摘要
耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)是医院感染的重要病原菌,也是引起烧伤、严重创伤后感染的最常见致病菌之一。在金黄色葡萄球菌感染中,MRSA所致的医院感染越来越流行,尤其在ICU、烧伤病房等,MRSA的检出率都有明显增高趋势,最高可达90%以上,给临床治疗护理工作带来了极大的困难。其中,老年患者、危重病患者、免疫功能低下者及新生儿等是MRSA感染的高危人群;长期使用抗生素,侵入性操作以及消毒隔离措施等医源性因素均可导致MRSA感染率增加。
MRSA的耐药机制主要与其mecA基因大量表达产生的一种特殊青霉素结合蛋白PBP2a有关,该蛋白由668个氨基酸组成,分子量为76.1 kDa,对所有β-内酰胺类抗生素亲和力低。水溶性的PBP2a为去除N-末端23个氨基酸后的蛋白质(不影响PBP2a与β-内酰胺类抗生素的结合),含有两个功能域:N-末端非青霉素结合域(24~326 aa),负责蛋白的延伸和锚定;C-末端功能域(327~668 aa),具有转肽酶活性和β-内酰胺酶的作用。当β-内酰胺类抗生素抑制了由敏感金葡菌产生的PBPs时,PBP2a可以替代其转肽酶功能,继续维持细菌细胞壁的合成,表现出耐药性。
MRSA临床分离株大多是多重耐药,仅对糖肽类抗生素如万古霉素敏感。然而,随着其广泛应用,已出现一些耐万古霉素金黄色葡萄球菌的报道。1996年在日本分离到万古霉素中度敏感的MRSA,随后2002年在美国发现了万古霉素耐药的MRSA,至今尚无其它治疗MRSA感染的有效药物。所以,更新研究策略、寻找新的药物来抑制PBP2a的活性,恢复MRSA对抗生素的敏感性或降低MRSA耐药性具有十分重要的临床意义。
目的
本研究基于PBP2a在MRSA耐药机制中的重要性,拟从MRSA临床分离株中获得多重耐药蛋白PBP2a,以此为靶点应用生物传感器技术筛选具有与PBP2a高结合力的中药,从中药中寻找抗菌增敏剂,恢复抗生素的敏感性,为MRSA感染的防治研究提供新的策略。
方法
1.可溶性重组蛋白PBP2a的表达、纯化与鉴定:采用PCR方法从MRSA临床分离株中扩增编码PBP2a可溶性全片段的mecA基因(76~2007 bp),构建原核表达载体pQE30-mecA,经酶切鉴定后进行DNA测序;将pQE30-mecA转化入大肠杆菌M15并诱导表达PBP2a可溶性全片段,对重组蛋白PBP2a进行Ni2+亲和层析纯化后,测定其纯度;并进行氨基酸测序与分子量鉴定。然后,对PBP2a的生物学活性与结合活性进行评价。采用分光光度法对PBP2a分别进行转肽酶及β-内酰胺酶活性测定,并通过二倍微孔稀释法与激光共聚焦技术观察PBP2a与抗生素的体外结合作用。
2.以PBP2a为靶点筛选拮抗MRSA的中药:将PBP2a包被于生物传感器的样品池作为固相配基,通过对26种中药水煎液与PBP2a结合活性的测定,选择结合力较高的10种中药,再结合直接抗菌实验,以及10种中药与苯唑西林的联合抗菌实验,筛选出对MRSA具有抗菌增敏作用的中药。
3.夏枯草中对MRSA抗菌增敏组分的分离提取及活性评价:首先通过生物传感器测定夏枯草不同部位水煎液与PBP2a的结合活性,再经联合抗菌实验对它们的抗菌效果进行初步测定;利用AB-8大孔吸附树脂分离富集夏枯草全草水煎液中的有效组分,追踪检测其与PBP2a的结合力,通过抗菌实验与联合抗菌实验确定抗菌增敏组分,并对其物质属性进行鉴定;观察其与苯唑西林联用的抑菌曲线、与β-内酰胺类抗生素的联合抗菌作用,然后通过透射电镜观察其对MRSA细菌形态的影响,评价可能的抗菌作用机制。
结果
1.成功扩增出mecA基因片段(76~2007bp),构建了重组质粒pQE30-mecA,基因序列分析表明,mecA基因片段的大小与预期一致,但序列中出现了9个碱基突变,所编码蛋白质与GenBank收录的蛋白质同源性为96%;在大肠杆菌M15中实现了可溶性PBP2a的高效表达,经分离纯化后得到的重组蛋白PBP2a纯度达到90.6%;蛋白质N端15个氨基酸测序结果为:MRGSH HHHHH GSKDK,与预期一致;质谱分析其相对分子质量约为74 kDa,与预期相符;所获得的重组蛋白PBP2a具有转肽酶活性与β-内酰胺酶活性,并且在体外与β-内酰胺类抗生素具有一定的结合活性。
2.成功地将PBP2a包被于生物传感器的羧甲基葡聚糖样品池上,从26种中药中筛选了10种与PBP2a有较高结合力的中药;体外抗菌实验显示,黄芩、夏枯草、黄连对MRSA的单独抗菌效果较好;10种中药与苯唑西林的联合抗菌实验显示,夏枯草与苯唑西林的联合抗菌效果最好。
3.夏枯草的全草与果穗中均含有与PBP2a具较高结合力的活性成分,其抗菌效果差别不明显;夏枯草全草水煎液经过AB-8大孔吸附树脂分离富集,得到SP2组分;SP2组分不仅与PBP2a具有较高的结合力,而且与苯唑西林联用能够明显抑制MRSA的生长速度;可以使苯唑西林、青霉素、舒氨西林、氨苄西林对MRSA的MIC分别降低256、8、16及4倍,SP2与苯唑西林、青霉素、舒氨西林、氨苄西林的FIC分别为0.007、0.151、0.064、0.276;SP2能够通过破坏MRSA的细胞壁结构发挥抗菌增敏作用;SP2为水溶性组分,含有大量的酚羟基类物质,其有效成分的分子量范围大约为3~5kDa。
结论
采用基因重组技术,成功实现了MRSA临床分离株中可溶性PBP2a的高效表达;以多重耐药蛋白PBP2a为靶点,应用生物传感器建立了筛选拮抗MRSA中药的技术平台。利用该平台,我们从26种中药中筛选出与PBP2a有较高结合力、与苯唑西林联合抗MRSA效果最好的夏枯草;进一步从夏枯草全草水煎液中分离得到水溶性有效组分SP2,SP2具有恢复β-内酰胺类抗生素对MRSA敏感性的作用;SP2组分中有效成分的分子量范围大约为3~5kDa,有望通过进一步的分离纯化得到有活性的小分子化合物作为抗菌增敏剂,为MRSA感染防治提供新的方法。
Methicillin-resistant Staphylococcus aureus (MRSA) is a very important pathogen in nosocomial infection, and one common cause of deep burns infections and severe trauma infections. Nosocomial infection caused by MRSA, among Staphylococcus aureus, become more and more popular, especially in intensive care units and burn wards. The detective rate of MRSA is increasing gradually, even up to over 90%. This issue brings great challenges to the clinical treatmant and nursing. Actually, the senile, the severe, the immunological- deficient and the neonatal, are high-risk groups with MRSA infection. The iatrogenic factors such as longtime administration of antibiotics, invading manipulation and sterilisation and isolation, can result in the growth of MRSA infection.
