Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Molecular understanding of aminoglycoside action and resistance

Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. They are particularly active against aerobic, Gram-negative bacteria and act synergistically against certain Gram-positive organisms. Aminoglycosides are used in the treatment of severe infections of the abdomen and urinary tract, bacteremia, and endocarditis. They are also used for prophylaxis, especially against endocarditis. Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat. This might be the reason for the interest in understanding the mechanisms of resistance. It is now clear that the resistance occurs by different mechanisms such as prevention of drug entry, active extrusion of drugs, alteration of the drug target (mutational modification of 16S rRNA and mutational modification of ribosomal proteins), and enzymatic inactivation through the expression of enzymes, which covalently modify these antibiotics. Enzymatic inactivation is normally due to acetyltransferases, nucleotidyltransferases, and phosphotransferases. In this review, we focus on the recent concept of molecular understanding of aminoglycoside action and resistance.     

Mechanisms of bacterial biocide and antibiotic resistance

Resistance to antibiotics is increasingly commonplace amongst important human pathogens. Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux. While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems. Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes. Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms. Still, target site mutations leading to biocide resistance, though rare, are known. Intriguingly, many multidrug efflux systems also accommodate biocides (e.g. triclosan) such that strains expressing these are both antibiotic- and biocide-resistant. Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics. Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan. These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two. Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems. Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.     

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.     

细菌抗菌肽耐药机制研究进展

细菌对传统抗生素的耐药程度十分严重,寻找克服耐药性的新型抗菌药物已成为当务之急。抗菌肽(antimicrobial peptides,AMPs)是当下较有前景的抗菌药物之一。虽然通常认为,AMPs优先攻击细胞膜的特点使其不会引起广泛的耐药性,但其对特定靶标的识别能力仍为基因突变和细菌耐药性的产生提供了可能。此外,一些细菌还显示出了抵御宿主AMPs的杀伤作用并与宿主细胞共存的能力,相应的细菌防御机制也使其对治疗性AMPs产生抗性,这种交叉抗性近年来也备受关注。这些耐药现象的发现均对AMPs的开发提出了新挑战。本综述就细菌对AMPs耐药的分子机制进行了研究进展的总结,并且对治疗性AMPs与宿主防御肽交叉抗性的相关机制研究进行了归纳,以期寻求新的对抗耐药性的策略。     

Predicting Therapeutic Template by Evaluating the Structural Stability of Anti-Cancer Peptides—A Computational Approach

Clinically significant antibiotic resistance has evolved against virtually every antibiotic deployed. Yet the development of new classes of antibiotics has lagged far behind our growing need for such drugs. Antimicrobial peptides (AMPs) have emerged as novel therapeutics hailed for their bactericidal and immunomodulatory properties. However, the process of optimizing antimicrobial peptide stability, using large peptide libraries is both tedious and expensive. The intent of this study is to analyze computationally the stability of anti-cancer peptides (ACPs) and to discover a potential template from a pool of ACPs for therapeutic use. Consequently we highlighted that ACP, NK-Lysin appears advantageous over the other ACPs with respect to stability, and may provide a convenient platform for the development of anticancer therapeutic peptide.     

A Window of Opportunity to Control the Bacterial Pathogen Pseudomonas aeruginosa Combining Antibiotics and Phages

The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.     

Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase.

The ErmE methyltransferase from the erythromycin-producing actinomycete Saccharopolyspora erythraea dimethylates the N-6 position of adenine 2058 in domain V of 23S rRNA. This modification confers resistance to erythromycin and to other macrolide, lincosamide, and streptogramin B antibiotics. We investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance to erythromycin and clindamycin. The degree of resistance corresponds to the level of ermE expression. In turn, ermE expression also correlates with the proportion of 23S rRNA molecules that are dimethylated at adenine 2058. The methyltransferase was isolated in an active, concentrated form from E. coli, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been disrupted by removal of magnesium ions. We conclude that the main features that are specifically recognized by the ErmE methyltransferase are displayed within the primary and secondary structures of 23S rRNA domain V.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.     

Selective agents and marker genes for use in transformation of monocotyledonous plants

Even the most successful transformation systems for monocotyledonous plants have a very low efficiency. To overcome this, the selection system is of utmost importance. Established selection systems based on kanamycin resistance are not generally applicable for monocotyledonous plants because monocot cells and tissues are relatively insensitive to kanamycin. Selection can be improved by using other antibiotics or herbicides and their respective resistance genes, which can lead to a substantial reduction in the number of untransformed regenerants. In choosing a selection system the resistance mechanism, either detoxification of the selective agent or modification of the target enzyme, should also be taken into account. Detoxification of the selective agent by expression of a modifying enzyme in transformed cells can enable untrasformed cells to escape. This does not happen when resistance is based on the production of a modified target enzyme. By use of vectors specifically designed for high expression in monocots, the level of resistance in transformed cells and tissues can be enhanced, resulting in an increased efficiency of selection. This paper presents an overview of the various antibiotics and herbicides available and the application of the related resistance genes to improve transformation of monocots.     

