CN111925959B - Multi-drug-resistant staphylococcus dolphin and application thereof - Google Patents
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Abstract
The invention discloses a multi-drug resistant staphylococcus dolphin and application thereof. Staphylococcus delphini 245-1, which was deposited at the Guangdong province microbial culture collection center (GDMCC) at 6/4/2020, with the deposit address of Guangdong province microbial research institute of No. 59, 5, Guangdong province, Miehuo No. 100, Guangzhou, and the deposit number is GDMCC NO: 61071. compared with the prior art, the invention has the beneficial effects that: the invention provides a multi-drug resistant staphylococcus dolphin carrying a multi-drug resistant gene cfr and application thereof. The strain is resistant to 6 antibiotics, particularly to linezolid, can be used as a model material for screening novel functional microorganisms/medicaments/antibacterial materials, and has a good application prospect.
Description
The technical field is as follows:
the invention belongs to the technical field of biomedicine, and particularly relates to staphylococcus dolphin carrying cfr multi-drug resistance genes and application thereof.
Background art:
staphylococci (Staphylococcus spp.) are among the most common pathogens in humans and animals. Almost all animals are susceptible to staphylococci, with intermediate staphylococci being a typical representative, often causing skin infections in companion and equine animals, and occasionally humans. In recent years, Staphylococcus intermedia comprise at least 3 different species, including Staphylococcus intermedia, Staphylococcus pseudointermedia, and Staphylococcus dolphin, according to the phenotypic identification classification, and these 3 bacteria together constitute the Staphylococcus intermedia group (Staphylococcus intermedia group). Among them, staphylococcus dolphin was first isolated from skin suppurative foci of dolphin in 1988, and it was confirmed by nucleic acid hybridization experiments that it is different from other coagulase-positive bacteria. Since the first discovery of Staphylococcus dolphin, there are Staphylococcus dolphin from different countries and different species that were isolated and identified one after another. The bacterium is separated from animals such as pigeons, horses, donkeys, camels, badgers, ferrets and the like, and is also separated and identified from nasal secretion of chronic nasosinusitis patients in rural areas in China.
With the use of antibiotics in large quantities, the problem of bacterial resistance is becoming more serious and a serious challenge facing the global public health system. By the day, the WHO has listed the problem of bacterial resistance as one of the most important factors threatening human health in the 21 st century. It is worth noting that the drug resistance gene is the main cause of the drug resistance of bacteria, such as the horizontal transfer of the vanA drug resistance gene, which causes the vancomycin resistance of methicillin-resistant staphylococcus aureus. The continuous aggravation of the drug resistance of bacteria deepens the difficulty of anti-infection treatment, and in order to find a solution, the research on novel drug-resistant bacteria resistant medicines and preparations is carried out at random. Drug-resistant strains obtained from the environment or clinic become necessary materials for searching new antibacterial drug research experiments, such as methicillin-resistant staphylococcus aureus, escherichia coli producing ESBLs or AmpC enzyme, klebsiella pneumoniae; multiple drug-resistant pseudomonas aeruginosa, candida tropicalis and the like. The cfr gene is the only found multiple drug resistance gene which can simultaneously mediate drug resistance to five antibacterial drugs (amide alcohols, lincomycins, pleuromutilins, oxazolidinones and streptogramins A) with different chemical structures, and is the first transferable drug resistance gene which can mediate drug resistance to oxazolidinones, which is found so far. Currently, staphylococci carrying cfr genes have been reported at home and abroad (Locke et al, 2012; Mendes et al, 2013; Sharkey et al, 2016; Su et al, 2018), but have never been found in Staphylococcus dolphin.
The invention content is as follows:
the invention aims to provide a multi-drug resistant Staphylococcus dolphin (Staphylococcus delphini)245-1, which is preserved in Guangdong province microbial culture collection (GDMCC) 6-4 days 2020, and the preservation address is the microbial research institute of Guangdong province, No. 59 building and No. 5 building of Guangdong province, Michelia Torrens 100, Guangzhou city, with the preservation number GDMCC NO: 61071.
the inventor discovers a multi-drug-resistant staphylococcus dolphin strain in research, the strain contains cfr-carrying genes, is not reported at home and abroad, is an important material for seeking a bacterial drug-resistant mechanism, and can be used for screening novel functional microorganisms/drugs/antibacterial materials.
The second purpose of the invention is to provide the application of the staphylococcus dolphin 245-1 in screening functional microorganisms, medicines or antibacterial materials.
The functional microorganism, medicine or antibacterial material is a functional microorganism, medicine or antibacterial material for inhibiting staphylococcus dolphin 245-1.