The mechanism of MRSA resistance is mainly associated with the abundant PBP2a, a special penicillin-binding protein that encoded by mecA gene. PBP2a is composed of 668 amino acids, and its molecular weight is 76.1 kDa. PBP2a has low affinity for all beta-lactam antibiotics. The soluble PBP2a (644 amino acids, 74 kDa) has two domains. One is an N-terminal lobe, a centralized non-penicillin binding domain of unknown function, the other is C-terminal transpeptidase domain. Of particular interest is the C-terminal transpeptidase domain, which has a folding pattern that is typical of the PBP transpeptidases and the serine beta-lactamases. PBP2a provides complementary transpeptidase activity for the cells so that cell walls can be synthesized although other PBPs are inhibited by beta-lactams, thus result in drug resistance. MRSA clinical isolates are typically multidrug resistant and could only be treated with glycopeptides such as vancomycin. However, vancomycin-resistant MRSA strain had emerged in Japan in 1996, followed by similar strains isolated in the United States in 2002, and no effective drug can cure this kind of infection caused by MRSA. Therefore, it is very important and significant to search for new agents that inhibit PBP2a in order to restore the susceptibility of antibiotics in MRSA or decrease MRSA resistance.
Objective
Considering that PBP2a plays an important role in MRSA resistance, we undertake the current study to express the soluble PBP2a from clinical MRSA isolate. Using the biosensor technology to screen traditional Chinese herbs with high affinity for PBP2a, and then search for anti-MRSA potentiator to restore antibiotics susceptibility in order to provide a new treatment for MRSA.
Methods
1. The expression, purification and identification of the soluble PBP2a.
The mecA gene fragments including bases 76 to 2007, encoding soluble PBP2a, were amplified by PCR to construct prokaryotic expression vector pQE30-mecA. After the positive recombinant identified by enzyme digestion was sequenced correctly, the recombinant was transformed into E.coli M15. The bacteria were induced with 1 mmol/L IPTG, and the soluble PBP2a was expressed. After the protein was purified with Ni-NTA agarose, its purity, amino acid sequences and molecular wieght were identified. Subsequently, biological activities of the PBP2a such as the transpeptidase and beta-lactamase activities were respectively detected by spectrophotometric method, and binding activities to antibiotics in vitro were observed through double micropore dilution method and confocal technology.
2. Screening of anti-MRSA traditional Chinese herbs targeting PBP2a.
The soluble PBP2a was immobilized onto the surface of carboxymethyl dextran cuvette as a target. According to the affinity capacities of 26 traditional Chinese herbs for PBP2a, 10 herbs were selected to test their antibacterial efficiency, then the specific anti-MRSA herb was chosen out.
3. Isolation of the anti-MRSA components from Spica Prunellae and evaluation of their activities.
The affinities of aqueous extractions for PBP2a from different parts of Spica Prunellae were firstly measured by biosensor. Their antibacterial effects were evaluated through synergetic antibacterial experiment in order to confirm the active components for the next study. The anti-MRSA components from entire plants of Spica Prunellae were isolated through macroporous adsorptive resins AB-8, followed by affinity tests, antibacterial and synergetic antibacterial experiments. At the same time, the material attribute was identified. Then the growth curves of MRSA and synergetic antibacterial effects of the active component combined with beta-lactams were observed respectively. Finally, the possible mechanism of the component, as anti-MRSA potentiator, was approached by observing the morphology of MRSA through transmission electron microscope.
Results
1. After the mecA gene fragment including bases 76 to 2007 had been amplified by PCR, the recombinant pQE30-mecA was successfully constructed. By gene sequencing it showed that the mecA fragments consisted of 1932 bp as expected, with 9 site-mutations. The recombinant protein was 96% homology with the one in GenBank. Then, soluble PBP2a was efficiently expressed in E.coli M15. The purity of recombinant PBP2a was up to 90.6%. The N-terminal amino acid sequences were MRGSH HHHHH GSKDK as expectated. Its molecular wieght was identified as 74 kDa by mass spectrography analysis. The recombinant PBP2a had transpeptidase and beta-lactamase activities, and possessed some binding effects to beta-lactam antibiotics in vitro.
2. The soluble PBP2a was successfully immobilized onto the surface of carboxymethyl dextran cuvette. 10 traditional Chinese herbs with high affinity for PBP2a were selected out from 26 herbs. Scutellaria, Spica Prunellae and Coptis exerted better anti-MRSA effects, while Spica Prunellae showed the best synergetic antibacterial effect with oxacillin among 10 herbs.
3. The active components of Spica Prunellae were mainly from the ear of plants, but there was no difference from the entire plants in anti-MRSA. Therefore, SP2 fraction was isolated from entire plants of Spica Prunellae through macroporous adsorptive resins AB-8. This fraction not only had high affinity for PBP2a, but also could decrease the MICs of oxacillin, penicilin, ampicillin and sulbactam sodium-ampicillin sodium on MRSA by 256, 8, 16 and 4 times respectively. Their FIC were 0.007, 0.151, 0.064 and 0.276. SP2 fraction was water-soluble and consisted of some phenolic hydroxyl groups, with the molecular wieght range of 3~5 kDa. SP2 could intensify antibacterial effects of antibiotics on MRSA by destroying their cell walls.
