细菌对氨基糖苷类抗生素的耐药机制

氨基糖苷类抗生素起源于1944年链霉素的发现,其主要抑制细菌蛋白质的合成,以及破坏细菌胞浆膜的完整性。具有抗菌谱广、杀菌完全、与β-内酰胺等抗生素有很好的协同作用,是最常用的抗感染药物。它依赖电子转运,通过细菌内膜而到达胞质溶胶中后,与核糖体30S亚基结合,但这种结合并不阻止起始复合物的形成,而是通过破坏控制翻译准确性的校读过程来干扰新生链的延长。随着临床的广泛和不科学使用,细菌对氨基糖苷类抗生素的耐药性逐年增高,其耐药机制也十分复杂,主要包括细菌产生使抗生素失活的修饰酶、细菌对药物的摄取和积累减少,以及核糖体结合位点的减少等;另外还发现有新的机制参与细菌对氨基糖苷类抗生素的耐药过程。现将细菌对氨基糖苷类抗生素的耐药机制进行综述,并探讨联合用药控制耐药。     

氨基糖苷类修饰酶引起的细菌耐药性机制的研究进展

氨基糖苷类抗生素是高效、广谱的杀菌药物。随着在临床的广泛应用,抗生素的抗药性日趋严重,这在很大程度上降低了其临床应用的潜力。其中,最主要的原因就是细菌产生了一系列修饰酶修饰抗生素的特定基团,使其失去药效。细菌产生的修饰酶种类众多,主要包括磷酸化、乙酰化和腺苷化修饰酶。研究发现,一种酶可以修饰多种抗生素,同时,一种抗生素也可以被多种修饰酶修饰。由于修饰酶底物的广谱性,使得细菌的耐药性难以克服。因此,本文就氨基糖苷类修饰酶和抗生素相互作用的热力学和动力学性质进行了详细的论述,试图找出不同修饰酶失活抗生素药物的共同作用机制。这将为设计新的抗生素药物及修饰酶抑制剂、克服细菌的耐药性,提供理论指导和技术支持。     

氨基糖苷类抗生素应用的新机遇

氨基糖苷类抗生素是较早被发现并应用于临床上的一类抗生素,虽然它们没有完全从市面上消失,但由于其他副作用较少且广谱的抗生素的出现,其重要性已经减弱。目前,随着由多药耐药性（MDR）细菌引起的感染急剧增加,作为为数不多的治疗选择,氨基糖苷类抗生素重新进入了人们的视野,特别是用于革兰氏阴性细菌感染。尽管病菌对氨基糖苷类抗生素的耐药机制已基本清楚,但对氨基糖苷类抗生素抗菌模式的认识还远未全面。面对越来越多几乎无法治疗的细菌感染,氨基糖苷类抗生素在对抗多药耐药性病原菌上显示出新的应用前景。     

氨基糖苷类抗生素钝化酶基因

氨基糖苷类抗生素的耐药性主要由细菌产生钝化酶所致,本文对其编码基因的起源,分布及调控,扩散机制作一介绍。     

金黄色葡萄球菌耐药性分析

目的 分析金黄色葡萄球菌的耐药性。方法 对临床各科室送检标本,用MicroScan全自动细菌鉴定仪鉴定细菌种类并检测该菌对抗生素的敏感性。结果 检出金黄色葡萄球菌378株,其中MRSA为197株,检出率为52.1%.MSSA对苯唑西林,阿莫西林-棒酸,第1、2、3代头孢菌素等β-内酰胺类以及喹诺酮类、氨基糖苷类均甚敏感。MRSA对万古霉素敏感,未检出对万古霉素耐药或敏感性降低的菌株。对利福平、呋喃妥因有较好的敏感性.而对β-内酰胺类青霉素、苯唑西林和氨苄西林是100%的耐药,对头孢类抗生索、氟喹诺酮、大环内酯类及氨基糖苷类药物耐药率高。结论 β-内酰胺类抗生素、喹诺酮类、氨基糖苷类抗生素对MSSA感染的治疗效果较好;MRSA感染首选万古霉素或替考拉宁;利福平不能单独用于MRSA感染的治疗;呋喃妥因可用于创面伤口MRSA感染的治疗。     

