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.     

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.     

Why are bacteria refractory to antimicrobials?

The incidence of antibiotic resistance in pathogenic bacteria is rising. Antibiotic resistance can be achieved via three distinct routes: inactivation of the drug, modification of the target of action, and reduction in the concentration of drug that reaches the target. It has long been recognized that specific antibiotic resistance mechanisms can be acquired through mutation of the bacterial genome or by gaining additional genes through horizontal gene transfer. Recent attention has also brought to light the importance of different physiological states for the survival of bacteria in the presence of antibiotics. It is now apparent that bacteria have complex, intrinsic resistance mechanisms that are often not detected in the standard antibiotic sensitivity tests performed in clinical laboratories. The development of resistance in bacteria found in surface-associated aggregates or biofilms, owing to these intrinsic mechanisms, is paramount.     

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.     

Biocide resistance mechanisms

Information about the mechanisms of action and resistance of biocides used as preservatives, sanitizers, and disinfectants continues to accumulate. While biocides are generally not as well-studied as antibiotics, it is becoming clear that bacteria employ the same major resistance strategies against both groups of antibacterial chemicals. The resistance strategies—inhibitor inactivation, target site alteration, and target site exclusion—are described in the context of the presumed mechanisms of action of the biocides using examples drawn from the literature.     

Bacterial multidrug efflux pumps: Mechanisms,physiology and pharmacological exploitations

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.     

Optimizing future treatment of enterococcal infections: attacking the biofilm?

Enterococcus faecalis and Enterococcus faecium are among the leading causative agents of nosocomial infections and are infamous for their resistance to many antibiotics. They cause difficult-to-treat infections, often originating from biofilm-mediated infections associated with implanted medical devices or endocarditis. Biofilms protect bacteria against antibiotics and phagocytosis, and physical removal of devices or infected tissue is often needed but is frequently not possible. Currently there are no clinically available compounds that disassemble biofilms. In this review we discuss all known structural and regulatory genes involved in enterococcal biofilm formation, the compounds directed against biofilm formation that have been studied, and potentially useful targets for future drugs to treat enterococcal biofilm-associated infections.     

Current updates on fungal endocarditis

Fungal endocarditis (FE) is a rare disease but in recent years its incidence as well as mortality is increasing particularly in developing nations. Candida and Aspergillus species occupy the prominent position as etiological agents of this invasive disease. Intravenous devices such as pacemakers, central line related thrombosis and prolonged use of antibiotics are major risk factors for FE. The epidemiology of endocarditis cases is also evolving over time with exceptionally rare species causing more invasive disease. Research over the last decade has also delineated the underlying pathogenic mechanism of FE. Improved understanding of these mechanisms will help to combat the increasing problem of antimicrobial drug resistance. The diagnosis of FE is dependent on the sensitivity and specificity of the method as fungi generally do not grow well in blood cultures. More advanced techniques including molecular and immunological assays now play a central role in accurate identification of causative fungal pathogens especially in culture negative scenario. In developing nations such as India, blood culture reports are generally negative due to prior antibiotic therapy. Echocardiography has emerged as the potential imaging technique for identifying invasive endocarditis including small masses of vegetation or abscess. Successful treatment often requires both the surgical interventions and prolonged antifungal therapy. In the present review, we briefly highlight the mechanisms of pathogenesis of this rare emerging disease along with the risk factors involved, the diagnostic criteria and the treatment strategy.     

Siderophores as drug delivery agents: application of the “Trojan Horse” strategy

The outer membrane permeability barrier is an important resistance factor of bacterial pathogens. In combination with drug inactivating enzymes, target alteration and efflux, it can increase resistance dramatically. A strategy to overcome this membrane-mediated resistance is the misuse of bacterial transport systems. Most promising are those for iron transport. They are vital for virulence and survival of bacteria in the infected host, where iron depletion is a defense mechanism against invading pathogens. We synthesized biomimetic siderophores as shuttle vectors for active transport of antibiotics through the bacterial membrane. Structure activity relationship studies resulted in siderophore aminopenicillin conjugates that were highly active against Gram-negative pathogens which play a crucial role in destructive lung infections in cystic fibrosis patients and in severe nosocomial infections. The mechanism of action and the uptake of the compounds via specific iron siderophore transport routes were demonstrated. The novel conjugates were active against systemic Pseudomonas aeruginosa infections in mice with ED₅₀ values comparable to the quinolone ofloxacin and show low toxicity.     

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.     

Antibiotic resistance is prevalent in an isolated cave microbiome

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.     

