CN111979292B - Application of MRSA (methicillin resistant staphylococcus aureus) simultaneously carrying multiple drug resistance genes cfr and lsa (E) - Google Patents
Application of MRSA (methicillin resistant staphylococcus aureus) simultaneously carrying multiple drug resistance genes cfr and lsa (E) Download PDFInfo
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Abstract
The invention discloses an application of MRSA simultaneously carrying multiple drug resistance genes cfr and lsa (E). The application of Staphylococcus aureus (Staphylococcus aureus) Sta2868B2 in screening functional microorganisms, medicines or antibacterial materials is disclosed, wherein the Staphylococcus aureus Sta2868B2 has a deposit number of: GDMCC No: 61037. the invention provides methicillin-resistant staphylococcus aureus Sta2868B2 simultaneously carrying multiple drug-resistant genes cfr and lsa (E). The strain is resistant to 11 types of antibiotics, particularly linezolid, can be used as a model material for screening novel functional microorganisms/medicaments/antibacterial materials, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of methicillin-resistant staphylococcus aureus simultaneously carrying multiple drug-resistant genes cfr and lsa (E).
Background
Staphylococcus aureus (Staphylococcus aureus) is a pathogen that can cause serious community and hospital-acquired diseases, including skin and soft tissue infections, infectious endocarditis, necrotizing pneumonia, life-threatening sepsis, and toxic shock syndrome (deep et al, 2006). In recent years, the problem of drug resistance of staphylococcus aureus has been highlighted. Among them, Methicillin-resistant staphylococcus aureus (MRSA) is a focus of attention at home and abroad. MRSA is resistant not only to beta-lactam antibacterial drugs, but also to a variety of antibacterial drugs commonly used in clinical practice, such as tetracyclines, aminoglycosides, macrolides, and the like, with morbidity and mortality exceeding aids, SARS, and avian influenza (Kluytmans, 2010). At the end of 2017, 2 months, the WHO even listed it in a world line of twelve superbacteria. In general, resistance to S.aureus can occur through point mutations or horizontal transfer of resistance genes mediated by mobile elements (e.g., plasmids, transposons or intervening sequence elements, etc.) that can be derived from horizontal transfer of the same species or other related species (Ferroro et al, 1995; Gao et al, 2012; Tsioras et al, 2001; Weigel et al, 2003; Weigel et al, 2007; Yan et al, 2016). Therefore, the emergence of drug resistance genes is the leading cause of bacterial drug resistance.
Among the many resistance genes, cfr and lsa (E) are very specific and mediate multiple resistance. Among them, cfr gene is the only one found multiple drug resistance gene that can mediate drug resistance to five antibacterial drugs (amide alcohols, lincomycins, pleuromutilins, oxazolidinones and streptogramins A) with different chemical structures at the same time, and is the first found transferable drug resistance gene that can mediate drug resistance to oxazolidinones. Linezolid (Linezolid) is the first oxazolidinone drug approved for treatment of infections caused by MRSA and VRE (vancomycin-resistant enterococci), and the effect has been significant in recent years, while the spread of Linezolid resistance has been greatly accelerated by the emergence of cfr genes. The lsa (E) gene belongs to ABC transporter gene, encodes ABC transporter, and can simultaneously mediate three types of antibacterial drugs with different chemical structures to generate drug resistance (lincosamines, pleuromutilins and streptogramins A), and the three types of antibacterial drugs are all very important anti-infective drugs in clinical practice of human medicine and veterinary medicine. Notably, the lsa (e) gene is often found in the multiple resistance cluster aadE-spc-lsa (e) -lnu (b) -tnp, which typically contains multiple resistance genes, aadE and spc, genes mediating aminoglycoside resistance, lsa (e) resistance genes of the lincosamide-pleuromutilin-streptogramin a class, lnu (b) genes mediating lincosamide resistance, and transposase tnp, respectively.
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.
Disclosure of Invention
The first purpose of the invention is to solve the defects of the prior art and provide the application of methicillin-resistant staphylococcus aureus simultaneously carrying multiple drug-resistant genes cfr and lsa (E), wherein the bacterium is first reported at home and abroad, is an important material for seeking a drug-resistant mechanism of the bacterium, and can be used for screening novel functional microorganisms/drugs/antibacterial materials.