Conclusions
The soluble PBP2a from clinical MRSA isolates has been successfully expressed by gene recombination technique. It is feasible to sreen anti-MRSA traditional Chinese herbs targeting the multidrug-resistant protein PBP2a through biosensor technology. Using this screening technique, Spica Prunellae, with high affinity for PBP2a and the best synergetic anti-MRSA effect with oxacillin, is selected out of 26 traditional Chinese herbs. Furthermore, water-soluble active component SP2 has been derived from entire plants of Spica Prunellae, which could enhance the antibacterial effects of beta-lactams on MRSA. Molecular weight of the active component in SP2 ranges from 3 to 5 kDa. The compound as an anti-MRSA potentiator may be discovered by further isolation and purification in the future.
引文
1 Styers D, Sheehan DJ, Hogan P, et al. Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States [J]. Ann Clin Microbiol Antimicrob.2006; 5:2.
2 Airesde SM, Crisostomo MI, Sanches IS, et al. Frequent recovery of a single clonal type of multidrug - resistant staphylococcus aureus from patients in two hospital in taiwan and China [J]. J Clin Microbiol. 2003; 41(1) :159 - 163.
3 Enright MC, Robinson DA, Randle G, et al. Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA) [J]. Proc Natl Acad Sci USA. 2002; 99(11):7687-7692.
4 Ayliffe GA. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus[J]. Clin Infect Dis. 1997; 24(1):74-79.
5 Gastmeier P, Sohr D, Geffers C, et al. Occurrence of methicillin-resistant staphylococcus aureus infections in German intensive care units[J]. Infection. 2002 ;30(4) :198-202.
6赖晓全,王洪源.重症监护病房MRSA感染及相关因素的研究[J].内科急危重症杂志, 2005;11(4 ):178-183.
7 Mizobuchi S, Minami J, Jin F, et al. Comparison of the virulence of methicillin- resistant and methicillin-sensitive Staphylococcus aureus [J] .Microbiol Immunol. 1995; 39(8):545-550.
8 Brown DF, Reynolds PE. Intrinsic resistance to beta-lactam antibiotics in Staphylococcus aureus[J]. FEBS Lett. 1980;122(2):275-278.
9 Hartman BJ, Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus[J]. J Bacteriol.1984; 158(2):513-516.
10 Goffin C, Ghuysen JM. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs[J]. Microbiol Mol Biol Rev. 1998; 62(4):1079-1093.
11 Murakami K, Fujimura T, Doi M. Nucleotide sequence of the structural gene for the penicillin- binding protein 2 of Staphylococcus aureus and the presence of a homologous gene in other staphylococci[J]. FEMS Microbiol Lett. 1994; 117 (2): 131-136.
12 Lim D, Strynadka NC. Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus[J]. Nat Struct Biol. 2002; 9(11):870-876.
13 Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution of methicillin-resistant Staphylococcus aureus[J]. Trends Microbiol.2001; 9(10):486-493.
14 Fuda CC, Fisher JF, Mobashery S. beta-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome[J]. Cell Mol Life Sci. 2005; 62(22): 2617-2633.
15 Massova I, Mobashery S. Kinship and diversification of bacterial penicillin-binding proteins and beta-lactamases[J]. Antimicrob Agents Chemother. 1998; 42(1):1-17.
16 Paetzel M, Danel F, de Castro L, et al. Crystal structure of the class D beta-lactamase OXA-10[J]. Nat Struct Biol. 2000; 7(10):918-925.
17 Pinho MG, de Lencastre H, Tomasz A. An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci[J]. Proc Natl Acad Sci USA. 2001; 98(19):10886-10891.
18 Fuda C, Hesek D, Lee M, et al. Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall[J]. J Am Chem Soc. 2005; 127(7):2056-2057.
19 Rohrer S, Berger-Bachi B. FemABX peptidyl transferases: a link between branched-chain cell wall peptide formation and beta-lactam resistance in gram-positive cocci[J]. Antimicrob Agents Chemother. 2003; 47(10):837-846.
20 Ryffel C, Strassle A, Kayser FH, et al. Mechanisms of heteroresistance in methicillin-resistant Staphylococcus aureus[J].Antimicrob Agents Chemother. 1994; 38(4):724-728.
21 Katayama Y, Zhang HZ, Chambers HF. PBP2a mutations producing very-high-level resistance to beta-lactams [J].Antimicrob Agents Chemother, 2004;48 (2) : 453-459.
22 Oldfield EC. No Mercy from MRSA [J]. Rev Gostroenterol Disord.2004;4 (2) : 95-96.
23 Walsh TR, Howe RA. The prevalence and mechanisms of vancomycin resistance in Staphylococcus aureus[J]. Annu Rev Microbiol. 2002; 56(30):657-675.
24 Schmidt-Ioanas M, de Roux A, Lode H. New antibiotics for the treatment of severe staphylococcal infection in the critically ill patient[J]. Curr Opin Crit Care. 2005; 11(5):481-486.
25 Iizawa Y, Nagai J, Ishikawa T, et al. In vitro antimicrobial activity of T-91825, a novel anti-MRSA cephalosporin, and in vivo anti-MRSA activity of its prodrug, AK-599[J]. J Infect Chemother. 2004; 10(3): 146-156.
26 Meng J, Hu B, Liu J,et al. Restoration of oxacillin susceptibility in methicillin -resistant Staphylococcus aureus by blocking the MecR1-mediated signaling pathway [J]. J Chemother. 2006;18(4): 360-365.
27 Li S, Roberts RW. A novel strategy for in vitro selection of peptide-drug conjugates[J]. Chem Biol. 2003; 10(3):233-239.
28 Therrien C,Levesque RC. Molecular basis of antibiotic resistance and beta-lactamase inhibition by mechanismbased inactivators: perspectives and future directions[J]. FEMS Microbiol Rev. 2000; 24(3): 251-262.
29陈文伟,肖丽英. 8种中草药对耐药金葡菌的最小抑菌浓度检测[J].广东药学2005; 15(3):72-73.
30 Genfa L, Jiang Z, Hong Z, et al. The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology[J]. Int Immunopharmacol. 2005 ; 5(6): 1007-1017.
31孙长贵译,抗微生物药物敏感性试验执行标准(CLSI)[M];第十六版信息增刊; 2006.
32 Zhao GS, Yeh WK, Carnahan RH, et al. Biochemical characterization of penicillin- resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics. J Bacteriology.1997, 179(15): 4901–4908.
33 Wang JT ,Chen YC ,Yang TL , et al . Molecular epidemiology and antimicrobial susceptibility of methicillin - resistant staphylococcus aureus in Taiwan [J] . Diagn Microbiol Infect Dis.2002;42(3):199 - 203.
34 Aires de SM, Crisostomo MI, Sanches IS, et al . Frequent recovery of a single clonal type of multidrug - resistant staphylococcus aureus from patients in two hospital in taiwan and China[J ] . J Clin Microbiol.2003;41(1):159 - 163.