Gatifloxacin Induces S and G2-Phase Cell Cycle Arrest in Pancreatic Cancer Cells via p21/p27/p53

Pancreatic cancer, despite being the most dreadful among gastrointestinal cancers, is poorly diagnosed, and further, the situation has been aggravated owing to acquired drug resistance against the single known drug therapy. While previous studies have highlighted the growth inhibitory effects of older generation fluoroquinolones, the current study aims to evaluate the growth inhibitory effects of newer generation fluoroquinolone, Gatifloxacin, on pancreatic cancer cell lines MIA PaCa-2 and Panc-1 as well as to elucidate the underlying molecular mechanisms. Herein, we report that Gatifloxacin suppresses the proliferation of MIA PaCa-2 and Panc-1 cells by causing S and G₂-phase cell cycle arrest without induction of apoptosis. Blockade in S-phase of the cell cycle was associated with increased TGF-β1 expression and translocation of Smad3-4 complex to the nucleus with subsequent activation of p21 in MIA PaCa-2 cells, whereas TGF-β signalling attenuated Panc-1 cells showed S-phase arrest by direct activation of p27. However, Gatifloxacin mediated G₂–phase cell cycle arrest was found to be p53 dependent in both the cell lines. Our study is of interest because fluoroquinolones have the ability to penetrate pancreatic tissue which can be very effective in combating pancreatic cancers that are usually associated with loss or downregulation of CDK inhibitors p21/p27 as well as mutational inactivation of p53. Additionally, Gatifloxacin was also found to synergize the effect of Gemcitabine, the only known drug against pancreatic cancer, as well as the broad spectrum anticancer drug cisplatin. Taken together our results suggest that Gatifloxacin possesses anticancer activities against pancreatic cancer and is a promising candidate to be repositioned from broad spectrum antibiotics to anticancer agent.     

基于翻译调控的细菌耐药研究进展

由于抗生素的大量使用,细菌耐药问题凸显,直接威胁人类生命健康和世界经济发展。过去对于细菌耐药的遗传和分子机制研究较为透彻,而对应的调控机制研究相对较少。翻译调控作为生命体最重要的调控方式之一,在细菌耐药研究领域的重要性尚未被学术界充分重视。本文介绍了影响翻译过程的抗生素的主要作用机制,重点从核糖体的修饰和突变、tRNA总量的动态调控、tRNA氨酰化、tRNA甲基化、核糖体保护蛋白和翻译因子这几个方面概述了基于翻译调控的细菌耐药研究进展,为研究者们提供了一个基于翻译调控角度研究细菌耐药的新视角,同时也为开发靶向细菌翻译调控的新型抗生素提供一些新思路。     

The emergence and evolution of methicillin-resistant Staphylococcus aureus

Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus (MRSA), which is resistant to practically all beta-lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta-lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.     

(p)ppGpp and drug resistance

In recent years, emerging and reemerging pathogens resistant to nearly all available antibiotics are on the rise. This limits the availability of effective antibiotics to treat infections, thus it is imperative to develop new drugs. The accumulation of alarmones guanosine tetraphosphate and guanosine pentaphosphate, collectively known as (p)ppGpp, is a global response of bacteria to environmental stress. (p)ppGpp has been documented to be involved in the resistance to β‐lactam and peptide antibiotics. Proposed mechanisms of action include occupation of drug targets, regulation of the expression of virulence determinants, and modification of protein activities. (p)ppGpp analogs might counteract these actions. Several such entities are being tested as new antibiotics. Further insights into the mechanisms of (p)ppGpp‐mediated drug resistance might facilitate the discovery and development of novel antibiotics. J. Cell. Physiol. 224: 300–304, 2010. © 2010 Wiley‐Liss, Inc.     

Mutational fitness landscape and drug resistance

Robust technology has been developed to systematically quantify fitness landscapes that provide valuable opportunities to improve our understanding of drug resistance and define new avenues to develop drugs with reduced resistance susceptibility. We outline the critical importance of drug resistance studies and the potential for fitness landscape approaches to contribute to this effort. We describe the major technical advancements in mutational scanning, which is the primary approach used to quantify protein fitness landscapes. There are many complex steps to consider in planning and executing mutational scanning projects including developing a selection scheme, generating mutant libraries, tracking the frequency of variants using next-generation sequencing, and processing and interpreting the data. Key experimental parameters impacting each of these steps are discussed to aid in planning fitness landscape studies. There is a strong need for improved understanding of drug resistance, and fitness landscapes provide a promising new approach.     

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.     

结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、药物外排泵系统、细菌细胞壁厚度的增加以及喹诺酮抗性蛋白MfpA介导的DNA旋转酶活性调控。其中在氟喹诺酮靶标DNA旋转酶功能活性改变的耐药机制方面,编码DNA旋转酶基因突变一直是研究的热点,但近年来发现DNA旋转酶的调控蛋白MfpA以及DNA旋转酶的修饰在细菌耐药性中起着重要的作用,相关机制还亟待发现。本文综述了当前结核分枝杆菌耐氟喹诺酮类药物的作用机制,旨在为研发精准诊断技术和药物发掘提供科学理论基础和参考。     

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.     

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.