The invention relates to methicillin-resistant Staphylococcus aureus simultaneously carrying multiple drug-resistant genes cfr and lsa (E), which belongs to Staphylococcus aureus (Staphylococcus aureus) and is named as Staphylococcus aureus (Staphylococcus aureus) Sta2868B2, and is preserved in Guangdong province microorganism culture Collection (GDMCC) at 5-30 days of 2020, and the address is as follows: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61037.
the invention provides application of staphylococcus aureus Sta2868B2 in screening functional microorganisms, medicines or antibacterial materials.
The functional microorganism, the medicine or the antibacterial material is the functional microorganism, the medicine or the antibacterial material for inhibiting staphylococcus aureus Sta2868B 2.
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.
The staphylococcus aureus Sta2868B2 is a typical beta-hemolysis, reduced potassium tellurite, a positive strain of plasma coagulase, and API STAPH is identified as staphylococcus aureus, and the coincidence rate is 77.7%. The biochemical characteristics are as follows: d-dextrose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol, potassium nitrate, beta-naphthyl phosphate, sodium pyruvate, D-Saccharum sinensis II, N-acetyl glucosamine, L-arginine and urea are positive, and the normal physiological and biochemical characteristics of staphylococcus aureus are realized. Drug resistance was identified by the antibiotic microdilution method specified in the American Clinical Laboratory Standards Institute (CLSI) 2015, which was found to be a Minimum Inhibitory Concentration (MIC) of 32. mu.g/mL for Staphylococcus aureus Sta2868B2, and the drug resistance was determined to be methicillin-resistant Staphylococcus aureus when the MIC of Staphylococcus aureus to Cephalosporium was not less than 8. mu.g/mL according to the CLSI (2015). Meanwhile, the staphylococcus aureus Sta2868B2 contains mecA gene through PCR confirmation, and the strain is further confirmed to be methicillin-resistant staphylococcus aureus.
Further, staphylococcus aureus Sta2868B2 of the present invention is resistant to penicillins (penicillin, amoxicillin/clavulanic acid, ampicillin, cefepime, ceftazidime), aminoglycosides (gentamicin, kanamycin, streptomycin), chloramphenicol (chloramphenicol, florfenicol), lincomamides (clindamycin), macrocyclic intralipoids (erythromycin, telithromycin), pleuromutilins (tiamulin), quinolones (ciprofloxacin, norfloxacin), tetracyclines (tetracycline), oxazolidinones (linezolid), sulfonamides (sulfamethoxazole), and streptogramins (quinupristin/dalfopristin), respectively.
The staphylococcus aureus Sta2868B2 simultaneously carries two multiple drug resistance genes cfr and lsa (E), the nucleotide sequence of the cfr is shown as SEQ ID NO1, and the nucleotide sequence of the lsa (E) is shown as SEQ ID No. 2.
Staphylococcus aureus Sta2868B2 can be cultured in TSA, BHI and NA media.
The applicant of the invention finds that a multi-drug resistant methicillin-resistant staphylococcus aureus (MRSA) -staphylococcus aureus Sta2868B2 resistant to 18 antibiotics contains a plasmid carrying cfr genes, and a chromosome carries a multi-drug resistant region, wherein the multi-drug resistant region contains lsa (E) drug resistant genes, so that the cfr and lsa (E) are found to exist in an MRSA isolate at the same time for the first time, and the strain is not reported at home and abroad.
Compared with the prior art, the invention has the beneficial effects that: the invention provides methicillin-resistant staphylococcus aureus Sta2868B2 simultaneously carrying multiple drug-resistant genes cfr and lsa (E). The strain is resistant to 11 types of antibiotics, particularly linezolid, can be used as a model material for screening novel functional microorganisms/medicaments/antibacterial materials, and has a good application prospect.
