Drug resistance marker of beta-lactam drug for RA and gene detection system

Abstract

The invention discloses a drug resistance marker for beta-lactam drugs for RA and a gene detection system, and relates to the field of gene detection. The drug resistance marker is located on a protein sequence of D type beta-lactamase of riemerella anatipestifer, and comprises the following combination: R2K, L10I, S24K, G36R, K43E, T66A, I70V, T95A, K105R, N108T, R141K, R149K, T155S, T170S and L181F. The drug resistance marker is accurate in prediction, convenient to detect and wide in application.

Description

Technical Field

[0001] This invention relates to the fields of gene detection and disease diagnosis technology, specifically to a drug resistance marker and gene detection system for β-lactam drugs against RA (Riemerella anatipestifer, abbreviated as RA). Background Technology

[0002] Anal rifamycosis (RAI), caused by Riemerella anatipestifer (RA), is an acute, contagious, and septicemic infectious disease characterized by fibrinous pericarditis, perihepatitis, air sacculitis, and meningitis. Clinical symptoms include cough, runny nose, torticollis, head tremors, and motor dysfunction. It primarily affects ducklings aged 1-8 weeks, causing acute and chronic septicemia. Widely distributed globally, it causes high mortality and poor growth in affected ducks, resulting in significant economic losses to the poultry industry. Epidemiological surveys show that approximately 40% of cases occur in ducklings aged 3-4 weeks. The mortality rate is highest in ducklings aged 3-8 weeks, while adult ducks generally present with latent infection or show no obvious symptoms after infection.

[0003] my country has the largest number of slaughtered ducks in the world, accounting for approximately 69% of the global total. Antimicrobial resistance mechanisms related to *Riemerella anatipestifer* are relatively few. Current research mainly focuses on the class D β-lactamase RAD-1, which, in addition to its hydrolytic activity against β-lactam antibiotics, also exhibits some hydrolytic activity against carbapenems. Furthermore, OXA-209 and OXA-347, located on plasmids, have been reported for the first time. However, the function of OXA-209 has not been fully verified; researchers have only conducted resistance studies on it against three β-lactam antibiotics. Whether it possesses resistance to other β-lactam antibiotics and carbapenems remains unknown. In addition, the extended-spectrum β-lactamase RAA-1 has also been discovered in recent years.

[0004] Therefore, there is an urgent need to develop a new resistance marker and gene detection system for β-lactam drugs targeting RA to address the current deficiencies and shortcomings. Summary of the Invention

[0005] In view of this, the main objective of the present invention is to provide a resistance marker and gene detection system for β-lactam drugs against RA, in order to at least partially solve the above-mentioned technical problems.

[0006] To achieve the above objectives, as a first aspect of the present invention, a resistance marker against β-lactam drugs for RA is proposed, located on the protein sequence of a class D β-lactamase of Riemerella anatipestifer, said resistance marker comprising the following protein molecule combination: R2K, L10I, S24K, G36R, K43E, T66A, I70V, T95A, K105R, N108T, R141K, R149K, T155S, T170S and L181F.

[0007] As a second aspect of the invention, a gene detection system for resistance to β-lactam drugs in RA is also proposed, comprising: A gene library containing the resistance markers for β-lactam drugs against RA as described above; Gene sequencing equipment.

[0008] Based on the above technical solution, it can be seen that the drug resistance marker and gene detection system for β-lactam drugs against RA of the present invention has at least one of the following beneficial effects compared with the prior art: (1) The drug resistance markers of the present invention have high prediction accuracy, are easy to detect, and are easy to make into detection kits for detection; (2) The gene detection system of the present invention has good reference value for predicting lesions and prognosis after infection through biomarker detection; (3) The present invention has broad application prospects and can be applied to fields such as risk assessment and drug development. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0010] Figure 1 This is a structural block diagram of the drug resistance gene detection system for β-lactam drugs targeting RA according to the present invention; Figure 2 A photograph showing the overall clinical symptoms of the sick ducks; Figure 3 A partial photograph of the brain lesions observed during a necropsy of a duckling; Figure 4 CFU counts were performed on the spleen, liver, brain, and heart tissues infected with strain 1. Data are expressed as mean ± 1 standard deviation. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0012] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of the invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0013] Many drug resistance modes have been summarized in existing technologies, such as: 1. Lamivudine / telbivudine resistance (LAM / LDT) This is the most basic resistance pattern. The sequence contains rtL180M (L10I) and rtM204V (M204V), which are classic "YIDD"->"YVDD" mutations. Almost all viral strains resistant to lamivudine (LAM) or telbivudine (LDT) must undergo these two mutations.12

[0014] 2. Adefovir dipivoxil resistance (ADV) The sequence contains rtN236T (N236T) and rtA181T (A181T). Among them, rtA181T is one of the main resistance sites to adefovir dipivoxil (ADV). When the virus possesses both L180M and A181T, drug resistance is significantly enhanced, which may lead to cross-resistance to entecavir (ETV).

