Chemical countermeasures for the control of bacterial biofilms: effective compounds and promising targets

The pathogenic nature of many infectious bacteria is enhanced by their ability to form surface-associated, protected communities known as "biofilms." Due to various factors, bacteria in biofilm communities display significantly greater resistance to traditional antimicrobial therapies than their planktonic brethren. This resistance complicates many common bacterial infections, resulting in recurrent ear infections, bacterial endocarditis, chronic lung infection in cystic fibrosis, infectious kidney stones, and surface infection of implanted medical devices. Owing to the serious nature of many biofilm-mediated infections and the near-complete dearth of effective strategies for treating them, efforts are underway to further understand the nature of bacterial infections involving biofilms and to discover and develop effective therapies to combat them. Particularly, several classes of chemical compounds have shown promise in combating biofilms when used in conjunction with traditional antimicrobials. The vast majority of these compounds exert their anti-biofilm properties through disruption of "quorum sensing," a common means of intercellular communication in bacterial communities that allows coordinated expression of virulence factors and facilitates formation of the oft-complex architecture of mature bacterial biofilms. Other new chemical entities are effective against biofilms without necessarily affecting quorum sensing. This review summarizes salient research in the development of effective chemical countermeasures for Gram-negative and Gram-positive bacterial infections involving biofilms.     

Antimicrobial strategies effective against infectious bacterial biofilms

Bacteria are able to adapt to undesirable changes in nutrient availability, environmental conditions and presence of antimicrobial products, as well as to immunological defenses. One particularly important example of bacterial adaptation is the ability to grow as part of a sessile community, commonly referred to as biofilm. It is a natural tendency of microorganisms to attach to biotic or abiotic surfaces, to multiply and to embed themselves in a slimy matrix, resulting in biofilms. Biofilms are the leading example of physiological adaptation and are one of the most important sources of bacterial resistance to antimicrobials. It is now recognized that most bacterial-associated infections, including endocarditis, dental caries, middle ear infections, osteomyelitis, medical device-related infections and chronic lung infections in cystic fibrosis patients are problematic because of biofilms. Bacteria in biofilms demonstrate intrinsic resistance to antimicrobial stress more effectively than the planktonic counterparts. Antimicrobial concentrations necessary to inhibit bacterial biofilms can be up to 10-1000 times higher than those needed to inhibit the same bacteria grown planktonically. Thus, in the presence of therapeutically available antibiotic concentrations biofilms remain viable after treatment. Therefore, the identification of new antimicrobials that inhibit or destroy biofilms is needed. The aim of this review is to cover the recent advances on the studies of antimicrobial strategies effective against infectious bacterial biofilms, including the current developments in the structure-activity relationship of those effective antimicrobials.     

Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms

Bacterial biofilms cause significant infections in the medical field. Antibiotics commonly used to treat these infections often do not achieve complete bacterial eradication. New approaches to eliminate biofilms have focused on dispersion compounds to entice the bacteria to actively escape or disperse from the biofilm, where the bacteria may become more susceptible to antibiotics. The aim of this study was to demonstrate that combining antibiotics with nutrient dispersion compounds can synergistically decrease the viability of Pseudomonas aeruginosa biofilms. The effects of various co-treatments were studied on mature biofilms through qualitative and quantitative confocal microscopy. Combined treatment of P. aeruginosa biofilms with antibiotic and dispersion compounds resulted in a significant reduction in the live bacterial population compared with the untreated control in all cases, with four combinations displaying synergistic action (citrate with amikacin disulphate, colistin methanesulphonate or erythromycin, and succinic acid with colistin methanesulphonate).     