Staphylococcus aureus Sta2868B2 was deposited at the guangdong province collection of microorganisms and cell cultures (GDMCC) at 30/5/2020, address: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61037.
description of the drawings:
FIG. 1 is a colony morphology of Sta2868B2 strain of methicillin-resistant Staphylococcus aureus;
FIG. 2 is a morphological diagram of the Sta2868B2 strain of methicillin-resistant Staphylococcus aureus under microscopic examination;
FIG. 3 is a schematic diagram of the biochemical identification of API Staph of methicillin-resistant Staphylococcus aureus Sta2868B2 strain;
FIG. 4 shows the KB method susceptibility results of the methicillin-resistant Staphylococcus aureus Sta2868B2 strain, A: AMP (ampicillin), AMC (amoxicillin/clavulanic acid), FEP (cefepime), FOX (cefoxitin); b: AK (amikacin), P (penicillin), CAZ (ceftazidime), CN (gentamicin); c: k (kanamycin), C (chloramphenicol), DA (clindamycin), S (streptomycin); d: CIP (ciprofloxacin), NOR (norfloxacin), E (erythromycin), TEL (telithromycin); e: RD (rifampin), TE (tetracycline), SXT (compound sulfamethoxazole), LZD (linezolid); f: TEC (teicoplanin), FD (fusidic acid), QD (quinupristin/dalfopristin), F (nitrofurantoin);
FIG. 5 is a diagram of the amplification of mecA gene of Sta2868B2 strain of methicillin-resistant Staphylococcus aureus by PCR;
FIG. 6 is a diagram of cfr gene and lsa (E) PCR amplification of methicillin-resistant Staphylococcus aureus Sta2868B2 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.
Example 1:
1. isolation, identification and cultivation of strains
Staphylococcus aureus Sta2868B2 is separated from quick-frozen dumplings of a certain supermarket brand in Hangzhou city, China, and qualitative and quantitative detection is carried out on collected samples at the same time, and the detection method is slightly adjusted on the basis of the national standard food microbiological inspection GB 4789.10-2010. Sampling 25g (mL) of the sample, adding 225mL of physiological saline into a sterile homogenizing bag, homogenizing and shaking uniformly to prepare a sample solution with a ratio of 1:10, taking 1mL of the sample solution, adding the sample solution into a test tube filled with 9mL of 10% sodium chloride tryptone soybean broth to prepare a sample solution with a ratio of 1:100, and preparing the sample solution with a ratio of 1:1000 according to the method; 3 gradients are parallel, the sample solution is placed in a constant temperature incubator at 37 ℃ for culturing for 48h, the sample solution with each gradient concentration is respectively streaked on a staphylococcus aureus chromogenic plate culture medium, and the culture is carried out for 24-48h at 37 ℃. Its typical staphylococcus aureus colonies were pink ball-shaped with smooth wet edges on the chromogenic plate (fig. 1). Target colonies were transferred from NA plates into brain heart infusion Broth (BHI) and allowed to resuscitate overnight at 37 ℃. The bacterial liquid was aseptically added to a glycerin tube having a final concentration of 40%, stored in a refrigerator at-40 ℃ and freeze-dried in the tube, thereby obtaining the strain Sta2868B 2.
The purified strain Sta2868B2 is subjected to identification in aspects of morphological characteristics, physiology and biochemistry, serotype, molecular biology and the like.
And (3) dyeing microscopic examination: and smearing the suspicious colonies, performing gram staining, and observing the morphology by microscopic examination. Staphylococcus aureus was gram positive and microscopically formed into prototypical staphylotric (FIG. 2).
Plasma coagulase assay: a single colony 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.
API Staph identification: pink single colonies were scraped from the chromogenic plates, prepared as cell suspensions of appropriate turbidity with physiological saline, and characterized using API Staph biochemical identification reagent strips (fig. 3).
The strain Sta2868B2 is a typical beta-hemolysis strain, reduces potassium tellurite, has positive plasma coagulase strain, is identified as staphylococcus aureus by API STAPH, and has a coincidence rate of 77.7%. The biochemical characteristics are as follows: d-dextrose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol, potassium nitrate, beta-naphthyl phosphate, sodium pyruvate, D-sugar two ponds, N-acetyl glucosamine, L-arginine and urea are positive, and the normal physiological and biochemical characteristics of staphylococcus aureus are realized. Drug resistance was identified by the antibiotic microdilution method specified in the Clinical and Laboratory Standards Institute (CLSI) 2015, where the Minimum Inhibitory Concentration (MIC) of cefoxitin to the bacterium was 32. mu.g/mL, and when the MIC of Staphylococcus aureus to cefoxitin was not less than 8. mu.g/mL according to the CLSI (2015), the bacterium was determined to be methicillin-resistant Staphylococcus aureus. Meanwhile, the strain contains mecA gene through PCR confirmation, and the strain is further confirmed to be methicillin-resistant staphylococcus aureus.