The functional microorganism, the medicine or the antibacterial material is the functional microorganism, the medicine or the antibacterial material for inhibiting linezolid drug-resistant bacteria.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a multi-drug resistant staphylococcus dolphin carrying a multi-drug resistant gene cfr and application thereof. The strain is resistant to 6 antibiotics, particularly to linezolid, can be used as a model material for screening novel functional microorganisms/medicaments/antibacterial materials, and has a good application prospect.
Staphylococcus delphini 245-1, which was deposited at the Guangdong province microbial culture collection center (GDMCC) at 6/4/2020, with the deposit address of Guangdong province microbial research institute of No. 59, 5, Guangdong province, Miehuo No. 100, Guangzhou, and the deposit number is GDMCC NO: 61071.
description of the drawings:
FIG. 1 is a colony morphology of multidrug-resistant Staphylococcus dolphin 245-1 strain;
FIG. 2 is a microscopic observation form diagram of a multidrug-resistant Staphylococcus dolphin 245-1 strain;
FIG. 3 is a schematic diagram of API Staph biochemical identification of the multi-drug resistant Staphylococcus dolphin 245-1 strain;
FIG. 4 is a 16srRNA sequence evolutionary tree analysis diagram of multi-drug resistant Staphylococcus dolphin 245-1 strain, in which lclQuery _129551 represents Staphylococcus dolphin 245-1.
FIG. 5 shows the results of the resistant phenotype of the multidrug resistant Staphylococcus dolphin 245-1 strain, A: k (kanamycin), C (chloramphenicol), DA (clindamycin), S (streptomycin); b: CIP (ciprofloxacin), NOR (norfloxacin), E (erythromycin), TEL (telithromycin); c: AMP (ampicillin), AMC (amoxicillin/clavulanic acid), FEP (cefepime), FOX (cefoxitin); d: AK (amikacin), P (penicillin), CAZ (ceftazidime), CN (gentamicin); e: RD (rifampin), TE (tetracycline), SXT (compound sulfamethoxazole), LZD (linezolid); f: TEC (teicoplanin), FD (fusidic acid), QD (quinupristin/dalfopristin), F (nitrofurantoin).
FIG. 6 is a PCR amplification plot of cfr gene of multidrug resistant Staphylococcus dolphin 245-1 strain;
FIG. 7 is a genetic background analysis diagram of cfr gene of multidrug-resistant Staphylococcus dolphin 245-1 strain.
The specific implementation mode is as follows:
it will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
A Staphylococcus dolphin resistant to multiple antibiotics is named as Staphylococcus dolphin (Staphylococcus aureus) 245-1 with multiple drug resistance; the strain is preserved in Guangdong province microorganism culture collection center (GDMCC) at 6-month and 4-month in 2020, the preservation address is No. 59 building No. 5 building No. Guangdong province microorganism research institute of Michelia Zhongluo No. 100, Guangzhou, Guangdong province, and the preservation number is GDMCC NO: 61071.
example 1 isolation, identification and culture of multiple drug-resistant Staphylococcus dolphin 245-1 Strain
First, isolation of the Strain
The method comprises the following steps of (1) separating staphylococcus dolphin 245-1 from quick-frozen duck leg food in a farmer market in the area of wine vinum of Guangzhou city, carrying out qualitative and quantitative detection on collected samples at the same time, sampling 25g (mL), adding 225mL of a sterile homogenizing bag containing normal saline, homogenizing and shaking uniformly to prepare 1:10 sample liquid, taking 1mL of the sample liquid, adding the sample liquid into a test tube containing 9mL of 10% sodium chloride tryptone soybean broth to prepare 1:100 sample liquid, and preparing the sample liquid into 1:1000 according to the method; 3 of each gradient are paralleled, the sample solution is placed in a constant temperature incubator for culturing for 48h at 37 ℃, and then the sample solution with each gradient concentration is respectively streaked on a staphylococcus chromogenic plate culture medium (figure 1) and cultured for 24-48h at 37 ℃. Target colonies were transferred from NA plates into brain heart infusion Broth (BHI) and allowed to resuscitate overnight at 37 ℃. Adding the bacterial liquid into a glycerin tube with the final concentration of 40% under the aseptic condition, storing in a refrigerator at the temperature of minus 40 ℃, and storing in a freeze-drying tube. Thus, strain 245-1 was obtained.
II, identifying the strain:
the purified strain 245-1 is subjected to identification in aspects of morphological characteristics, physiology, biochemistry, serotype, molecular biology and the like.