大肠埃希菌生物膜形成与耐药机制的研究进展

生物膜细菌与浮游菌相比有着其独特的生理学特性、毒力作用及耐药机制,对其耐药机制及治疗成为近年来的研究热点。大肠埃希菌(Escherichia coli)是医院感染的重要病原菌之一,也是生物膜感染的常见病原菌。形成生物膜的大肠埃希菌具有高度的耐药性并能逃避免疫系统的攻击,其感染易慢性化并难于控制。本文通过对大肠埃希菌生物膜形成与耐药机制研究特点进行阐述,为寻找有效的控制手段,指导抗生素合理使用提供理论依据。     

Candida biofilms: antifungal resistance and emerging therapeutic options

Intravascular catheter infections are a major cause of morbidity and mortality in hospitalized patients, accounting for the majority of the 200,000 nosocomial bloodstream infections occurring in the US annually. Of the intravenous lines that are culture-positive for Candida, 40% actually represent fungemia, which generally necessitates systemic treatment and line removal to affect cure. Until recently, the reason for the need for device removal was unclear. However, our research group and others have demonstrated a near-total resistance to antifungals by biofilm-associated Candida. Similar to bacterial species, Candida biofilm formation proceeds through early, intermediate and maturation phases. This process is associated with the generation of a polysaccharide extracellular matrix (ECM). Mature C. albicans biofilms have a heterogeneous architecture, in terms of distribution of fungal cells and ECM, and exhibit broad antimicrobial resistance. The mechanisms causing such profound antifungal resistance are beginning to be understood. Recent data indicate that resistance is phase-specific and multifactorial, involving efflux pumps and sterol synthesis (at early and mature biofilm phases, respectively). Neither metabolic quiescence nor the ECM appear to contribute substantially. Susceptibility testing and confocal scanning laser microscopy demonstrated that azoles failed to exert activity against mature Candida biofilms. However, sub-inhibitory concentrations of voriconazole impaired biofilm formation and caused cell morphological aberrations. In contrast, lipid-formulation amphotericins and the echinocandins uniquely exhibited activity against mature biofilms. The mechanisms underlying this ability are unknown. The role of other pharmacological (eg, catheter coatings, antimicrobial peptides and antibiotic locks) and non-pharmacological methods in the prevention and treatment of device-related biofilms is discussed in this review.     

Biofilms and antibiotic therapy: is there a role for combating bacterial resistance by the use of novel drug delivery systems?

The conventional view of antibiotic resistance is one where bacteria exhibit significantly reduced susceptibility to antimicrobials in laboratory tests by mechanisms such as altered drug uptake, altered drug target and drug inactivation. Whilst these mechanisms undoubtedly make a major contribution to antibiotic failure in the clinic, the phenomenon of clinical failure in spite of sensitivity in laboratory tests is also well recognised. It is in this context that attention has focussed on bacteria growing as adherent biofilms, not only as the mode of growth of device-related infections associated for example with artificial joints and venous catheters, but also with other chronic infections such as those occurring in the respiratory tract. Growth as a biofilm almost always leads to a significant decrease in susceptibility to antimicrobial agents compared with cultures grown in suspension and, whilst there is no generally agreed mechanism for the resistance of biofilm bacteria, it is largely phenotypic. That is, when biofilm bacteria are grown in conventional laboratory suspension culture they become susceptible to antimicrobials. A number of elements in the process of biofilm formation have been studied as targets for novel drug delivery technologies. These include surface modification of devices to reduce bacterial attachment and biofilm development as well as incorporation of antimicrobials-again to prevent colonisation. Electrical approaches have been used either to release antimicrobials from device surfaces or to drive antimicrobials through the biofilm. Other technologies not specifically focussed on biofilms include aerosolized delivery of antibiotics to the lung and formulation into liposome and polymer-based vehicles. Liposomal systems have been widely studied, either to target antibiotics to the surface of bacterial biofilms, or by virtue of their property of being taken up cells of the reticuloendothelial system, to target antibiotics towards intracellular bacteria. Many polymer-based carrier systems have also been proposed, including those based on biodegradable polymers such as poly(lactide-co-glycolide) as well as thermoreversible hydrogels. Their contribution to the prevention or resolution of infection is reviewed.     