The appearance, the morphology, the gram stain and the biochemical reaction of the strain Sta2868B2 can be comprehensively judged to identify the strain Sta2868B2 as Staphylococcus aureus, which is named as Staphylococcus aureus (Staphylococcus aureus) Sta2868B2, and is preserved in Guangdong province microbial culture Collection (GDMCC) 5-30 days 2020, and the address: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61037.
2. drug sensitivity profile analysis
The drug sensitivity of the staphylococcus aureus strain Sta2868B2 strain is confirmed by using 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 L of MH broth medium was added to the first well of each row as a negative control; adding 100 μ L of Sta2868B2 bacterial liquid of Staphylococcus aureus and 100 μ L of MH broth into each row of second wells as positive control; adding MH broth culture medium 100 μ L into each row of 3-12 wells, adding test drug 100 μ L into each row of 3 rd well, and mixingMixing, taking out 100 μ L, transferring to 4 th well, repeating until 12 th well, mixing, sucking 100 μ L, discarding, and adding mixed Staphylococcus aureus Sta2868B2(5 × 10) 5 cfu/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: the strain (Staphylococcus aureus Sta2868B2) is activated by NA plate, and diluted with normal saline to final concentration of 1 × 10 7 cfu/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.
Selecting antibiotics AMOCILLIN-VIAC, ampicillin, cefepime, penicillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, linezolid, rifampin, sulfamethoxazole, quinupramine, quinupristin/dalfopristin, tiazem, nitrofurantoin, fuscin, florfenicol, tiazem, vancomycin and daptomycin for drug sensitivity confirmation, wherein AMOCILLIN-VIAC, ampicillin, cefepime, penicillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, sulfamethoxazole, quinupristin/dalfopristin, tiazem, nitrofuradapton and streptomycin are performed by KB method (FIG. 4), the rest of the antibiotics were performed by gradient dilution. Experiments prove that the Staphylococcus aureus Sta2868B2 cefoxitin has the MIC value of 32 mu g/mL, is methicillin-resistant Staphylococcus aureus, and is resistant to 11 types of antibiotics, particularly linezolid. The results of the specific drug sensitivity of Sta2868B2 are shown in Table 1.
TABLE 1 drug resistant phenotype of methicillin resistant Staphylococcus aureus Sta2868B2 strain
Note: FFC, florfenicol; CHL, chloramphenicol; CLI, clindamycin; TIA, peptidomimeticin; LZD, linezolid; ERY, erythromycin; FOX, cefoxitin; VAN, vancomycin; RIP, rifampin; DAP, daptomycin; AMP, ampicillin; FEP, cefepime; CAZ, ceftazidime; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; TEL, telithromycin; CIP, ciprofloxacin; NOR, norfloxacin; TET, tetracycline; SXT, compound sulfamethoxazole; QD, quinupristin/dalfopristin
3. Detection of methicillin-resistant staphylococcus aureus Sta2868B2 strain drug-resistant gene
Since staphylococcus aureus Sta2868B2 has drug resistance to selected beta-amide antibiotics, in order to further confirm whether staphylococcus aureus is methicillin-resistant staphylococcus aureus, a PCR method is adopted to confirm whether staphylococcus aureus Sta2868B2 contains mecA/mecC genes. The mecA/mecC gene encodes penicillin binding protein 2a (PBP2a), making the relevant strain very low in affinity for beta-lactam antibiotics. When other PBP is inhibited by beta-lactam antibiotics, PBP2a is not inhibited and replaces other PBP to play a role in catalyzing cell wall synthesis, so that bacteria can live. The primers and amplification method of mecA and mecC genes are reported in the prior literature. The primer sequences are shown in Table 2 by the method of single PCR. The PCR amplification conditions were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 50s, for 35 cycles; finally, extension is carried out for 8min at 72 ℃. The reaction system (25. mu.l) contained: 12.5. mu.L of 2 XDreamTAq mastermix, 9.5. mu.L of ultrapure water, 40ng of template DNA, and 0.5. mu. mol/L of upstream and downstream primers. Mu.l of the PCR product was applied to a 2.0% agarose gel for electrophoretic separation (120V, 25min) using a 2000bp DNAmarker (FIG. 5).