利用糖芯片技术检测氨基糖苷类抗生素与RNAs和蛋白质之间的相互作用

氨基糖苷类抗生素是一类广谱型抗细菌感染药物,其不断增加的细菌耐药性很大程度上限制了它的临床应用,研究和开发新型氨基糖苷类抗生素具有重要意义。将氨基糖苷类抗生素固定到玻璃片基上,制成糖芯片,再分别与荧光标记的RNAs和蛋白质杂交,通过分析杂交后的荧光信号强度检测它们之间的相互作用。结果显示,氨基糖苷类抗生素芯片可以特异性地与r RNA的A位点模拟物、I型核酶和蛋白酶结合。因此糖芯片技术不仅可以检测氨基糖苷类抗生素与r RNAs的特异性结合,而且可以应用于寻找新型RNA结合配体的研究,为快速鉴定和筛选可紧密结合RNA靶标且毒性较低的新型氨基糖苷类抗生素奠定了一定的基础。     

外源化学物质对副溶血性弧菌药物敏感性的影响

【目的】细菌对抗生素的耐药性已成为全球公共卫生问题关注的热点。有研究表明外源添加化学物质可以增强耐药细菌对抗生素的敏感性。本研究比较了3种化学物质葡萄糖、丙氨酸、甘油对增强副溶血性弧菌抗生素敏感性的作用效果。【方法】在亚抑菌浓度抗生素胁迫条件下,通过比较副溶血性弧菌在添加终浓度为10 mmol/L葡萄糖、丙氨酸、甘油后细菌存活率随时间的变化水平,来观察弧菌对亚抑菌浓度抗生素敏感性作用效果的改变,并采用氧化磷酸化解偶联剂CCCP对实验结果进行验证。【结果】发现3种外源化学物质均能增强亚抑菌浓度氨基糖苷类抗生素对副溶血性弧菌的杀菌能力,其中外源添加葡萄糖对增强亚抑菌浓度卡那霉素的杀菌能力最为显著,而对其他种类抗生素的杀菌能力则无明显增强作用。加入氧化磷酸化解偶联剂CCCP后可消除由外源化学物质引发的弧菌抗生素敏感性作用增强的现象。【结论】通过调节细菌细胞代谢水平可提高耐药副溶血性弧菌对氨基糖苷类抗生素的敏感性,对多重耐药副溶血性弧菌的防控具有一定的实际应用价值。     

细菌对大环内酯类抗生素耐药的机制及控制策略

随着大环内酯类抗生素在临床的广泛使用,细菌对大环内酯类抗生素的耐药性日趋严重。细菌对大环内酯类药物耐药的机制,包括靶位(核糖体)的改变,细菌对大环内酯类抗生素的主动外排,细菌产生灭活大环内酯的酶等。本文就大环内酯类药物的耐药机制作一综述,并根据其耐药机制,从大环内酯类抗生素的结构修饰和主动外排抑制剂的应用等方面讨论了控制耐药性的策略。     

一种新的氨基糖苷类耐药决定因子：质粒介导的16S rRNA甲基化酶

氨基糖苷类抗生素在治疗革兰阳性和阴性细菌引起的危重感染中起着重要的作用。该抗生素通过与细菌30S 核糖体亚基的16S rRNA 的A 位点结合而阻碍蛋白质的合成。耐该类抗生素的机制主要包括产氨基糖苷修饰酶、作用靶位改变、膜通透性降低和外排系统导致的细胞内药物浓度降低。质粒介导的16S rRNA甲基化酶是近年来新发现的一种耐药决定因子, 可导致4, 6-二取代基-脱氧链霉胺类氨基糖苷类高水平耐药。该类甲基化酶编码基因常位于细菌特异性重组系统中( 如转座子) , 使得其可在细菌不同种属间广泛传播。在致病性革兰阴性菌中发现的甲基化酶基因的G+C含量与其推测的起源菌——放线菌中的G + C 含量存在较大差异, 因此其真正的起源有待进一步研究。由于16S rRNA 甲基化酶在临床上的重要性, 为引起医务人员的重视, 本文就其耐药机制、分类、基因背景以及流行病学特征等方面的研究进展作一综述。     