Virulence‐targeted Antibacterials: Concept,Promise, and Susceptibility to Resistance Mechanisms

In view of the relentless increase in antibiotic resistance in human pathogens, efforts are needed to safeguard our future therapeutic options against infectious diseases. In addition to regulatory changes in our antibiotic use, this will have to include the development of new therapeutic compounds. One area that has received growing attention in recent years is the possibility to treat or prevent infections by targeting the virulence mechanisms that render bacteria pathogenic. Antivirulence targets include bacterial adherence, secretion of toxic effector molecules, bacterial persistence through biofilm formation, quorum sensing and immune evasion. Effective small‐molecule compounds have already been identified that suppress such processes. In this review, we discuss the susceptibility of such compounds to the development of resistance, by comparison with known resistance mechanisms observed for classical bacteriostatic or bacteriolytic antibiotics, and by review of available experimental case studies. Unfortunately, appearance of resistance mechanisms has already been demonstrated for some, showing that the quest of new, lasting drugs remains complicated.     

抗生物膜治疗黏液型铜绿假单胞菌感染的研究进展

摘要：铜绿假单胞菌是临床上常见的重要条件致病菌,常引起慢性囊性肺纤维化患者、慢性创伤患者及滞留导尿管患者感染,当感染未得到有效控制时,菌体形成生物膜,包裹于外周,转化成黏液型铜绿假单胞菌,对抗菌药物极不敏感,疗效不佳,感染难以治愈。因此,防止生物膜的形成、破坏已形成的生物膜成为治疗黏液型铜绿假单胞菌的关键。本文就抗生物膜治疗相关的研究进行汇总概述,为临床有效治疗黏液型铜绿假单胞菌感染提供依据。     

Metal nanobullets for multidrug resistant bacteria and biofilms

Infectious diseases were one of the major causes of mortality until now because drug-resistant bacteria have arisen under broad use and abuse of antibacterial drugs. These multidrug-resistant bacteria pose a major challenge to the effective control of bacterial infections and this threat has prompted the development of alternative strategies to treat bacterial diseases. Recently, use of metallic nanoparticles (NPs) as antibacterial agents is one of the promising strategies against bacterial drug resistance. This review first describes mechanisms of bacterial drug resistance and then focuses on the properties and applications of metallic NPs as antibiotic agents to deal with antibiotic-sensitive and -resistant bacteria. We also provide an overview of metallic NPs as bactericidal agents combating antibiotic-resistant bacteria and their potential in vivo toxicology for further drug development.     

细菌生物膜形成与细菌耐药机制研究进展

细菌生物膜是一个具有结构性、协调性和功能性的高度组织群体,其相关感染因为对抗生素的耐药性成为临床难治性感染的重要原因之一。本文参阅近年来国内外研究结果,介绍了细菌生物膜的结构及特性,重点讨论其形成发展,抗生素耐药机制及防治策略方面的研究进展,对于寻找有效控制手段,指导临床合理用药和开发新药有重要意义。     

Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates

Efflux mechanisms protect bacterial cells by pumping out toxic compounds and actively contribute to bacterial multidrug resistance. Agents inhibiting efflux pumps are of interest for the control of multidrug-resistant bacterial infections. Herein we report the effects of new chloroquinoline derivatives that render resistant Enterobacter aerogenes isolates noticeably more susceptible to structurally unrelated antibiotics. In addition, some of these chloroquinolines increase the intracellular concentration of chloramphenicol. Some of the molecules tested in this work are able to inhibit the main efflux pump (AcrAB-TolC), which is involved in E. aerogenes antibiotic resistance.     

A cathelicidin-2-derived peptide effectively impairs Staphylococcus epidermidis biofilms

Staphylococcus epidermidis is a major cause of nosocomial infections owing to its ability to form biofilms on the surface of medical devices. Biofilms are surface-adhered bacterial communities. In mature biofilms these communities are encased in an extracellular matrix composed of bacterial polysaccharides, proteins and DNA. The antibiotic resistance of bacteria present in biofilms can be up to 1000-fold higher compared with the planktonic phenotype. Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel antibiotics. Recently, we demonstrated that a short variant of the HDP chicken cathelicidin-2, peptide F(2,5,12)W, has potent antibacterial and lipopolysaccharide-neutralising activities. This study reports on the antibiofilm activity of peptide F(2,5,12)W against two strains of S. epidermidis, including a multiresistant strain. Peptide F(2,5,12)W potently inhibited the formation of bacterial biofilms in vitro at a low concentration of 2.5 μM, which is below the concentration required to kill or inhibit growth (minimal inhibitory concentration=10 μM). Moreover, peptide F(2,5,12)W also impaired existing S. epidermidis biofilms. A 4-h challenge of pre-grown biofilms with 40 μM F(2,5,12)W reduced the metabolic activity of the wild-type strain biofilm completely and reduced that of the multiresistant strain biofilm by >50%. It is concluded that F(2,5,12)W prevents biofilm formation and impairs mature S. epidermidis biofilms.     