TABLE 2 Staphylococcus aureus mecA/mecC primers and amplified fragments
Meanwhile, multiple drug resistance genes cfr and lsa (E) are selected to carry out PCR detection on the staphylococcus aureus strain Sta2868B2 according to the multiple drug resistance characteristics. 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 3 for the sequence of the primers). The reaction system (25. mu.L) contained: 12.5. mu.L of 2 XDreamTAq mastermix, 8.5. mu.L of ultrapure water, 80ng of template DNA, and 0.5. mu. mol/L of upstream and downstream primers. The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 65 [. DELTA.T-1 ℃ ] for 45s, extension at 72 ℃ for 90s, and performing 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 (FIG. 6).
TABLE 3 primers and amplified fragments for multiple drug resistance genes cfr and lsa (E)
Experiments prove that the methicillin-resistant staphylococcus aureus Sta2868B2 strain simultaneously carries the two multi-drug-resistant genes, the nucleotide sequence of the cfr is shown as SEQ ID NO. 1, and the nucleotide sequence of the lsa (E) is shown as SEQ ID NO. 2.
4. Application of methicillin-resistant staphylococcus aureus Sta2868B2 strain
The specific application method of the staphylococcus aureus Sta2868B2 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 streptogramin A) with different chemical structures, particularly Linezolid (Linezolid) belonging to oxazolidinone antibiotics is considered as a good drug for treating infection caused by MRSA and VRE (vancomycin-resistant enterococcus), the effect is always remarkable in recent years, and the diffusion of the drug resistance of the Linezolid is greatly accelerated due to the appearance of the cfr gene. The lsa (E) gene belongs to ABC transport genes, encodes ABC transport proteins, and can simultaneously mediate three types of antibacterial drugs with different chemical structures to generate drug resistance (lincomamines, pleuromutilins and streptogramins A), and the three types of antibacterial drugs are all very important anti-infective drugs in clinical practice of human medicine and veterinary medicine. The appearance of the two multiple drug resistance genes in the same strain has important guiding significance for discussing the drug resistance generation mechanism of MRSA and researching a new strategy for treating MRSA infection, and can be used as an important material for seeking the drug resistance 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 aureus Sta2868B2 stored in a refrigerator at the temperature of 4 ℃ is taken out one day before the experiment, a small number of bacterial colonies are respectively picked by using inoculating loops after the staphylococcus aureus Sta2868B2 is placed at the room temperature, and the bacterial colonies are respectively inoculated on MH agar culture media and cultured in a constant temperature box at the temperature of 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 × 10 8 cfu/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, the prepared standard bacteria with concentration of 1.5 × 10 is dipped with sterile cotton swab 8 cfu/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 staphylococcus aureus Sta2868B2 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 respectively added to the Oxford cup, and the inhibition effect of the probiotic strain on the growth of the 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 staphylococcus aureus Sta2868B2 stored in a refrigerator at the temperature of 4 ℃ is taken out, when the staphylococcus aureus Sta2868B2 is placed at room temperature, a small number of bacterial colonies are respectively picked by inoculating loops, and are respectively inoculated on MH agar culture media and cultured in a thermostat at the temperature of 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 × 10 8 cfu/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 10 5 cfu/mL. When used in KB paper diffusion method, the concentration is 1.5 × 10 8 cfu/mL of bacterial suspension.
The test was carried out according to the method and standard of the American Committee for Clinical Laboratory Standards (CLSI), and the standard bacteria to be tested were inoculated on MH agar medium the day before the test, and cultured in a 35 ℃ incubator for 18-24 hours 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. By using sterilityDipping cotton swab with prepared standard bacteria at a concentration of 1.5 × 10 8 cfu/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 the zone of inhibition. 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 Sta2868B2 bacterial liquid of staphylococcus aureus 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 hole 5 cfu/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. The inorganic antibacterial agent can be mainly classified into heavy metals (Au, Ag, Hg, Cu, Zn, Ba, Bi, Ta, and their compounds), and photocatalysis (TiO 2, ZnO, CdS, WO, etc.) 3 、Fe 2 O 3 、PnS、SnO2、ZnS、SiO 2 Etc.) two broad categories; 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 comprises a carrier-type antibacterial agent containing one or more of silver, copper and zinc ions, or inorganic metal oxide or sulfide such as TiO with photocatalytic activity 2 And the like.