1. And (3) dyeing microscopic examination: the strain 245-1 was smeared, gram stained, and the morphology was visualized by microscopic examination. Staphylococcus dolphin 245-1 of the present invention was shown to be gram positive, microscopically in the form of a prototype staphyloccous (FIG. 2).
2. Plasma coagulase assay: a single colony of strain 245-1 was inoculated into 5mLBHI medium and cultured at 37 ℃ for 18-24 hours. The culture medium was aspirated by 1mL, added to plasma coagulase, and cultured at 37 ℃. After 2.5h, whether coagulation occurs or not is observed once per hour, and if coagulation does not occur after 6h, the culture is carried out overnight and then observed and verified.
3. API Staph identification: single colonies of the pink strain 245-1 were scraped from the chromogenic plate, prepared in normal saline into cell suspensions of appropriate turbidity, and identified using the API Staph biochemical identification reagent strip (FIG. 3).
4. 16srDNA analysis: the nucleic acid of strain 245-1 was extracted with a bacterial genomic DNA extraction kit (Takara, Japan), and then PCR amplification was carried out using 16s rRNA gene PCR and the universal bacterial primer 27F-1492R primer (5'-AGA GTT TGA TCC TGG CTC AG-3' and 5 '-TAC GGY TAC CTT GTT ACG ACT T-3') followed by sequencing. The sequencing result is shown in SEQ ID NO. 1.
A phylogenetic tree based on 16S rRNA genes is established, 16S rRNA sequences of adjacent strains are downloaded in an NCBI nucleic acid database (https:// www.ncbi.nlm.nih.gov/nucleotide /), genetic distances are calculated, phylogenetic trees of related species are established by an N-J method, the phylogenetic tree shows that 245-1 has a closest genetic relationship with Staphylococcus delphini NCTC12225, and the similarity is 99.6%, as shown in FIG. 4.
The bacterial colony is comprehensively judged to have appearance, shape, gram stain and 16s result, the bacterial strain 245-1 can be identified to be staphylococcus dolphin, and the bacterial strain is named as: staphylococcus dolphin (Staphylococcus delphini)245-1, deposited in the Guangdong provincial collection of microorganisms and cell cultures (GDMCC), with the accession number GDMCC NO: 61071; the preservation date is 6 months and 4 days in 2020, and the preservation address is the microbiological research institute of Guangdong province of No. 59 building, 5 building, Guangdong province of Mirabi Zhonglu No. 100 college, Guangzhou, Guangdong province.
Example 2 drug sensitivity characterization of multiple drug-resistant Staphylococcus dolphin 245-1 Strain
The drug sensitivity of the staphylococcus dolphin 245-1 strain is confirmed by a gradient dilution method and a KB method. The gradient dilution method uses Staphylococcus aureus ATCC29213 as a quality control strain, and the KB method uses Staphylococcus aureus ATCC25923 as a quality control strain. Both methods are referenced to assays and standards according to the clinical laboratory standards Committee (CLSI) in the united states.
Gradient dilution method: each group was run in parallel 3 times to give an average concentration. Sterile 96-well flat-bottom microplates were selected, and 200 μ LMH broth medium was added to the first well of each row as a negative control; adding 100 mu L of the staphylococcus dolphin 245-1 drug-resistant bacteria liquid and 100 mu L of LMH broth culture medium into each row of second holes to serve as positive control; adding MH broth culture medium 100 μ L into each row of 3-12 holes, adding drug to be tested 100 μ L into each row of 3-3 holes, mixing well, taking out 100 μ L, transferring to 4 th hole, repeating until 12 th hole is mixed well, sucking 100 μ L, discarding, and adding mixed Staphylococcus dolphin 245-1(5 × 10) into each hole5cfu/mL) of the suspension. The plates were incubated at 37 ℃ for 18h to observe the results and the MIC values were determined. The black background is selected for observation, and the concentration of the drug in the lowest concentration well with clear and bright solution is taken as MIC.
KB method: activating by NA plate, adding physiological saline, and diluting to final concentration of 1 × 107cfu/mL was spread on MH plates, and after the bacterial solution had dried, antibiotic paper sheets were attached to the surface of the medium and cultured at 37 ℃ for 24 h. And measuring the size of the bacteriostatic zone by using a vernier caliper to be accurate to 0.01 mm.