Antibiotic resistance of bacterial biofilms

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the body's defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms. Conventional resistance mechanisms such as chromosomal β-lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase) as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.     

Shortened Courses of Antibiotics for Bacterial Infections: A Systematic Review of Randomized Controlled Trials

Commonly prescribed durations of therapy for many, if not most, bacterial infections are not evidence‐based. Misunderstandings by clinicians and patients alike influence perspectives on antibiotic use, including duration of therapy and its role in antibiotic resistance. To demonstrate that shorter durations of antibiotic therapy are as efficacious as longer durations for many infections, a systematic review was undertaken of English‐language articles by using PubMed to identify articles for inclusion. Additionally, infection‐specific guidelines were identified for review of recommendations. Search terms included specific infection types, randomized controlled trial (RCT), duration of therapy, treatment duration, short course, and long course. Only RCTs of single‐agent antibiotic therapy for the treatment of bacterial infections in adults were included. Independent data extraction of articles was conducted by two authors by using predefined guidance for article inclusion. In total, 23 RCTs met our criteria for inclusion. All trials compared single‐agent antibiotics for a short and long antibiotic course in six common infections: community‐acquired pneumonia, ventilator‐associated pneumonia, intraabdominal infections, skin and soft tissue infections, uncomplicated cystitis, and complicated cystitis or pyelonephritis. Clinicians can decrease net antibiotic use by recommending shorter courses where evidence supports them. Antimicrobial stewardship programs that systematically address treatment duration may significantly affect institutional antibiotic use without negatively affecting patient care.     

非编码sRNA在细菌耐药机制方面的研究进展

近年来的研究发现,细菌应答抗生素压力时会产生特异的非编码小RNA(small RNA, sRNA)谱,进而可能调控下游基因的表达,帮助细菌克服抗生素压力。sRNA以各种方式调控细菌耐药相关基因(如抗生素转运蛋白、药物外排泵、细胞被膜的合成与修饰),参与细菌耐药网络。因此,sRNA及其相关因子(如Hfq)可能被用作抗菌治疗的靶标。本文将从sRNA应答抗生素压力并产生抗生素耐药及其作为药物靶点的前景等方面,综述sRNA在细菌耐药调控方面的研究进展。     

Use of antimicrobial peptides against microbial biofilms: advantages and limits

The formation of surface-attached cellular agglomerates, the so-called biofilms, contributes significantly to bacterial resistance to antibiotics and innate host defenses. Bacterial biofilms are associated to various pathological conditions in humans such as cystic fibrosis, colonization of indwelling medical devices and dental plaque formation involved in caries and periodontitis. Over the last years, natural antimicrobial peptides (AMPs) have attracted considerable interest as a new class of antimicrobial drugs for a number of reasons. Among these, there are the broad activity spectrum, the relative selectivity towards their targets (microbial membranes), the rapid mechanism of action and, above all, the low frequency in selecting resistant strains. Since biofilm resistance to antibiotics is mainly due to the slow growth rate and low metabolic activity of bacteria in such community, the use of AMPs to inhibit biofilm formation could be potentially an attractive therapeutic approach. In fact, due to the prevalent mechanism of action of AMPs, which relies on their ability to permeabilize and/or to form pores within the cytoplasmic membranes, they have a high potential to act also on slow growing or even non-growing bacteria. This review will highlight the most important findings obtained testing AMPs in in vitro and in vivo models of bacterial biofilms, pointing out the possible advantages and limits of their use against microbial biofilm-related infections.     