Subsistence and complexity of antimicrobial resistance on a community-wide level

There are a multitude of resistance strategies that microbes can apply to avoid inhibition by antimicrobials. One of these strategies is the enzymatic modification of the antibiotic, in a process generally termed inactivation. Furthermore, some microorganisms may not be limited to the mere inactivation of the antimicrobial compounds. They can continue by further enzymatic degradation of the compounds' carbon backbone, taking nutritional and energetic advantage of the former antibiotic. This driving force to harness an additional food source in a complex environment adds another level of complexity to the reasonably well-understood process of antibiotic resistance proliferation on a single cell level: It brings bioprotection into play at the level of microbial community. Despite the possible implications of a resistant community in a host and a lurking antibiotic failure, knowledge of degradation pathways of antibiotics and their connections is scarce. Currently, it is limited to only a few families of antibiotics (e.g. β-lactams and sulfonamides). In this article, we discuss the fluctuating nature of the relationship between antibiotic resistance and the biodegradation of antibiotics. This distinction mainly depends on the genetic background of the microbe, as general resistance genes can be recruited to function in a biodegradation pathway.     

The basis for resistance to beta-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus

Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.     

Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents

Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.     

The characteristics of the microflora isolated in bovine endometritis]

117 pure bacterial cultures of numerous species isolated by the authors at acute postnatal pyo-catarrhal cattle endometritis have been studied for their virulence and resistance to 18 antibiotics with the aim to determine their etiologic role in the development of the given pathology and to make a prediction estimation of possible therapeutic and preventive efficiency of the antibiotics. The experiments on white mice have revealed pathogenic properties and virulence in most studied strains of bacteria of different species. The studied microflora is, mainly, resistant practically to all 18 tested antibiotics. A direct dependence is established between virulence of the microflora, isolated in case of endometritis, and its resistance to antibiotics.     

Bacterial efflux systems and efflux pumps inhibitors

It is now well established that bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterial agents and that manifests in all fields of their application. Among the three main mechanisms involved in bacterial resistance (target modification, antibiotic inactivation or default of its accumulation within the cell), efflux pumps, responsible for the extrusion of the antibiotic outside the cell, have recently received a particular attention. Actually, these systems, classified into five families, can confer resistance to a specific class of antibiotics or to a large number of drugs, thus conferring a multi-drug resistance (MDR) phenotype to bacteria. To face this issue, it is urgent to find new molecules active against resistant bacteria. Among the strategies employed, the search for inhibitors of resistance mechanisms seems to be attractive because such molecules could restore antibiotic activity. In the case of efflux systems, efflux pump inhibitors (EPIs) are expected to block the pumps and such EPIs, if active against MDR pumps, would be of great interest. This review will focus on the families of bacterial efflux systems conferring drug resistance, and on the EPIs that have been identified to restore antibiotic activity.     

细菌固有耐药的研究进展

人们以往大多只关注由敏感细菌通过基因水平转移和自发突变方式获得的耐药性,而忽略了细菌对某类抗生素天然耐药的重要特性,细菌的这种特性又被称为固有耐药。固有耐药由固有耐药基因决定,这类基因是指存在于某类细菌染色体上位置保守的与耐药相关的一类基因。近年来,对固有耐药基因的研究已经越来越受到重视。固有耐药基因的发现不仅可以为新药研制提供药物作用靶标,而且通过阻断病原菌固有耐药基因还可使以往对该类菌不起作用的抗生素药物重新焕发抗菌活性。此外,已有研究表明固有耐药基因能够被移动元件捕获进而可水平转移至其他细菌,因此通过监测固有耐药基因可以预测耐药菌的出现。本文对传统的细菌固有耐药机制包括细胞膜的低渗透性和多药外排泵系统,以及已知重要病原菌的转移酶和代谢相关酶的固有耐药机制进行了介绍。同时,进一步对隐性固有耐药基因的特性进行了阐释,最后探讨了固有耐药与获得性耐药的进化关系,指出固有耐药基因很可能是一些获得性耐药基因的来源。     

Olaquindox resistance in the coliform flora of pigs and their environment: an ecological study

Faecal samples were taken weekly from a chosen pen on each of four commercial pig farms, two of which used olaquindox as a feed additive. Coliform bacteria were isolated from the samples and the incidence and level of resistance to olaquindox and to four therapeutic antibiotics determined. The coliforms isolated were biotyped to follow the emergence of drug-resistant sub-populations. The results showed that the coliform flora was complex and that a turnover of biotypes was associated with changes in the occupancy of the pen and, possibly, diet. This turnover led to large fluctuations in both incidence and level of resistance to olaquindox and the antibiotics. Olaquindox resistance was not specifically linked with resistance to any therapeutic antibiotic, and the possible origin of olaquindox-resistant strains is discussed in relation to the biotypes.