The staphylococcus aureus Sta2868B2 is used for screening antibacterial materials according to a GB15979 annex C test method for product performance, antibacterial performance and stability. The specific method comprises the following steps: the Staphylococcus aureus Sta2868B224 h slant culture of the invention is washed by PBS to prepare bacterial suspension (the required concentration is 100 mul is dripped on a control sample or in a sample liquid, the number of recovered bacteria is 1 × 10 4 -9×10 8 cfu/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 putting the samples into test tubes containing 5mL of corresponding neutralizer with sterile forceps, mixing well, diluting properly, taking 2-3 dilutions, sucking 0.5mL respectively, placing in two plates, cooling to 40-45 deg.CThe nutrient agar culture medium is poured by 15mL, the plate is rotated to be fully and uniformly, the plate is turned over after the agar is solidified, the culture is carried out for 48 hours at the temperature of 35 +/-2 ℃, and the bacterial colony count of viable bacteria is carried out.
The test was repeated 3 times, and the sterilization rate was calculated according to the 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 10 4 -9×10 8 The 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 a sample: and (3) placing the original sample at room temperature for at least 1 year, and carrying out bacteriostatic or bactericidal performance test every half year.
2) And (3) accelerated test: and (3) placing the original sample in a constant temperature cabinet at 54-57 ℃ for 14 days or a constant temperature cabinet 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> application of MRSA simultaneously carrying multiple drug resistance genes cfr and lsa (E)
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1050
<212> DNA
<213> Staphylococcus aureus Sta2868B2(Staphylococcus aureus)
<400> 1
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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
<210> 2
<211> 804
<212> DNA
<213> Staphylococcus aureus Sta2868B2(Staphylococcus aureus)
<400> 2
atgttaaaac aaaaagaatt aattgcaaac gttaagaatc ttactgagtc agatgaacga 60
attacagctt gtatgatgta tggatcgttt accaaaggag aaggtgacca atactctgat 120
atagagttct atatattttt gaaagatagt ataacctcga actttgattc atccaactgg 180
ttgtttgacg tagctccgta cttgatgctt tataaaaatg agtacggaac agaggtagtt 240
atttttgata atcttatacg tggggaattt catttccttt ctgaaaaaga tatgaacata 300
atcccctcgt ttaaagattc aggttatatt cctgatacga aggctatgct tatttacgat 360
gaaacagggc aattagaaaa ttatttatca gagataagtg gtgcaagacc aaatagactt 420
actgaagaaa atgctaattt tttgttgtgt aatttctcta atctatggtt gatgggaatc 480
aacgttctaa aaagaggaga atatgctcgt tcattagaac tcttatcaca acttcaaaaa 540
aatacactac aacttatacg tatggcagaa aaaaatgctg ataattggct aaacatgagt 600
aaaaaccttg aaaaagaaat tagccttgaa aattataaaa aatttgcaaa gaccactgct 660
cgattagata aggtagaatt atttgaagcc tataaaaatt ctttgctatt agttatggat 720
ttgcaaagtc accttattga acaatacaac ttaaaagtta cacatgacat tttagaaaga 780
ttgttgaatt acattagtga atag 804
Claims (1)
1. Staphylococcus aureus (1)Staphylococcus aureus) The application of Sta2868B2 in screening functional microorganisms, medicines or antibacterial materials is disclosed, wherein Sta2868B2 of Staphylococcus aureus has a preservation number: GDMCC No: 61037; the functional microorganism, the medicine or the antibacterial material is the functional microorganism, the medicine or the antibacterial material for inhibiting the staphylococcus aureus Sta2868B 2.
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| JP2011526887A (en) * | 2008-07-01 | 2011-10-20 | タイゲン バイオテクノロジー カンパニー，リミテッド | Treatment of antibiotic-resistant bacterial infections |
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