铜绿假单胞菌新基因PA0058插入失活对氨基糖苷类抗生素耐药性的影响

【目的】铜绿假单胞菌是一种重要的条件致病菌,临床上常引起难治性和顽固性感染,随着各种抗生素的广泛使用,该菌对多种抗生素呈现耐药性,研究其耐药性机理有着重要意义。【方法】以一株临床分离株Pseudomonas aeruginosa PA68作为出发菌株,应用人工Mu转座技术构建突变文库并从中筛选得到一株对链霉素抗性明显增强的菌株M122,并对突变株M122进行测序分析及表型检测。通过Southern杂交实验证实转座子是否为单拷贝插入,对突变株M122的基因表达谱与野生型PA68菌株进行对比分析。【结果】确定了Mu转座子在M122基因组上为单拷贝插入,插入位点为基因PA0058的第214 bp处。对M122进行表型检测,发现其对多种氨基糖苷类抗生素的耐药性均得到增强,通过转入携带完整基因PA0058的表达质粒可以使突变株M122的耐药性有所降低,利用同源重组的方法,在模式菌株P.aeruginosa PAK中进行PA0058基因敲除,得到的敲除株具有链霉素耐药性升高的表型。基因PA0058的缺失引起多种基因表达水平改变,尤其是katB、ahpC、ahpF等抗氧化酶基因转录表达显著增高。【结论】首次发现铜绿假单胞菌PA0058基因的插入失活提高了细菌对氨基糖苷类抗生素的耐药性,且导致突变株M122中抗氧化酶基因转录表达水平的上调。     

Aminoglycoside resistance rates, phenotypes, and mechanisms of Gram-negative bacteria from infected patients in upper Egypt

With the re-emergence of older antibiotics as valuable choices for treatment of serious infections, we studied the aminoglycoside resistance of Gram-negative bacteria isolated from patients with ear, urinary tract, skin, and gastrointestinal tract infections at Minia university hospital in Egypt. Escherichia coli (mainly from urinary tract and gastrointestinal tract infections) was the most prevalent isolate (28.57%), followed by Pseudomonas aeruginosa (25.7%) (mainly from ear discharge and skin infections). Isolates exhibited maximal resistance against streptomycin (83.4%), and minimal resistance against amikacin (17.7%) and intermediate degrees of resistance against neomycin, kanamycin, gentamicin, and tobramycin. Resistance to older aminoglycosides was higher than newer aminoglycosides. The most common aminoglycoside resistance phenotype was that of streptomycin resistance, present as a single phenotype or in combination, followed by kanamycin-neomycin as determined by interpretative reading. The resistant Pseudomonas aeruginosa strains were capable of producing aminoglycoside-modifying enzymes and using efflux as mechanisms of resistance. Using checkerboard titration method, the most frequently-observed outcome in combinations of aminoglycosides with β-lactams or quinolones was synergism. The most effective combination was amikacin with ciprofloxacin (100% Synergism), whereas the least effective combination was gentamicin with amoxicillin (53.3% Synergistic, 26.7% additive, and 20% indifferent FIC indices). Whereas the studied combinations were additive and indifferent against few of the tested strains, antagonism was never observed. The high resistance rates to aminoglycosides exhibited by Gram-negative bacteria in this study could be attributed to the selective pressure of aminoglycoside usage which could be controlled by successful implementation of infection control measures.     

Aminoglycoside resistance in gramnegative pathogens of nosocomial infections in Russia]

The study of the mechanisms of aminoglycoside resistance in gramnegative pathogens of nosocomial infections in 14 hospitals of Russia showed that the basic mechanism was production of aminoglycoside modifying enzymes, mainly adenylyl transferase ANT(2"), acetyl transferases AAC(3)-V and ACC(6)-I, and phosphotransferases APH(3')-I and APH(3')-VI. In all the hospitals enzymes modifying gentamicin and tobramycin were wide spread while the resistance phenotypes to aminoglycosides were different in separate hospitals. Isepamycin proved to be the most active aminoglycoside. Recommendations for the use of antibiotics in hospital formulas and empiric therapy should be developed on the basis of the local specific features of the resistance in nosocomial pathogens to aminoglycosides.     

Comparing aminoglycoside binding sites in bacterial ribosomal RNA and aminoglycoside modifying enzymes

Aminoglycoside antibiotics are used against severe bacterial infections. They bind to the bacterial ribosomal RNA and interfere with the translation process. However, bacteria produce aminoglycoside modifying enzymes (AME) to resist aminoglycoside actions. AMEs form a variable group and yet they specifically recognize and efficiently bind aminoglycosides, which are also diverse in terms of total net charge and the number of pseudo‐sugar rings. Here, we present the results of 25 molecular dynamics simulations of three AME representatives and aminoglycoside ribosomal RNA binding site, unliganded and complexed with an aminoglycoside, kanamycin A. A comparison of the aminoglycoside binding sites in these different receptors revealed that the enzymes efficiently mimic the nucleic acid environment of the ribosomal RNA binding cleft. Although internal dynamics of AMEs and their interaction patterns with aminoglycosides differ, the energetical analysis showed that the most favorable sites are virtually the same in the enzymes and RNA. The most copied interactions were of electrostatic nature, but stacking was also replicated in one AME:kanamycin complex. In addition, we found that some water‐mediated interactions were very stable in the simulations of the complexes. We show that our simulations reproduce well findings from NMR or X‐ray structural studies, as well as results from directed mutagenesis. The outcomes of our analyses provide new insight into aminoglycoside resistance mechanism that is related to the enzymatic modification of these drugs. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Aminoglycosides versus bacteria – a description of the action, resistance mechanism, and nosocomial battleground