Further, the antibiotics of amoxicillin clavulanic acid, ampicillin, cefepime, penicillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, linezolid, rifampin, compound sulfamethoxine, quinupristin/dalfopristin, tiazem, nitrofurantoin, fuscin, florfenicol, peptidomimeticin, vancomycin and daptomycin are selected for drug sensitivity confirmation, wherein amoxicillin clavulanic acid, ampicillin, cefepime, penicillin, ceftazidime, amikacin, gentamikacin, kanamycin, streptomycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, compound neomycin, quinupristin/dalfopristin, nitrovin, nitrofuratin, furadaptorin and fuscin are selected for confirmation by KB method, the remaining antibiotics were performed by gradient dilution.
The results of the KB experiment are shown in FIG. 5. Through experiments, the staphylococcus dolphin 245-1 can generate drug resistance to chloramphenicol (MIC 128 mu g/mL), florfenicol (MIC 256 mu g/mL), peptide miacin (MIC 128 mu g/mL), clindamycin (MIC 4 mu g/mL), kanamycin (MIC 16 mu g/mL) and linezolid (MIC 8 mu g/mL), and is judged to be multidrug-resistant staphylococcus dolphin.
Example 3 detection of drug resistance genes of multidrug-resistant Staphylococcus dolphin 245-1 Strain
Aiming at the characteristics of the multiple drug resistance spectrum of the staphylococcus dolphin 245-1, the multiple drug resistance genes cfr are selected to carry out PCR detection on the staphylococcus dolphin 245-1. The primers and amplification method are described in the literature. The primers used were synthesized by Beijing Liuhe Dagen science and technology Co., Ltd (see Table 1 for the sequence of the primers). The reaction system (25. mu.L) contained: 12.5. mu.l of 2 XDreamtaq mastermix, 9.5. mu.l of ultrapure water, 1. mu.l of template DNA, and 1. mu.l of each of the upstream and downstream primers. The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45s, annealing at 65 [. DELTA.T-1 ℃ ] for 45s, and extension at 72 ℃ for 90s, for 15 cycles; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 90s, for 20 cycles; finally, extension is carried out for 10min at 72 ℃. mu.L of the PCR product was applied to a 2.0% agarose gel for electrophoretic separation (120V, 40min) using a 2000bp DNA Marker.
TABLE 1 primers and amplified fragments for multiple drug resistance genes cfr and lsa (E)
Through experiments, the multi-drug resistant staphylococcus dolphin 245-1 carries a multi-drug resistant gene cfr (figure 6), and the nucleotide sequence of the multi-drug resistant gene cfr is shown as SEQ ID No. 2.
Example 4 genetic background analysis of cfr multidrug resistance genes of multidrug resistant Staphylococcus dolphin 245-1 Strain
Third-generation whole genome sequencing analysis is carried out on the staphylococcus dolphin 245-1, and a chromosome contains a plurality of Antibiotic Resistance Genes (ARGs), including fexA (chloramphenicol resistance), aacA-aphD and aadD (aminoglycoside resistance), ble (bleomycin resistance) and cfr genes, and the result is consistent with the drug resistance phenotype result. Further analysis of these ARGs revealed that cfr and another 4 ARGs mapped on a novel Tn558 mutant transposon. This Tn558 mutant transposon (20,264bp) was formed by the insertion of a region (13,611bp) into Tn558 (FIG. 7). The insertion region consists of multiple drug resistant regions (12,287-bp) and a 1,326-bp repeat (duplicated region, DR)B) Composed of Tn558 inserted into the transposase gene tnPC of Tn 558. No repetitive target sequence was found at the junction of the left and right of this insertion region. The cfr gene IS located in the multiple drug resistant region of the insertion sequence, and comprises ARGs (aacA-aphD, aadD and ble), insertion sequences (IS257 and IS21-558), resolvase gene (res), transposase gene (tnp) and two orfs (orf1 and orf 2).