细菌药物主动外排系统在生物被膜耐药中的作用

摘要：细菌生物被膜的产生使传统抗菌药物难以对其进行有效的清除,进而导致严重感染的复发和持续性感染, 是人类目 前面临的又一新的挑战。生物被膜细菌中由于营养物质和代谢产物的积累,促使药物主动外排系统相关基因表达明显增加, 使 外排泵转运多种不同类型的化合物能力增强,从而产生多重耐药表型,是引起细菌耐药的主要机制之一;外排泵基因的表达对 生物被膜细菌的生长和耐药性增强方面有着重要作用。本文从生物被膜耐药的影响因素、药物主动外排系统参与生物被膜形成 及其影响、外排泵抑制剂对生物被膜耐药的影响几个方面对细菌药物主动外排系统在生物被膜耐药中的作用进行综述。     

海洋环境中细菌耐药性研究进展

细菌对抗生素的耐药性作为一个潜在威胁健康的问题已成为严重的全球性公共卫生问题，细菌耐药性持续存在是在相当长的时间内必须面对和认真研究解决的难题。海洋环境也已逐步成为耐药性细菌的重要储存库。因此，本文针对海洋环境中细菌耐药性这一问题，结合国内外最新文献，对海洋环境中细菌耐药性研究进展进行了综述，简单介绍了细菌耐药性在海洋环境中的产生原因、流行现状、传播途径及影响因素，并对未来重点研究方向进行了展望。     

Antibiotics as both friends and foes of the human gut microbiome: The microbial community approach

The exposure of the human gut to antibiotics can have a great impact on human health. Antibiotics pertain to the preservation of human health and are useful tools for fighting bacterial infections. They can be used for curing infections and can play a critical role in immunocompromised or chronic patients, or in fighting childhood severe malnutrition. Yet, the genomic and phylogenetic diversity of the human gut changes under antibiotic exposure. Antibiotics can also have severe side effects on human gut health, due to the spreading of potential antibiotic resistance genetic traits and to their correlation with virulence of some bacterial pathogens. They can shape, and even disrupt, the composition and functioning diversity of the human gut microbiome. Traditionally bacterial antibiotic resistances have been evaluated at clone or population level. However, the understanding of these two apparently disparate perspectives as both friends and foes may come from the study of microbiomes as a whole and from the evaluation of both positive and negative effects of antibiotics on microbial community dynamics and diversity. In this review we present some metagenomic tools and databases that enable the studying of antibiotic resistance in human gut metagenomes, promoting the development of personalized medicine strategies, new antimicrobial therapy protocols and patient follow-up.     

Antimicrobial and biofilm inhibiting diketopiperazines

Diketopiperazines are the smallest cyclic peptides known. 90% of Gram-negative bacteria produce diketopiperazines and they have also been isolated from Gram-positive bacteria, fungi and higher organisms. Biosynthesis of cyclodipeptides can be achieved by dedicated nonribosomal peptide synthetases or by a novel type of synthetases named cyclopeptide synthases. Since the first report in 1924 a large number of bioactive diketopiperazines was discovered spanning activities as antitumor, antiviral, antifungal, antibacterial, antiprion, antihyperglycemic or glycosidase inhibitor agents. As infections are of increasing concern for human health and resistances against existing antibiotics are growing this review focuses on the antimicrobial activities of diketopiperazines. The antibiotic bicyclomycin is a diketopiperazine and structure activity studies revealed the unique nature of this compound which was finally developed for clinical applications. The antimicrobial activities of a number of other diketopiperazines along with structure activity relationships are discussed. Here a special focus is on the activity-toxicity problem of many compounds setting tight limitations to their application as drugs. Not only these classical antimicrobial activities but also proposed action in modulating bacterial communication as a new target to control biofilms will be evaluated. Pathogens organized in biofilms are difficult to eradicate because of the increase of their tolerance for antibiotics for several orders. Diketopiperazines were reported to modulate LuxR-mediated quorum-sensing systems of bacteria, and they are considered to influence cell-cell signaling offering alternative ways of biofilm control by interfering with microbial communication. Concluding the review we will finally discuss the potential of diketopiperazines in the clinic to erase biofilm infections.