[0015] 3. Entecavir / Tenofovir resistance (ETV / TDF) The sequence contains rtT184G (T184G), rtS202G (S202G), and rtM250V (M250V). These three sites (T184, S202, and M250) are high-barrier resistance sites for entecavir (ETV). If T184G+S202G+M250V are present simultaneously, the virus will be highly resistant to ETV; if they are only partially present, it may lead to low-level resistance to tenofovir (TDF / TAF).

[0016] Based on the isolated *Riemerella anatipestifer*, the inventors conducted gene detection and drug susceptibility testing. Specifically, through gene detection and examination of the resistance rates of all isolates to antibiotics such as penicillin, ampicillin, amoxicillin, piperacillin, cefoperazone, cefoxitin, ceftazidime, cefotaxime, and ceftazidime, several protein molecule combinations that can serve as markers of drug resistance were identified.

[0017] Specifically, this invention proposes a resistance marker for β-lactam drugs against RA, wherein the resistance marker is located on the protein sequence of class D β-lactamase of Riemerella anatipestifer, and the resistance marker comprises the following protein molecule combination: R2K, L10I, S24K, G36R, K43E, T66A, I70V, T95A, K105R, N108T, R141K, R149K, T155S, T170S and L181F.

[0018] This invention also proposes a gene detection system for resistance to β-lactam drugs in RA, comprising: A gene library containing the aforementioned resistance markers to β-lactam drugs targeting RA; Gene sequencing equipment.

[0019] The gene sequencing equipment employs, for example, the following detection methods: SANGER sequencing (gold standard, directly performing bidirectional sequencing on amplified DNA fragments), NGS (high-throughput sequencing), and qPCR / TAQMAN probe detection system (using allele-specific probes ASO combined with CastPCR technology).

[0020] like Figure 1 As shown, the gene detection system for resistance to β-lactam drugs in RA also includes a data processing module for generating a resistance analysis report based on the detection results from the gene sequencing equipment.

[0021] The data processing module can execute the following control logic: (1) Whether there are corresponding drug resistance markers. If there are, output the corresponding antibiotic resistance prompt and proceed to the next step. If there are no markers, do not perform serum type judgment and output the result directly.

[0022] If serum type is also detected during gene sequencing, the data processing module can further execute the following control logic: (2) Determine the serum type. If the serum type is serum type 2, 6, or 7, the pathogenicity is less than 48 hours after the challenge and the mortality rate is higher than 50%, so the highest risk warning is given. If the serum type is type 1, the pathogenicity is greater than 48 hours after the challenge and the mortality rate is 10% to 20%, so the medium risk warning is given.

[0023] The present invention will be further illustrated below through specific embodiments. It should be noted that the following embodiments are merely illustrative and not intended to limit the present invention.

[0024] Materials and Methods (1) Ethical Statement Based on 120 strains of Riemerella anatipestifer isolated from a region in Anhui Province between 2017 and 2023, the inventors conducted gene testing and drug susceptibility testing on them. All animal infection experiments were carried out in accordance with the "Guidelines for the Management of Laboratory Animals" issued by the Ministry of Science and Technology of China (Beijing) and under the supervision of the Animal Protection and Utilization Committee of Henan Agricultural University.

[0025] (2) Bacterial strains, plasmids and experimental animals The strains 1 and 2 used were isolated from goose brain tissue from a goose farm in Anhui, China. Strain 1 contained the combination of drug resistance markers described in this invention, while strain 2 did not. DH5α competent cells (Qingdao Biotechnology Co., Ltd.), BL21(DE3) cells (Qingdao Biotechnology Co., Ltd.), ATCC11854 (provided by the College of Veterinary Medicine, Yangzhou University), MH broth (Haibo Biotechnology Co., Ltd.), antibiotics (Beijing Solarbio Biotechnology Co., Ltd.), and plasmid pET28a were used in the experiment.

[0026] (3) Antibiotic susceptibility testing Antibiotic susceptibility testing was performed using the microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). Different concentrations of β-lactam antibiotics were prepared through serial dilutions and added to sterile 96-well polystyrene plates. 10 μL of antibiotic solution was added to wells 1 through 11, with well 12 serving as a growth control without antibiotics. Subsequently, a bacterial suspension equivalent to 0.5 McFarland turbidity standard was added, diluted 1:1000 with MH broth, 100 μL per well. The plates were sealed and incubated at 37°C for 20 hours, after which the results were evaluated. Lactobacillus acidophilus ATCC11854 was used for quality control.

[0027] (4) Whole genome sequencing Genomic DNA was extracted using a modified version of the cetyltrimethylammonium bromide (CTAB) method. DNA concentration, quality, and integrity were then determined using a Qubit fluorometer (Ingenieur, Inc.) and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Sequencing libraries were constructed using the TruSeq DNA Sample Preparation Kit and Template Preparation Kit (Pacific Biosciences, Inc.).