Example 5 application of multidrug-resistant Staphylococcus dolphin 245-1 Strain
The specific application method of the staphylococcus dolphin 245-1 is mainly embodied in two aspects: (1) the cfr gene can simultaneously generate drug resistance to five antibacterial drugs (amide alcohols, lincomycins, pleuromutilins, oxazolidinones and streptogramins A) with different chemical structures, particularly Linezolid (Linezolid) belonging to oxazolidinone antibiotics is considered to be a good drug for treating infection caused by staphylococcus, particularly drug-resistant gram-positive bacteria (such as methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus), the effect is always remarkable in recent years, and the diffusion of the Linezolid drug resistance is greatly accelerated due to the occurrence of the cfr gene. The appearance of the multi-drug resistant gene in the staphylococcus dolphin has important guiding significance for researching a new strategy for discussing a drug resistant generation mechanism of the staphylococcus dolphin and treating infection caused by the multi-drug resistant staphylococcus dolphin, and can be used as an important material for seeking a drug resistant mechanism of bacteria. (2) The other purpose is to screen functional microorganism/drug/antibacterial material, and the screening method is as follows:
1) screening of functional microorganisms
The staphylococcus dolphin 245-1 is taken out one day before the experiment and placed in a refrigerator for storage at-4 ℃, a small number of colonies are picked up by using inoculating loops after the experiment is placed at room temperature, the colonies are respectively inoculated on MH agar culture media, and the culture is carried out for 18 to 24 hours in a constant temperature box at 35 ℃. Selecting a small amount of activated colony with inoculating loop on MH agar culture medium with newly grown colony, placing in sterilized dry test tube, diluting with sterile normal saline to obtain 1.5 × 108cfu/mL of bacterial suspension (standard turbiditube control) for use. Meanwhile, selecting a proper culture medium to activate probiotics, selecting 1-2 single colonies, putting the single colonies into a 5mLBHI culture medium, carrying out shake cultivation for 18h at 37 ℃, preparing a stock solution, and storing the stock solution in a refrigerator at-4 ℃ for later use. In application, sterile cotton swab is dipped to obtain prepared bacteria with standard concentration of 1.5 × 108cfu/mL of bacterial suspension was spread evenly onto the corresponding nutrient agar plates using a spreading bar. Placing the sterilized Oxford cup on the coated MH agar culture medium, and adding 200 mu L of the cultured probiotic stock solution into the Oxford cup; an equal volume of BHI medium was taken as a negative control. The plate is cultured for 24 hours at 37 ℃ and under the condition of nutrient agar culture medium pH of 7.2-7.4 and keeping out of the sun, and the diameter of the generated antibacterial ring is measured.
Inoculating functional bacteria with good bacteriostatic effect on corresponding culture medium, and making into stock solution by the same method as above. Subpackaging the autoclaved BHI liquid culture medium into a plurality of triangular flasks, and adding the subpackaged BHI liquid culture medium into hydrochloric acid and sodium hydroxide for a plurality of times in small amount to adjust the pH value of the culture medium. 3-level 4-factor orthogonal experiments were performed with the temperatures (. degree. C.) set at 32, 35 and 37 ℃, initial pHs set at 4, 5.5 and 7, inoculum sizes set at 10%, 25% and 50%, and incubation times set at 24, 36 and 48h, respectively. And (3) culturing the functional probiotic strain by using the optimal culture condition selected by the orthogonal experiment, and taking the culture stock solution as the seed solution. Meanwhile, according to the antagonistic experiment method, the drug-resistant strain staphylococcus dolphin 245-1 diluted bacterial liquid is prepared and added to a nutrition plate to be evenly coated, the sterilized oxford cup is placed on the nutrition plate, 200 mu L of cultured functional probiotic strain seed liquid is added to the oxford cup respectively, and the inhibition effect of the probiotic strain on the growth of staphylococcus aureus is detected. BHI culture medium is adopted as a reference, and a preparation method of a seed solution of the reference strain is the same as that of an antagonistic primary screening experiment.
2) Drug screening
Precisely weighing a certain amount of the drug to be detected, dissolving with a suitable solvent (such as DMSO with a mass concentration of 5% or DMF with a mass concentration of 5%), and preparing into a solution of 30mg/mL for use. Then, a solution with a concentration of 2000 μ g/mL is prepared with a suitable solvent (e.g., 5% by mass of DMSO or 5% by mass of DMF), and is used to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) and stored in a refrigerator at-4 ℃ for further use. One day before the experiment, the drug-resistant strain staphylococcus dolphin 245-1 stored in a refrigerator at-4 ℃ is taken out, a small number of bacterial colonies are respectively picked by using inoculating loops after the bacterial colonies are placed at room temperature, and the bacterial colonies are respectively inoculated on MH agar culture media and cultured in a thermostat at 35 ℃ for 18-24 hours. Selecting a small amount of activated colony with inoculating loop on MH agar culture medium with newly grown colony, placing in sterilized dry test tube, diluting with sterile normal saline to obtain 1.5 × 08cfu/mL of bacterial suspension (standard turbiditube control) for use. When MIC value was measured, the sample was diluted again with sterile physiological saline to a concentration of 5.0X 105cfu/mL. When used in KB paper diffusion method, the concentration is 1.5 × 108cfu/mL of bacterial suspension.
