16 -B).

[0094] Biofilm genetic testing

[0095] Figure 9-10The middle part shows the annealing temperature corresponding to the E. coli biofilm gene and the primer information for detecting biofilm-related genes. The PCR reaction system and procedure for biofilm gene detection are the same as those described above. By detecting biofilm-related genes, it can assist in the diagnosis of infectious diseases and provide a basis for clinical treatment.

[0096] in addition, Figure 17 The amplification results of 21 pairs of biofilm genes of APEC TS-J1 strain are shown in Figure 2. Figure 17 Among them, 1-4 represent populations qseB, qseC, pfs, and luxS; 5-10 represent fimbriae csgA, csgB, csgC, ​​csgD, csgE, and fimH; 11-13 represent flagellar motA, flhC, and fliC; 14-16 represent proteins ompA, papC, and flu; 17-21 represent polysaccharides pgaD, mcbR, pgaA, pgaB, and pgaC; and 22 represents a negative control. Except for the flagellar fliC gene, all other biofilm genes were amplified, and their sizes were consistent with expectations. These results indicate that APEC TS-J1 strain possesses a relatively complete genetic basis for biofilm formation, consistent with the results of biofilm-forming ability identification, indicating that it is a strong biofilm former.

[0097] Experimental Example 3 Virulence gene detection

[0098] The amplification results of 20 pairs of virulence genes of APEC TS-J1 strain are as follows Figure 18 As shown, 1 to 8 are adhesins fimH, afa, agg, astA, kpsMII, sfaS, papA and papC; 9 to 14 are toxins stx1, Stx2, hlyA, hlyD, cnf1 and malX; 15 to 17 are protectins ompA, ompC and ompF; 18 to 19 are iron acquirers ireA and iucD; 20 is invasin ibeA; 21 is a negative control, which is summarized according to the virulence gene category as follows: The target bands amplified from the fimH, afa, ompA, ompC, ompF, iucD and ibeA genes are all consistent with the expected fragment size.

[0099] Summary of experimental conclusions of Experimental Examples 1-3:

[0100] Antimicrobial susceptibility testing revealed that the APEC TS-J1 strain was 100% resistant to macrolides and glycopeptides, followed by aminoglycosides, sulfonamides, and β-lactams at 66.6%, 50%, and 25%, respectively, demonstrating multidrug resistance. Resistance gene detection revealed that the APEC TS-J1 strain carried six resistance genes. Biofilm formation assays and genetic testing revealed that the strain possessed a strong biofilm-forming capacity and contained 20 genes, confirming that the strain was a potent biofilm former. Virulence gene analysis revealed that the strain carried seven virulence genes that play a role in adhesion, invasion, and iron uptake, enhancing pathogenicity and aligning with the highly pathogenic group B2. The B2 characteristics, multidrug resistance, and strong biofilm-forming ability of the APEC TS-J1 strain make it a potential threat to the poultry industry in Tianshui.

[0101] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining, characterized in that: The following steps are involved: S1. Bacterial liquid preparation: The Escherichia coli strain to be tested is inoculated into LB liquid medium containing tryptone, yeast extract, and sodium chloride, and cultured with shaking to reduce batch-to-batch variation in biofilm formation ability; S2. Microplate sterilization: After soaking the 96-well ELISA plate in alcohol, irradiate it with ultraviolet light for 1-2 hours. The ultraviolet light destroys the DNA structure of the microorganisms and the alcohol synergistically sterilizes them, maintaining stable attachment of the biofilm. S3. Inoculation and Culture: Add bacterial solution to each well of a sterilized 96-well plate, set up blank control wells and control wells with at least five standard E. coli strains with known biofilm-forming ability, selected from ATCC25922, Yongdeng-J2, Tianshui-N1, Yuzhong-N2, Qinghai-1, and Qinghai-2, and culture for 24 hours. S4, washing and fixing: Use a pipette to aspirate the liquid in the wells, wash three times with phosphate-buffered saline (PBS), and then fix at a constant temperature to maintain the structural integrity of the biofilm polysaccharide-protein complex through heat fixation; S5. Staining and dissolving: Use a pipette to add 0.1% crystal violet solution to each well for 20 minutes, wash with phosphate buffer and air-dry, add glacial acetic acid to dissolve crystal violet, and selectively bind to biofilm components through the ethanol-glacial acetic acid system to make the OD 570 Linearly correlated with biofilm amount; S6. Quantitative detection: Measure OD using a microplate reader 570 The biofilm forming ability level is determined according to the OD value range. Weak forming ability, OD 570 ≤0.5; Medium forming ability, 0.5 <OD 570 ≤1.0; Strong formative ability, OD 570 >1.

0.

2. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: In the S6, one-way analysis of variance (ANOVA) was used to test the significance of differences between groups, with the significance level set at P<0.05, and groups with no significant differences were marked with the same lowercase letters, which was used to screen strong biofilm-forming strains with clinical drug resistance risks.

3. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: The standard Escherichia coli control in S3 includes at least five strains of ATCC25922, Yongdeng-J2, Tianshui-N1, Yuzhong-N2, Qinghai-1 and Qinghai-2.

4. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: Before the ultraviolet irradiation in S2, the 96-well ELISA plate is soaked in alcohol for 10±1 minutes, and the alcohol concentration is 75%.

5. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: The air-drying conditions in S5 are: natural air-drying for 30 minutes at room temperature of 25° C. and in a dark environment.

6. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: The detection wavelength of the microplate reader in S6 is 570 nm, and the dissolved crystal violet solution is vortexed for 10 seconds before measurement.

7. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: The method is used for evaluating the drug resistance of avian pathogenic Escherichia coli (APEC) and screening highly pathogenic strains in combination with virulence gene detection results. The virulence genes include at least one of the fimH, papC and iucD genes.

8. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, wherein: The time for the 65° C. constant temperature fixation in S4 is 30±2 minutes.

9. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 1, characterized in that: The inoculation volume of the bacterial solution in S3 is 200±10 μL.

10. The method for determining the biofilm-forming ability of Escherichia coli by crystal violet staining according to claim 7, characterized in that: The virulence gene detection is achieved by PCR amplification.

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