[0028] After removing adapter contamination, data was filtered using Adapter Removal and SOAPec. Filtered sequencing reads were assembled using SPAdes and A5-miseq to construct scaffolds and contigs. Data obtained from sequencing on the Nanopore platform was assembled using Flye and Unicycler software. All assembly results were then integrated to generate the complete sequence. Finally, the genome sequence was obtained after correction using Pilon software.

[0029] The R. anatipestifer genome was functionally annotated using RAST (https: / / rast.nmpdr.org / ). Antibiotic resistance genes (ARGs) in the bacterial genome were identified using tools such as NCBI, AMRfinderPlus, MEGARes, and ABRicate.

[0030] (5) Infection with R. anatipestifer All animal experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee. Twenty-day-old female Cherry Valley ducks, purchased from Anhui Qiangying Food Co., Ltd., were used in the experiments. The female ducks were randomly divided into three groups. The bacterial strain was cultured to the logarithmic growth phase, washed with physiological saline, and the bacterial suspension was concentrated to 5 × 10⁹ CFU / mL. Each female duck was inoculated with 0.2 mL of the bacterial suspension (1 × 10⁹ CFU). Twenty-four hours after infection with *R. anatipestifer*, ceftiofur was administered subcutaneously at a dose of 1 mg / kg. Two days later, all surviving female ducks (control and experimental groups) were deeply anesthetized, euthanized by exsanguination via the carotid artery, and dissected. Liver, spleen, brain, and heart tissues were collected. Each organ portion was aseptically transferred to 1 mL of sterile physiological saline. After tissue homogenization, the CFU of bacteria per gram of tissue was calculated.

[0031] Example 1 Sixty female ducks were randomly divided into three groups of 20 each: strain 2 (control group), strain 1 (treatment group), and saline inoculation group (NC group).

[0032] Bacterial cultures of strain 1 were cultured to the logarithmic growth phase and then infected with *R. anatipestifer* using the method described above. Liver, spleen, brain, and heart tissues from the infected organisms were collected and aseptically transferred to 1 mL of sterile physiological saline. After tissue homogenization, the CFU of bacteria per gram of tissue was calculated.

[0033] like Figure 2-4As shown, the results indicate that, compared with reference strain 2, strain 1 exhibited a significant increase in drug resistance, with resistance to penicillin, ampicillin, amoxicillin, piperacillin, cefoperazone, cefoxitin, ceftazidime, cefotaxime, and ceftazidime increasing by more than 16 times. Furthermore, strains with the aforementioned drug resistance markers showed stronger resistance to the three third-generation cephalosporins: cefotaxime, ceftazidime, and ceftiofur sodium.

[0034] Example 2 Another 60 female ducks were randomly divided into three groups of 20 each: strain 2 (control group), strain 1 (treatment group), and saline inoculation group (NC group), and the above experiment was repeated.

[0035] The results once again demonstrated that, compared with reference strain 2, strain 1 showed a significant increase in drug resistance, and strains with the above-mentioned drug resistance markers had stronger resistance to the three third-generation cephalosporins: cefotaxime, ceftazidime, and ceftiofur sodium.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A marker for resistance to β-lactam drugs against rheumatoid arthritis (RA), characterized in that, The drug resistance markers are located on the protein sequences of D-type β-lactamases of Riemerella anatipestifer, and the drug resistance markers include the following protein molecule combinations: R2K, L10I, S24K, G36R, K43E, T66A, I70V, T95A, K105R, N108T, R141K, R149K, T155S, T170S and L181F.

2. A gene detection system for β-lactam drug resistance in rheumatoid arthritis (RA), characterized in that, include: A gene library containing the resistance markers for β-lactam drugs against RA as described in claim 1; Gene sequencing equipment.

3. The gene detection system according to claim 2, characterized in that, The gene sequencing equipment employs the following detection methods: SANGER sequencing, NGS (high-throughput sequencing), or QPCR / TAQMAN probe method.

4. The gene detection system according to claim 2, characterized in that, The gene detection system also includes a data processing module for generating a drug resistance analysis report based on the detection results from the gene sequencing equipment.

5. The gene detection system according to claim 4, characterized in that, The data processing module executes the following control logic: (1) Whether there are corresponding drug resistance markers. If there are, output the corresponding antibiotic resistance prompt and proceed to the next step. If there are no markers, do not perform serum type judgment and output the result directly.

6. The gene detection system according to claim 5, characterized in that, If serum type is also detected during gene sequencing, the data processing module further executes the following control logic: (2) Determine the serum type. If the serum type is serum type 2, 6, or 7, the pathogenicity is less than 48 hours after the challenge and the mortality rate is higher than 50%, so the highest risk warning is given. If the serum type is type 1, the pathogenicity is greater than 48 hours after the challenge and the mortality rate is 10% to 20%, so the medium risk warning is given.

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