The assay was performed according to the methods and standards of the clinical laboratory standards Committee (CLSI), the day before the assay, and the standards to be assayedThe quasi-bacteria are inoculated on MH agar medium and cultured in a thermostat at 35 ℃ for 18-24h to activate. Dissolving the drug to be tested in a suitable solvent (such as DMSO with a mass concentration of 5% or DMF with a mass concentration of 5%), and standing. Dipping prepared standard bacteria with a concentration of 1.5 × 10 with a sterile cotton swab8cfu/mL of bacterial suspension, using a coating rod to evenly spread to the corresponding MH agar medium. Placing the sterilized Oxford cup on the coated MH agar culture medium, and adding 50 microliter of prepared liquid medicine (30mg/mL) into the Oxford cup by using a micropipettor; culturing in 35 deg.C incubator for 18-24 hr, and measuring the size of antibacterial zone. Equal volume of BHI medium was taken and added with vehicle as a blank control.
Selecting a drug to be tested which is sensitive to a drug-resistant strain (the diameter of a bacteriostatic circle is not less than 10mm), determining MIC values of the strain to different drugs according to a micro liquid-based dilution method recommended by the American Clinical and Laboratory Standards Institute (CLSI), and performing parallel operation for 3 times in each group to obtain a concentration average value. Sterile 96-well flat-bottom microplates were selected, and 200 μ L of MH broth medium was added to each row of the first well as a negative control; adding 100 mu L of the drug-resistant bacterium liquid and 100 mu L of MH broth culture medium into each row of second wells as positive control; adding 100 mu L of MH broth culture medium into each row of 3 rd to 12 th holes, then adding 100 mu L of the drug to be detected into each row of 3 rd holes, uniformly mixing, taking out 100 mu L, transferring to the 4 th hole, repeating the steps until the 12 th hole is uniformly mixed, sucking 100 mu L, discarding, and finally adding the uniformly mixed drug-resistant bacteria (5 multiplied by 10) of the invention into each hole5cfu/mL) of the suspension. The plates were incubated at 35 ℃ for 18h to observe the results and the MIC values were determined. The black background is selected for observation, and the concentration of the drug in the lowest concentration well with clear and bright solution is taken as the MIC value.
When the MIC value is determined, the mixture of bacteria and MH broth culture medium 3-5 holes before the MIC value is taken and inoculated on MH agar medium, the mixture is placed in an incubator at 35 ℃, the mixture is taken out after being cultured for 22h and observed, and the lowest drug concentration with the average value less than 5 is determined as the MBC of the compound.
3) Antimicrobial material screening
The antibacterial material depends on the selected antibacterial agent, and currently, the antibacterial agent can be classified into three major classes, i.e., inorganic, organic and composite, according to its chemical composition. Inorganic antibacterial agents are mainly classified into heavy metals (metals such as Au, Ag, Hg, Cu, Zn, Ba, Bi, Ta, and compounds thereof), photocatalysts (e.g., TiO2, ZnO, CdS, WO3, Fe2O3, PnS, SnO2, ZnS, SiO2, and the like); the organic antibacterial agent includes two major types of natural antibacterial agent and chemical synthetic organic antibacterial agent, the natural antibacterial agent mainly comprises chitosan, protamine, cassia oil, Thujopsis Dolabrata oil and the like, most of the natural antibacterial agent is refined from animals and plants, and has the advantages of excellent weather resistance, low toxicity, safe use and the like, and the main defects of the organic antibacterial agent are poor heat resistance and short duration of drug effect. The chemically synthesized organic antibacterial agent mainly comprises quaternary ammonium salts, biguanides, alcohols, phenols, aldehydes, organic acids, esters, ethers, peroxides, halogens, imidazoles, thiazoles, nitriles, pyridines and the like. In order to overcome the defects of toxicity of the inorganic antibacterial agent, easy elution of the organic antibacterial agent and heat resistance, a method of compounding organic and/or inorganic and hydrophilic substances is adopted. In a complex system, the inherent antibacterial activity of organic and inorganic antibacterial agents and the like may be significantly improved. Generally, the organic antimicrobial agent may form a copolymer with the hydrophilic material. The inorganic antibacterial agent includes a carrier type antibacterial agent containing one or more of silver, copper and zinc ions, or an inorganic metal oxide or sulfide having photocatalytic activity such as TiO2 or the like is used.