35 Tenover FC, Weigel LM, Appelbaum PC, et al. Vancomycin– resistant staphylococcus aureus isolate from a patient in pennsylvania[J]. Antimicrob Agents Chemother. 2004; 48(1):275 - 280.
36 CDC. Brief report: vancomycin-resistant S taphy lococcus aureus-New York, 2004[J] . Morb Mortal Wkly Rep.2004;53(15) :322-323.
37 Lim D, Strynadka NC. Structural basis for theβ-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus[J]. Nat Struct Biol. 2002; 9(11): 870–876.
38 ErnieWU, Joann H, Larry C, et al. Construction of a water-soluble form of Penicillin- binding protein 2a from a methicillin-resistant Staphylococcus aureus isolates[J]. Antimicrobial Agents and Chemotherapy.1992;36(3):533-539.
39 Bujard H,Gentz R,Lanzer M,et al.A T5 promotor-based transcription-translation system for the analysis of proteins in vitro and in vive.Methods Enzymol.1987;155:416-433.
40 Casey JL,Keep PA,Chester KA,et al.Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography[J].Immunol Methods.1995;179(1):105–116.
41 Will CL, Schneider C, Reed R,et al.Indentification of both shared and distinct proteins in the major and minor.Science.1999; 284(5422):2003-2005.
42 Skerra A, Pfitzinger I, Pluckthun A.The functional expression of antibody Fv fragments in Escherichia coli:improved vectors and a generally applicable purification technique. Bio Technol.1991;9(3):273– 278.
43 Mark P, Franck D, Liza de C,et al. Crystal structure of the class Dβ-lactamase OXA-10. Nature Structural Biology. 2000;7(10):918-925.
44 Massova I, Mobashery S. Kinship and Diversification of Bacterial Penicillin-Binding Proteins and b-Lactamases. Antimicrob Agents Chemother. 1998;42(1):1-17.
45 Girlich D, Poirel L,Leelaporn A, et al. Molecular epidemiology of the integron - located veb - 1 extended spectrum - lactamase in nosocomial enterobacterial isolates in bangkok, thainland. J Clin Microbiol.2001; 39(1):175 - 182.
46 Patzer J, Toleman MA, Deshpande LM, et al. Peseudomonas aeruqinose strains harbouring an unusual bla vim - 4gene cassette isolated from hospitalized children in poland ( 1998 - 2001 ) . J Antimicrob Chemother. 2004;53(3): 451 - 456.
47 Frank LJ, Wisniewski D, Hammond GG, et al. High-yield expression,refold,and purification of penicillin-binding protein 2a from methicillin-resistant staphylococcus aureus strain 27R. Protein Expression and Purification. 1995;6(5):671-678.
48 Baird CL, Myszka DG. Current and emerging commerical optical biosensor[J]. J MolRecognit. 2001;14(5):261-268.
49 Gaudin V, Fomtaine J, Maris P. Screening of penicillin residues in milk by a surface plsamon resonance-baced biosensor assay: comparison of chemical and enzymatic sample pre-treatment[J]. Anal chim Acta. 2001;436:191-198.
50 Baxter GA, Ferguson JP, O’connor MC, et al. Dectection of streptomycin residue in whole milk using an optical immunobiosensor[J]. J Agric Food Chem. 2001;49(7):3204-3207.
51 Abad LW, Neumann M, Tobias L, et al. Development of a biosensorbased method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors[J]. Anal Biochem. 2002;310(1):107–113.
52 Barrington RA, Pozdnyakova O, Zafari MR, et al. B lymphocyte memory: role of stroma cell complement and Fc_RIIB receptors[J]. J Exp Med. 2002;196(9):1189–1199.
53 Rebecca LR, David GM. A survey of the year 2002 commercial optical biosensor literature [J]. Mol Recognit. 2003; 16(6): 351–382.
54 Rebecca LR, David GM. Survey of the year 2001 commercial optical biosensor literature [J]. Mol Recognit. 2002; 15(6): 352–376.
55 Lowe P, Clark T, Davies R, et al. New approaches for the analysis of molecular recognition using Iasys evanescent wave biosensor[J]. J Mol Recognit.1998; 11(1-6):194-199.
56 Edwards P, Maule C, Leatherbarrow R, et al. Second-order .kinetic analysis of IAsys biosensor data: its use and applicability[J]. Anal Biochem. 1998;263(1):1-12.
57吴翀,蒋栋能,周红等.大蒜抗内毒素组分的分离及活性分析.重庆医学, 2006;35 (7): 622-629.
58 Wu Z, Wang B, Dong S, et al. Amperometric glucose biosensor based on lipid film[J]. Biosens Bioelectron. 2000; 15(3-4): 143-147.
59 Yoon HC, Hong MY, Kim HS, et al. Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode[J]. Anal Biochem. 2000; 282(1):121-128.
60 Rogers KR. Principles of affinity based biosensors[J]. Mol Biotechnol. 2000;14(2): 109-129.
61 Ito H, Naito S, Kato N. Hexagonal assembly of the magnesium salt of an R form lipopolysaccharide from Klebsiella pneumoniae: its lowered stability compared withoriginal non-electrodialyzed preparation[J]. Microbiol Immunol. 2000; 44(5): 395-400.
62程娟,郑江,周红,等.以CpG ODN为靶点应用生物传感器技术筛选抗炎中药[J].中国临床药理学与治疗学,2005;10(11): 1240-1244.
63 Magdalena Labieniec, Teresa Gabryelak. Effects of tannins on Chinese hamster cell line B14[J]. Mutation Research/Genetic Toxicology and Environmental Mutagenesis.2003; 539(1-2): 127-135.
64 Rich RL, Myszka DG. Survey of the year 2004 commercial optical biosensor literature[J]. J Mol Recognit. 2005; 18(6): 431-478.
65 Kim KJ,Yu HH,Jeong SI,et al.Inhibitory effects of Caesalpinia sappan on growth and invasion of methicillin-resistant Staphylococcus aureus[J].J Ethnopharmacol. 2004;91(1):81-87.
66平松启一.MC-200616 potentiates the activity ofβ-lactamantibiotics against MRSA[J].日化杂志,1997;45:801.
67 Shibata H, Shirakata C, Kawasaki H, et al.Flavone markedly affects phenotypic expression of beta-lactam resistance in methicillin-resistant Staphylococcus aureus strains isolated clinically[J].Biol Pharm Bull.2003;26(10):1478-1483.