Since 1944, we have come a long way using aminoglycosides as antibiotics. Bacteria also have got them selected with hardier resistance mechanisms. Aminoglycosides are aminocyclitols that kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane. Aminoglycoside resistance mechanisms include: (a) the deactivation of aminoglycosides by N-acetylation, adenylylation or O-phosphorylation, (b) the reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport, active efflux, and drug trapping, (c) the alteration of the 30S ribosomal subunit target by mutation, and (d) methylation of the aminoglycoside binding site. There is an alarming increase in resistance outbreaks in hospital setting. Our review explores the molecular understanding of aminoglycoside action and resistance with an aim to minimize the spread of resistance.     

Aminoglycoside-modifying enzymes.

Bacterial resistance to the aminoglycoside antibiotics is most frequently associated with the expression of modifying enzymes that can phosphorylate, adenylate or acetylate these compounds. The recent availability of representative crystal structures for all three classes of modifying enzymes has greatly expanded our knowledge of enzyme function, and has revealed unexpected and exciting connections to other families of enzymes. Furthermore, the complete genome sequences for several bacteria have revealed many potential aminoglycoside-resistance elements.     

Kinetic mechanism of enterococcal aminoglycoside phosphotransferase 2"-Ib

The major mechanism of resistance to aminoglycosides in clinical bacterial isolates is the covalent modification of these antibiotics by enzymes produced by the bacteria. Aminoglycoside 2'-Ib phosphotransferase [APH(2')-Ib] produces resistance to several clinically important aminoglycosides in both Gram-positive and Gram-negative bacteria. Nuclear magnetic resonance analysis of the product of kanamycin A phosphorylation revealed that modification occurs at the 2'-hydroxyl of the aminoglycoside. APH(2')-Ib phosphorylates 4,6-disubstituted aminoglycosides with kcat/Km values of 10(5)-10(7) M-1 s-1, while 4,5-disubstituted antibiotics are not substrates for the enzyme. Initial velocity studies demonstrate that APH(2')-Ib operates by a sequential mechanism. Product and dead-end inhibition patterns indicate that binding of aminoglycoside antibiotic and ATP occurs in a random manner. These data, together with the results of solvent isotope and viscosity effect studies, demonstrate that APH(2')-Ib follows the random Bi-Bi kinetic mechanism and substrate binding and/or product release could limit the rate of reaction.     

Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.

BACKGROUND: The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes. RESULTS: The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type li (AAC(6')-li) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 A resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1. CONCLUSIONS: Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.     

An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases

The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5′ leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches.     

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2′) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2′). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2′) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.     

Aminoglycoside resistance patterns in clinical isolates of E. coli and Klebsiella sp. from Czechoslovakia and the United States

Resistance of gram-negative bacilli to aminoglycoside antibiotics differs by region and country. Previous studies have demonstrated predominance of the nucleotidyltransferase ANL(2") as the mechanism of enzymatic resistance to gentamicin in the United States and many European countries (Federal Republic of Germany, Switzerland, Greece, Turkey) whereas the acetylating enzymes AAC(6') and AAC(3) were the principal causes of resistance to aminoglycosides in Japan and Chile. In the present comparison of 18 drug resistant isolates of E. coli and Klebsiella sp. from Czechoslovakia and the United States, with aminoglycoside-inactivating enzymes, ANT(2") characterized the most strains from both countries. In a higher number of isolates from Czechoslovakia however, the aminoglycoside resistance was mediated by AAC(3). In the majority of strains a simultaneous occurrence of two gentamicin-inactivating enzymes i.e. ANT(2"), plus AAC (2'), or AAC(6') or AAC(3) was observed. In amikacin resistant E. coli strains the mechanism of resistance was represented by production of AAC(6') or AAC*--an acetyltransferase with uncommon substrate profile. In all E. coli and K. pneumoniae strains from the United States apart from ANT(2") also AAC(2') was detected. This represents a broadening of the host range of aac(2') gene, the occurrence of which has been limited only to Providencia and Proteus strains.