The screening of the bacterial strain for the antibacterial material is carried out according to a test method of performance, antibacterial performance and stability of product in appendix C of GB 15979. The specific method comprises the following steps: the Staphylococcus dolphin 245-124h slant culture of the present invention was washed with PBS to prepare a bacterial suspension (the required concentration: 100. mu.L was dropped on the control sample or in the sample solution, the number of recovered bacteria was 1X 104-9×108cfu/tablet or mL). 4 pieces of each of the test sample (2.0 cm. times.3.0 cm) or the sample solution (5 mL) and the control sample (the same material as the test sample, the same size, but containing no antibacterial material, and subjected to sterilization treatment) were taken and placed in 4 sterilized dishes (or 4 tubes) in 4 groups. Dropping 100 μ L of the above bacterial suspension on each sample and control sample, uniformly coating, timing, acting for 2, 5, 10, 20min, respectively adding sample into test tube containing 5mL of corresponding neutralizer with sterile forceps, mixing well,diluting properly, taking 2-3 dilutions, sucking 0.5mL respectively, placing in two plates, pouring 15mL of nutrient agar culture medium cooled to 40-45 ℃, rotating the plates to make the plates fully uniform, turning over the plates after agar solidification, culturing at 35 +/-2 ℃ for 48h, and counting viable bacteria colonies.
The experiment was repeated 3 times and the inhibition was calculated according to formula (C1):
X＝(A-B)/A×100％....................(Cl)
in the formula: x bacteriostatic rate,%; a-average colony number of control samples; b-average colony number of test sample.
For the dissoluble antibacterial material, when the bacteriostasis rate is more than or equal to 50-90 percent, the material has the bacteriostasis function; when the bacteriostasis rate is more than 90 percent, the relevant materials have stronger bacteriostasis. For the non-leaching antibacterial material, the number of colonies in the sample-free group was 1X 104-9×108The cfu/mL, and the difference of the average colony number before and after sample oscillation is within 10 percent, so that the test is effective; difference value of bacteriostatic rate of tested sample slice group and bacteriostatic rate of control sample slice group>26%, the sample has antibacterial effect.
The stability test method of the antibacterial material comprises the following steps:
1) and (4) naturally reserving the sample, namely placing the original sample at room temperature for at least 1 year, and testing the bacteriostasis or sterilization performance every half year.
2) And (3) an accelerated test, namely placing the original sample in a constant temperature box at 54-57 ℃ for 14 days or a constant temperature box at 37-40 ℃ for 3 months, keeping the relative humidity more than 75%, and carrying out bacteriostatic or bactericidal performance test.
After the antibacterial material is subjected to natural sample retention, the sterilization rate or the bacteriostasis rate of the antibacterial material reaches the specified standard value, and the retention time of the sterilization or bacteriostasis of the product at room temperature is the natural sample retention time.
The product is subjected to an accelerated test at 54 ℃, the sterilization rate or the bacteriostasis rate reaches the specified standard value, and the sterilization or bacteriostasis effect of the product is kept for at least one year at room temperature.
The product is tested at 37 ℃ in an acceleration way, the sterilization rate or the bacteriostasis rate of the product reaches the specified standard value, and the sterilization or bacteriostasis effect of the product is kept for at least two years at room temperature.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> multi-drug-resistant staphylococcus dolphin and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1454
<212> DNA
<213> Staphylococcus dolphin (Staphylococcus delphini)
<400> 1
gggcaggggc ggtgctatac atgcagtcga gcgaacagat aaggagcttg ctcctttgac 60
gttagcggcg gacgggtgag taacacgtgg gtaacctacc tataagactg gaataactcc 120
gggaaaccgg ggctaatgcc ggataacatg ttgaaccgca tggttctaca gtgaaagacg 180
gtcttgctgt cacttataga tggacccgcg ccgtattagc tagttggtgg ggtaacggcc 240