68王莉娟,关显智,李菁华.中药双花对金黄色葡萄球菌R质粒消除作用的实验研究[J].武警医学,1996;7(6):345.
69林杉,李仲昆,赵云,等.双黄连粉针与头孢唑啉伍用效果机理研究[J].中国药房,2000;11(1):18-19.
70李仝,胡凯文,陈信义,等.浙贝母对呼吸系统耐药金黄色葡萄球菌逆转作用的临床研究[J].北京中医药大学学报, 2001;24(5):51-52.
71 Liu IX(杨媛译).黄芩苷与β-内酰胺类抗生素对β-内酰胺耐药性金黄色葡萄球菌的协同作用[J].国外医药·抗生素分册, 2001;22(3):142.
72 Hatano T,Kusuda M,Hori M,et al.Theasinensin A, a tea polyphenolformed from epigallocatechin gallate, suppresses antibiotic resistance of methicillin-resistant Staphylococcus aureus[J].Planta Med.2003;69(11):984-989.
73 Li RC.New pharmacodynamic parameters for antimicrobial agents[J].Int JAntimicrob Agent.2000; 13(4):229-235.
74刘成梅主编.天然产物有效成分的分离与应用[M].北京:化学工业出版社.2003;第一版.
75屠万倩,李昌勤.大孔吸附树脂在医药研究领域中应用的进展[J].中医研究,2006;19(4):61-64.
76何伟,李伟.大孔树脂在中药成分分离中的应用[J].南京中医药大学学报,2005; 21(2):134-136.
77林秋凤,王凌云,李药兰,等. AB - 8大孔吸附树脂分离长瓣金莲花总黄酮.暨南大学学报,2003;24(1):55-58.
78刘悦,宋少江,徐绥绪.夏枯草的化学成分及生物活性研究进展[J].沈阳药科大学学报, 2003; 20 (1) : 55-59.
79刘敬顺.夏枯草药理作用实验研究简况[J].山西中医, 2002;18(2) : 52-53.
80 Li J, GuoWJ, Yang QY. Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15 [ J ]. World J Gastroenterol.2002;8 (3) : 493-495.
81李晓蒙,廖华卫.夏枯草中熊果酸的含量测定[J].广东药学院学报, 2002;18 (1): 31-32.
82许斌,李莉,徐焰,等.植物消毒剂对悬液中细菌和病毒的灭活作用[J].第三军医大学学报,2005;27 (9) : 907– 909.
83 Chattopadhyay D, Arunachalam G, Mandal AB, et al. Antimicrobial and anti- inflammatory activity of folklore:Mallotus peltatus leaf extract[J].J Ethnopharmacol. 2002;82 (2 /3) : 229– 237.
84 Kim SY, Kim SH, Shin HY, et al.Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production[J].Exp Biol Med (Maywood). 2007;232(7): 921-926.
85 Harpu US, Saracoglu I, Ogihara Y. Effects of two Prunella species on lymphocyte proliferation and nitric oxide production[J]. Phytother Res. 2006, 20(2): 157-159.
86 Sun HX, Qin F, Pan YJ. In vitro and in vivo immunosuppressive activity of Spica Prunellae ethanol extract on the immune responses in mice[J]. J Ethnopharmacol. 2005;101(1-3): 31-36.
87 Nolkemper S, Reichling J, Stintzing FC, et al. Antiviral effect of aqueous extracts from species of the Lamiaceae family against Herpes simplex virus type 1 and type 2 in vitro[J] . Planta Med. 2006;72(15): 1378-1382.
88 Fang X, Yu M M,Yuen WH,et al.Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages [J ]. Int J Mol Med. 2005;16(6): 1109-1116.
89肖丽英,黄焯坡. 3种中草药对耐药金葡菌的敏感性评价[J].时珍国医国药,2001; 12(10):878-879.
90董剑英,杨静,赵春芝等.夏枯草提取物的薄层层析及体外抗菌活性研究[J].军事医学科学院院刊,2005;29(5):450-452.
91 Gibbons S. Anti-staphylococcal plant natural products[J]. Nat Prod Rep. 2004;21(2): 263-277.
92 Kim L, Frederick MA. Prospects for plant-derived antibacterials[J]. Nat Biotech. 2006;24(12):1504-1507.
1 Oldfield EC. No Mercy from MRSA[J].Rev Gostroenterol Disord. 2004;4 (2) : 95-96.
2陈蕾,邓诗琳,梁建伟等.烧伤重症监护病房细菌学调查及其药物敏感性分析[J].中华烧伤杂志, 2005;21(4):270-272.
3魏迪南,刘军.烧伤患者细菌学调查及耐药性分析[J].中华烧伤杂志, 2006;22(2): 92 -95.
4 Asoh N,Masaki H,Watanabe H, et al. Molecular characterization of the transmission between the colonization of methicillin-resistant Staphylococcus aureus to human and environmental contamination in geriatric[J]. Intern Med. 2005, 44(1) : 41-45.
5 Salmenlinna S,Lyytikainen O, Vuopio VJ. Community-acquired methicillin- resistant Staphylococcus aureus ,Finland[J]. Emerg Infect Dis.2002;8 (6) :602-607.
6 Shopsin B, Barry N. Kreiswirth. Molecular epidemiology of methicillin- resistant Staphylococcus aureus[J]. Emerg Infect Dis. 2001;(2) :323-326.
7李家泰, Weinstein AJ,杨敏.中国细菌耐药监测研究[J].中华医学杂志,2001;81 (1) :8-16.
8 CDC. Brief report : vancomycin-resistant S taphy lococcus aureus-New York , 2004[J ] . Morb Mortal Wkly Rep.2004;53(15) :322-323.
9马越,陈鸿波,姚蕾等.耐甲氧西林金黄色葡萄球菌对万古霉素敏感性的变迁[J].中华内科杂志,2002;41 (1) :31-33.
10 Rosato AE, Kreiswirth BN, Craig WA, et al. mecA-blaZ corepressors in clinical Staphylococcus aureus isolates[J]. Antimicrob Agents Chemother. 2003;47(4): 1460–1463.
11 Lowy FD. Antimicrobial resistance: the example of Staphylococcus aureus[J]. J Clin Invest. 2003;111(9):1265-1273.
12 Hiramatsu K, Asada K, Suzuki E, et al. Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) [J]. FEBS Lett. 1992; 298(2-3): 133–136.
13 Clarke SR, Dyke KG. Studies of the operator region of the Staphylococcus aureus beta-lactamase operon[J]. J Antimicrob Chemother. 2001;47(4): 377–389.
14 McKinney TK, Sharma VK, Craig WA, et al. Transcription of the gene mediatingmethicillin resistance in Staphylococcus aureus (mecA) is corepressed but notcoinduced by cognate mecA andβ-lactamase regulators[J]. J Bacteriol. 2001; 183(23): 6862–6868.
15 Ryffel C., Kayser F. H. and Berger-Bachi B. Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci[J]. Antimicrob. Agents. Chemother. 1992; 36: 25–31
16 Melckebeke HV, Vreuls C, Gans P, et al. Solution structural study of BlaI: implications for the repression of genes involved in b-lactam antibiotic resistance[J]. J Mol Biol. 2003;333(4): 711–720.
17 Wilke MS, Hills TL, Zhang HZ, et al. Crystal structures of the Apo and penicillinacylated forms of the BlaR1β-lactam sensor of Staphylococcus aureus[J]. J Biol Chem. 2004;279(45): 47278–47287.
18 Moews PC, Knox JR, Dideberg O, et al. Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution[J]. Proteins .1990;7(2): 156–171.
19 Kerff F, Charlier P, Colombo ML, et al. Crystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis[J].Biochemistry.2003; 42(44): 12835–12843.
20 Safo MK, Zhao Q, Ko TP, et al. Crystal structures of the BlaI repressor from Staphylococcus aureus and its complex with DNA: insights into transcriptional regulation of the bla and mec Operons[J].J Bacteriol. 2005;187(5): 1833–1844.
21 Rowland SJ, Dyke KG. Tn552, a novel transposable element from Staphylococcus aureus[J]. Mol Microbiol. 1990;4(6): 961–975.
22 Hackbarth CJ, Chambers HF. blaI and blaR1 regulate beta-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus[J]. Antimicrob. Agents. Chemother. 1993;37(5): 1144–1149
23 Wang PZ, Projan SJ, Novick RP. Nucleotide sequence ofβ-lactamase regulatory genes from staphylococcal plasmid pI258[J]. Nucleic Acids Res.1991;19(14): 4000.
24 Herzberg O. Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution[J]. J Mol Biol. 1991;217(4): 701–719.
25 Massova I, Mobasherv S. Kinship and diversification of bacterial penicillin- binding proteins andβ-lactamases[J]. Antimicrob Agents Chemother.1998;42(1): 1–17.
26 Ghuysen JM. Molecular structures of penicillin-binding proteins andβ-lactamases [J]. Trends Microbiol. 1994;2(10): 372–380
27 Zhang HZ, Hackbarth CJ, Chansky KM, et al. A proteolytic transmembrane signaling pathway and resistance to beta-lactams in staphylococci[J]. Science.2001;291(5510): 1962–1965.
28 Archer GL, Bosilevac JM. Signaling antibiotic resistance in staphylococci[J]. Science. 2001;291(5510): 1915–1916.
29 Herzberg O, Moult J. Bacterial resistance to blactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution[J]. Science. 1987;236(4802): 694–701.
30 Chen CC, Herzberg O. Relocation of the catalytic carboxylate group in class Aβ-lactamase: the structure and function of the mutant enzyme Glu166Gln:Asn170Asp [J]. Protein Eng. 1999;12(7): 573–579.
31 Birck C, Cha JY, Cross J, et al. X-ray crystal structure of the acylatedβ-lactam sensor domain of BlaR1 from Staphylococcus aureus and the mechanism of receptor activation for signal transduction[J]. J Am Chem Soc. 2004;126(43): 13945–13947.
32 Hardt K, Joris B, Lepage S, et al. The penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four-alpha-helix bundle[J]. Mol Microbiol. 1997; 23(5): 935–944.
33 Hanique S, Colombo ML, Goormaghtigh E, et al. Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction[J]. J Biol Chem.2004;279(14): 14264–14272.
34 Lewis RA, Dyke KG. MecI represses synthesis from the beta-lactamase operon of Staphylococcus aureus[J]. J Antimicrob Chemother. 2000;45(2): 139–144.
35 Gregory PD, Lewis RA, Curnock SP, et al. Studies of the repressor (BlaI) of b-lactamase synthesis in Staphylococcus aureus[J]. Mol. Microbiol. 1997;24(5): 1025–1037.
36 Sharma VK, Hackbarth CJ, Dickinson TM, et al. Interaction of native and mutant MecI repressors with sequences that regulate mecA, the gene encoding penicillin binding protein 2a in methicillin-resistant staphylococci[J]. J Bacteriol. 1998; 180(8):2160–2166.
37 Garcia-Castellanos R, Mallorqui-Fernandez G, Marrero A, et al. On the transcriptional regulation of methicillin resistance: MecI repressor in complex with its operator[J]. J Biol Chem. 2004;27917): 17888–17896.
38 Golemi-Kotra D, Cha JY, Meroueh SO, et al. Resistance to beta-lactam antibiotics and its mediation by the sensor domain of the transmembrane BlaR signaling pathway in Staphylococcus aureus[J]. J Biol Chem. 2003;278(20): 18419–18425.
39 Ryffel C, Kayser FH, Berger-Bachi B. Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci[J]. Antimicrob Agents Chemother. 1992;36(1): 25–31.
40 Berger-Bachi B, Rohrer S. Factors influencing methicillin resistance in staphylococci [J]. Arch Microbiol. 2002;178(3): 165–171.
41 Clarke SR, Dyke KG. Studies of the operator region of the Staphylococcus aureus beta-lactamase operon[J]. J Antimicrob Chemother. 2001;47(4):377-389.
42 Linares J. The VISA/ GISA problem: therapeutic implications[J] Clin Microbiol Infect.2001;7 (Suppl 4) :8-15.
43 Couto I, Wu SW, Tomasz A,et al. Development of methicillin resistance in clinical isolates of Staphylococcus sciuri by transcriptional activation of the mecA homologue native to the species[J]. J. Bacteriol. 2003;185(2): 645–653.
44 Wu SW, Lencastre H, Tomasz A. Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus[J]. J Bacteriol. 2001;183(8): 2417–2424.
45 Fuda C, Suvorov M, Shi Q, et al. Shared functional attributes between the mecA gene product of Staphylococcus sciuri and penicillin-binding protein 2a of methicillin -resistant Staphylococcus aureus [J]. Biochemistry.2007;46(27): 8050-8057.
46 Fuda CC, Fisher JF, Mobashery S. b-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome[J]. Cellular Molecular Life Sciences. 2005; 62(22) :2617–2633.