taccaaggcg acgatacgta gccgacctga gagggtgatc ggccacactg gaactgagac 300
acggtccaga ctcctacggg aggcagcagt agggaatctt ccgcaatggg cgaaagcctg 360
acggagcaac gccgcgtgag tgatgaaggt cttcggatcg taaagctctg ttgttaggga 420
agaacaaatg tgtaagtaac tgtgcacatc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttatccgg aattattggg 540
cgtaaagcgc gcgtaggcgg ttttttaagt ctgatgtgaa agcccacggc tcaaccgtgg 600
agggtcattg gaaactggaa aacttgagtg cagaagagga aagtggaatt ccatgtgtag 660
cggtgaaatg cgcagagata tggaggaaca ccagtggcga aggcggcttt ctggtctgca 720
actgacgctg atgtgcgaaa gcgtggggat caaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
gacccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccaa 960
atcttgacat cctttgacaa ctctagagat agagctttcc tcttcggagg acaaagtgac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgaacttagt tgccatcatt cagttgggca ctctaagttg actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga tttgggctac 1200
acacgtgcta caatggacaa tacaaagggc agcaaaaccg cgaggtcaag caaatcccat 1260
aaagttgttc tcagttcgga ttgtagtctg caactcgact acatgaagct ggaatcgcta 1320
gtaatcgtag atcagcatgc tacggtgaat acgttcccgg gtcttgtaca caccgcccgt 1380
cacaccacga gagtttgtaa cacccgaagc cggtggagta accatttgga gctagccgtc 1440
gaaggtgaca aagt 1454
<210> 2
<211> 1050
<212> DNA
<213> Staphylococcus dolphin (Staphylococcus delphini)
<400> 2
atgaatttta ataataaaac aaagtatggt aaaatacagg aatttttaag aagtaataat 60
gagcctgatt atagaataaa acaaataacc aatgcgattt ttaaacaaag aattagtcga 120
tttgaggata tgaaggttct tccaaaatta cttagggagg atttaataaa taattttgga 180
gaaacagttt tgaatatcaa gctcttagca gagcaaaatt cagagcaagt tacgaaagtg 240
ctttttgaag tatcaaagaa tgagagagta gaaacggtaa acatgaagta taaagcaggt 300
tgggagtcat tttgtatatc atcacaatgc ggatgtaatt ttgggtgtaa attttgtgct 360
acaggcgaca ttggattgaa aaaaaaccta actgtagatg agataacaga tcaagtttta 420
tacttccatt tattaggtca tcaaattgat agcatttctt ttatgggaat gggtgaagct 480
ctagccaacc gtcaagtatt tgatgctctt gattcgttta cggatcctaa tttatttgca 540
ttaagtcctc gtagactttc tatatcaacg attggtatta tacctagtat caaaaaaata 600
acccaggaat atcctcaagt aaatcttaca ttttcattac actcacctta tagtgaggaa 660
cgcagcaaat tgatgccaat aaatgataga tacccaatag atgaggtaat gaatatactc 720
gatgaacata taagattaac ttcaaggaaa gtatatatag cttatatcat gttgcctggt 780
gtaaatgatt ctcttgagca tgcaaacgaa gttgttagcc ttcttaaaag tcgctataaa 840
tcagggaagt tatatcatgt aaatttgata cgatacaatc ctacaataag tgcacctgag 900
atgtatggag aagcaaacga agggcaggta gaagcctttt acaaagtttt gaagtctgct 960
ggtatccatg tcacaattag aagtcaattt gggattgata ttgacgctgc ttgtggtcaa 1020
ttatatggta attatcaaaa tagccaatag 1050
Claims (4)
1. Staphylococcus dolphin (Staphylococcus delphini)245-1, accession number GDMCC NO: 61071.
2. use of staphylococcus dolphin 245-1 as claimed in claim 1 in screening functional microorganisms, drugs or antibacterial materials.
3. The use according to claim 2, wherein the functional microorganism, drug or antimicrobial material is a functional microorganism, drug or antimicrobial material that inhibits staphylococcus dolphin 245-1.
4. The use according to claim 2, wherein the functional microorganism, drug or antimicrobial material is a functional microorganism, drug or antimicrobial material that inhibits linezolid-resistant bacteria.
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| CN107435034A (en) * | 2017-07-18 | 2017-12-05 | 余巍 | A kind of methicillin-resistant staphylococcus aureus and application to drug resistance of vancomycin |
| WO2018086516A1 (en) * | 2016-11-09 | 2018-05-17 | 湖南科技学院 | Antibacterial peptide of inhibiting drug-resistant bacteria and application thereof |
| CN108368079A (en) * | 2015-12-02 | 2018-08-03 | 优拓制药公司 | Treat the compound and method of bacterium infection |
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| WO2018086516A1 (en) * | 2016-11-09 | 2018-05-17 | 湖南科技学院 | Antibacterial peptide of inhibiting drug-resistant bacteria and application thereof |
| CN107435034A (en) * | 2017-07-18 | 2017-12-05 | 余巍 | A kind of methicillin-resistant staphylococcus aureus and application to drug resistance of vancomycin |
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| Antimicrobial resistance among pathogenic bacteria from mink (Neovison vison) in Denmark;Nanett Kvist Nikolaisen等;《Acta Vet Scand》;20170913;第59卷(第1期);参见全文 * |
| 赛鸽源致病性多重耐药海豚葡萄球菌分离鉴定;李博等;《吉林农业大学学报》;20171231;第39卷(第2期);参见全文 * |
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