47 Pinho MG, Filipe SR, de Lencastre H, et al. Complementation of the essential peptidoglycan transpeptidase function of penicillin-binding protein 2 (PBP2) by the drug resistance protein PBP2A in Staphylococcus aureus[J]. J Bacteriol.2001;183(22):6525-6531.
48 Katayama Y, Zhang HZ, Chambers HF. PBP2a mutations producing very-high-level resistance to beta-lactams [J].Antimicrob Agents Chemother. 2004; 48 (2) : 453-459.
49 Noriko K, Kyoko KA, Hiroko KM, et al. Eagle-Type Methicillin Resistance : New Phenotype of High Methicillin Resistance under mec Regulator Gene Control[J]. Antimicrobial Agents Chemother. 2001;45 (3) :815-824.
50 Berger BB, Strassle A,Gustafson JE, et al. Mapping and characterization of mutiple chromosome factors involved in methincillin resistance in Staphyloccus aureus[J]. Antimicrob Agents Chemother. 1992; 36 (7):1367-1373.
51 Rohrer S, Ehlert K, Tschierske M, et al. The essential Staphy lococcus aureus gene fm hB is involved in the firststep of peptidoglycan pentaglycine interpeptide formation[J]. Proc Natl Acad Sci USA. 1999, 96(16): 9351-9356.
52 Berger BB, Rohrer S. Factors influencing methicillin resistance in staphylococci[J]. Arch Microbiol. 2002;178(3):165-171.
53 Noguchi N, Suwa J, Narui K, et al . Susceptibilities to antiseptic agents and dist ribution of antiseptic-resistance genes qacA/ B and smr of methicillin-resistant Staphy lococcus aureus isolated in Asia during 1998 and 1999[J]. J Med Microbiol. 2005;54 (6) : 557-565.
54 Kristiansen MM, Leandro C, Ordway D, et al . Phenothiazines alter resistance of methicillin-resistant strains of S taphy lococcus aureus (MRSA) to oxacillin in vitro[J] . Int J Antimicrob Agents. 2003;22(3) :250-253.
55 Palumbi SR. Humans as the world’s greatest evolutionary force[J]. Science. 2001;293(5536): 1786–1790.
56 Fung TJC, Clark J, Minassian B, et al. In vit ro and in vivo activities of a novel cephalosporin,BMS-247243,against methicillin-resistant and susceptible staphylococci [J ] .Antimicrob Agents Chemother. 2002;46 (4) :971-976.
57 Li Q, Lee JY, Castillo R, et al. NB2001 , a novel antibacterial agent with broad - spectrum activity and enhanced potency againstβ- lactamase - producing strains [J]. Antimicrob Agents Chemother. 2002;46 (5) :1262-1268.
58 Sunagawa M, Itoh M , Kubota K, et al . New anti - MRSA andanti - VRE carbapenem: synthesis and structure - activity relationships of realtionships of 1β-methyl- 2-( thiazol-2-ylthio)carbapenems[J]. J Antibiot. 2002 ;55 (8) :722-757.
59 Guignard B, Entenza JM, Moreillon P. Beta-lactams against methicillin-resistant Staphylococcus aureus[J]. Curr Opin Pharmacol. 2005;5(5): 479-489.
60 Wang J, Kodali S,Lee SH,et al. Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties[J]. Proc Natl Acad Sci USA. 2007; 104(18): 7612-7616.
61 Yamamoto Y, Kurazono M. A new class of anti-MRSA and anti-VRE agents: preparation and antibacterial activities of indole-containing compounds[J]. Bioorg Med Chem Lett. 2007; 17(6): 1626-1628.
62 Turos E, Shim JY, Wang Y,et al. Antibiotic-conjugated polyacrylate nanoparticles: new opportunities for development of anti-MRSA agents[J]. Bioorg Med Chem Lett. 2007;17(1): 53-56.
63 Shinabarger DL, Marotti KR, Murray RW, et al. Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions[J]. Antimicrob Agents Chemother. 1997; 41(10): 2132-2136.
64 Cercenado E, Garcia GF, Bouza E. In vitro activity of linezolid against multiply resistant Gram-positive clinical isolates[J]. J Antimicrob Chemother. 2001;47(1): 77-81.
65 Tsiodras S, Gold HS, Sakoulas EGM, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus[J]. Lancet. 2001;358(9277): 207-208.
66 Good L, Awasthi SK, Dryselius R, et al. Bactericidal antisense effects of peptide-PNA conjugates[J]. Nat Biotechnol. 2001;19(4): 360-364.
67 Torres VC, Tsiodras S, Gold HS, et al. Restoration of Vancomycin susceptibility in Enterococcus faecalis by antiresistance determinant gene transfer[J]. Antimicrob Agents Chemother. 2001;45(3): 973-975.
68 Rapaport E, Levina A, Metelev V, et al. Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains[J]. Proc Natl Acad Sci USA. 1996;93(2): 709-713.
69 Harth ZPC, Tang JY, Tabatadze D, et al. Treatment of Mycobaterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate /glutamine cell wall struture,and bacterial replication[J]. Proc Natl Acad Sci USA. 2000;97(1): 418-423.
70 Meng J, Hu B, Liu J,et al. Restoration of oxacillin susceptibility in methicillin -resistant Staphylococcus aureus by blocking the MecR1-mediated signaling pathway [J]. J Chemother. 2006;18(4): 360-365.
71刘杰,罗晓星,孟静茹等.脱氧核酶抑制耐甲氧西林金黄色葡萄球菌耐药基因的表达[J].中国临床药理学与治疗学,2004; 9 (6 ): 641-645.
72侯征,孟静茹,扈本荃等.脱氧核酶抑制耐药基因mecR1的表达逆转MRSA耐药性[J].中国抗生素杂志,2006;31(3):144-148.
73 Roth DM, Senna J P, Machado DC. Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti-methicillin-resistant Staphylococcus aureus[J].Genet Mol Res. 2006; 5(3): 503-512.
74王汉敏,陈林娜,周立勤等.电镜下观察中药制剂对耐药菌株的抑菌作用[J].国际检验医学杂志,2007;28(1):84-86.
75杨明炜,陆付耳,徐丽君等. 20种清热解毒中药对耐甲氧西林金黄色葡萄球菌体外抑菌的初步观察[J].中国药师,2006;9(2):141-142.
76陈文伟,肖丽英. 8种中草药对耐药金葡菌的最小抑菌浓度检测[J].广东药学,2005;15(3):72-73.
77左国营,王根春,徐贵丽等. 30种中草药提取物体外抗MRSA的筛选研究[J].中国现代应用药学,2006;23(4):293-295.
78左国营,余巍,徐贵丽等. 18种中草药提取物抗金葡菌作用的筛选研究[J].中国药师,2005;8(7):606-608.
79李仲兴,王秀华,张明明等.五倍子乙醇提取物对金葡菌的体外抗菌研究[J].中国新药与临床药理, 2005;16(2):103-105.
80 Kawazoe K, Tsubouchi Y, Abdullah N, et al. Sesquiterpenoids from Artemisia gilvescens and an anti-MRSA compound[J]. J Nat Prod. 2003;66(4): 538–539 .
81 Shibata H, Shirakata C, Kawasaki H, et al.Flavone markedly affects phenotypic expression of beta-lactam resistance in methicillin-resistant Staphylococcus aureus strains isolated clinically[J].Biol Pharm Bull. 2003;26(10):1478-1483.
82 Kim KJ, Yu HH, Jeong SI, et al. Inhibitory effects of Caesalpinia sappan on growth and invasion of methicillin-resistant Staphylococcus aureus[J].J Ethnopharmacol. 2004;91(1):81-87.
83 Kubo I, Xiao P, Fujita K. Anti-MRSA activity of alkyl gallates[J]. Bioorg Med Chem Lett. 2002;12(2): 113–116.
84王莉娟,关显智,李菁华.中药双花对金黄色葡萄球菌R质粒消除作用的实验研究[J].武警医学,1996;7(5):345-347.
85林杉,李仲昆,赵云等.双黄连粉针与头孢唑啉伍用效果机理研究[J].中国药房,2000;11(1):18-19.
86李仲昆,林杉,李海蜀等.双黄连粉针与4种抗生素伍用的体外最小抑菌浓度研究[J].中成药,1999;21(3):137-139.
87李仝,胡凯文,陈信义等.浙贝母对呼吸系统耐药金黄色葡萄球菌逆转作用的临床研究[J].北京中医药大学学报,2001;24(5):51-52.
88 Liu IX(杨媛译).黄芩苷与β-内酰胺类抗生素对β-内酰胺耐药性金黄色葡萄球菌的协同作用[J].国外医药·抗生素分册,2001;22(3):142.
89 Hatano T,Kusuda M,Hori M,et al.Theasinensin A, a tea polyphenolformed from epigallocatechin gallate,suppresses antibiotic resistance of methicillin-resistant Staphylococcus aureus[J]. Planta Med.2003;69(11):984-989.
90山口晃史(张来彪,赛敏摘译).难治性呼吸系统感染性疾病与儿茶精吸入疗法[J].日本医学介绍.2001;22(9):410～411.
91 Li RC . New pharmacodynamic parameters for antimicrobial agents[J] . Int J Antimicrob Agent.2000;13(4):229-235.
92 Kim SY, Kim SH, Shin HY, et al.Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production[J].Exp Biol Med (Maywood). 2007;232(7): 921-926.
93 Harpu US, Saracoglu I, Ogihara Y. Effects of two Prunella species on lymphocyte proliferation and nitric oxide production[J]. Phytother Res. 2006;20(2): 157-159.
94 G.ibbons S. Anti-staphylococcal plant natural products[J]. Nat Prod Rep. 2004;21(2) :263-277.
95 Kim L, Frederick MA. Prospects for plant-derived antibacterials[J]. Nature Biotechnology, 2006;24(12):1504-1507.
1 Styers D, Sheehan DJ, Hogan P, et al. Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States[J]. Ann Clin Microbiol Antimicrob.2006;5:2.
2 Airesde SM, Crisostomo MI, Sanches IS, et al. Frequent recovery of a single clonal type of multidrug - resistant staphylococcus aureus from patients in two hospital in taiwan and China[J]. J Clin Microbiol. 2003;41(1) :159 - 163.
3 Lim D, Strynadka NC. Structural basis for theβ-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus[J]. Nat Struct Biol,2002,9(11): 870 - 876.
4 Sambrook J,Russell DW. Molecular Cloning: A Laboratory Manual,3rd ed[M].New York: Cold Spring Harbor Laboratory.2001;1228-1232.
5 Zhao GS, Yeh WK, Carnahan RH, et al. Biochemical characterization of penicillin- resistant and sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics[J]. J Bacteriology. 1997; 179(15): 4901 - 4908.
6 Ehlert K. Methicillin-resistance in Staphylococcus aureus- molecular basis, novel targets and antibiotic therapy[J]. Curr Pharm Des.1999;5(2):45-55.
7 Enright MC, Robinson DA, Randle G, et al. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA)[J]. Proc Natl Acad Sci USA. 2002;99(11):7687-7692.
8 Oldfield EC 3 rd. No Mercy from MRSA. Rev Gostroenterol Disord. 2004; 4 (2): 95.
9 Walsh TR, Howe RA. The prevalence and mechanisms of vancomycin resistance in Staphylococcus aureus[J]. Annu Rev Microbiol. 2002; 56:657-675.
10 McDonald LC, Hageman JC.Vancomycin intermediate and resistant Staphylococcus aureus. What the nephrologist needs to know[J]. Nephrol News Issues. 2004;18(11): 63-72.
11 Daniel L, Natalie CJS. Structural basis for the b-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus[J]. Nature struct boil. 2002; 9(11): 870-876.
12 Ito T, Okuma K, Ma XX, et al. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC[J]. Drug Resist Updat. 2003; 6: 41-52.
13 Ma XX, Ito T, Tiensasitorn C, et al. Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains[J]. Antimicrob Agents Chemother. 2002; 46:1147-1152.
14 Leatherbarrow JR, Edwards PR. Analysis of molecular recognition using optical biosensors[J]. Curr Opin Chem Biol.1999;3(5):544 - 547.
15 Lowe P, Clark T, Davies R,et al. New approaches for the analysis of molecular recognition using iasys evanescent wave biosensor[J]. J Mol Recognit. 1998; 11(1-6): 194-199.
16 Genfa L, Jiang Z, Hong Z, et al. The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology[J]. Int Immunopharmacol. 2005; 5(6): 1007-1017.
17 Wenwei C, Liying X. Detection of MIC of 8 Chinese Herbage Medicines against MRSA[J].Guangdong Pharmaceutical Journal. 2005;15(3):72-73.
18 Liying X, Chaopo H. A Discussion on Sensitivity of 23 Chinese Herbage Medicines against MRSA[J]. Lishizhen Medicine and Medical Research. 2001;12(10):878-879.