Antimicrobial resistance,respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients

Lung infection is the main cause of morbidity and mortality in patients with cystic fibrosis and is mainly dominated by Pseudomonas aeruginosa. The biofilm mode of growth makes eradication of the infection impossible, and it causes a chronic inflammation in the airways. The general mechanisms of biofilm formation and antimicrobial tolerance and resistance are reviewed. Potential anti-biofilm therapeutic targets such as weakening of biofilms by quorum-sensing inhibitors or antibiotic killing guided by pharmacokinetics and pharmacodynamics of antibiotics are presented. The vicious circle of adaptive evolution of the persisting bacteria imposes important therapeutic challenges and requires development of new drug delivery systems able to reach the different niches occupied by the bacteria in the lung of cystic fibrosis patients.     

葡萄球菌属细菌生物被膜研究进展

细菌生物被膜是细菌为适应自然环境有利于生存而特有的生命现象,广泛存在于自然界,现代医学材料和许多慢性细菌感染性疾病的器官组织表面均发现有细菌生物被膜。近年来,国内外研究发现,细菌生物被膜与生物医学材料相关感染和某些慢性顽固性感染疾病反复发作有关,尤其是葡萄球菌属细菌生物被膜已成为引起很多严重感染久治不愈和反复发作的重要原因之一。通过查阅国内外文献,我们对葡萄球菌属细菌生物被膜研究进展进行综述。     

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.     

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.     

Pharmaceutical strategies for the treatment of bacterial biofilms in chronic wounds

Biofilms are sessile communities of microorganisms, mainly bacteria, that grow on biotic and abiotic surfaces. These microorganisms are embedded within an extracellular polymeric substance that provides enhanced protection from antimicrobials. Chronic wounds provide an ideal habitat for biofilm formation. Bacteria can easily attach to wound debris and can infect the wound due to an impaired host immune response. This review highlights the mechanism of biofilm formation and the role of biofilms in the pathophysiology of chronic wounds. Our major focus is on various formulation strategies and delivery systems that are employed to eradicate or disperse biofilms, thereby effectively managing acute and chronic wounds. We also discuss clinical research that has studied or is studying the treatment of biofilm-infected chronic wounds.     

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.     

铜绿假单胞菌中乙酰化褐藻胶的生物合成和功能研究进展

铜绿假单胞菌(Pseudomonas aeruginosa)是临床上三大机会致病菌之一,可导致多种急慢性感染。铜绿假单胞菌感染人体后,往往会转变成过量产生乙酰化褐藻胶的粘液型细菌,并形成生物被膜(biofilm)。一旦形成生物被膜,细菌将具有极强的抗生素耐药性和逃避机体免疫系统攻击的能力,造成临床上难治性、持续性感染。乙酰化褐藻胶是P. aeruginosa 生物被膜的主要成分,决定了生物被膜的结构与功能。因此,本文从乙酰化褐藻胶的生物合成途径及调控机制、在细菌中的生物学功能及作为抗菌药物开发靶点等几个方面进行了综述。     

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。     

铜绿假单胞菌生物被膜研究进展

铜绿假单胞菌是临床上常见的机会致病菌和耐药菌之一,它的感染常伴随着生物被膜的产生,进而造成严重的慢性感染和持续性感染。由于铜绿假单胞菌生物被膜的产生使其产生耐药性,并能够逃避宿主免疫系统的攻击,因此传统治疗方法很难将其去除。以生物被膜为靶标开发新型抗感染药物可以降低病原菌的耐药性,有望发展成1种新的治疗方法。本文总结了铜绿假单胞菌生物被膜的形成过程、主要成分的结构、生物合成过程及其功能,并对已经报道的靶向生物被膜的新型药物进行了总结。     

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).     

Intestinal flora and Crohn's disease

The pathogenesis of inflammatory bowel diseases (IBD) proceeds through stages of initiation, amplification and healing. Abundant clinical and experimental data incriminate luminal bacteria or bacterial products in both the initiation and perpetuation of chronic intestinal inflammation. Macrophage and T-cell activation with accompanying inflammatory cytokine production appears to be an early event. Studies of lymphocyte responsiveness to autologous and heterologous intestinal bacteria have suggested that this activation may result from a breakdown in tolerance to the enteric flora in IBD. This lack of tolerance might be due to an imbalance between protective and aggressive commensal luminal bacterial species (dysbiosis), a decreased barrier function and/or an impaired mucosal clearance allowing the access of bacteria to the mucosal immune system and lack of regulatory mediators or cells. There is still controversy over whether the virulence traits of bacteria are expressed broadly or just in a small subset of bacteria. Individual bacterial species within the indigenous flora vary in their capacity to drive intestinal inflammation. In experimental models, some bacteria such as Bacteroides vulgatus can cause colitis alone when monoassociated in the HLA-B27 transgenic rat model. Others, including Lactobacillus and Bifidobacterium species have no proinflammatory capacity and have been used as probiotics. In patients with IBD, systematic approach to this issue is hampered by the limited knowledge of intestinal flora. Adherent-invasive Escherichia coli are a possible candidate for the onset and/or persistence of intestinal inflammation in patients with Crohn's disease, since they possess all the virulence factors that allow the bacteria to cross the intestinal barrier, to move to deep tissues, and to continuously activate macrophages. The recent identification of NOD2/CARD15 as a susceptibility gene for Crohn's disease has provided another link between the immune response to enteric bacteria and the development of mucosal inflammation. NOD2/CARD15 is composed of two caspase recruitment domain (CARD), a nucleotide-binding domain (NBD) and a leucin-rich-repeat (LRR) region. The LRR domain of NOD2/CARD15 has binding activity for bacterial peptidoglycans and its deletion stimulates the NF-kappaB pathway. The most frequent variants of NOD2/CARD15 observed in Crohn's disease tend to cluster in the LRR and its adjacent regions. This suggests that the LRR domain of CD-associated variants is likely to be impaired in its recognition of microbial components. Continuing studies are investigating the pathophysiological mechanisms induced by NOD2/CARD15 variants in the intestinal mucosa.     

中药对细菌生物被膜作用机制的研究进展

如今越来越多耐药细菌的出现,对于细菌耐药的研究显得更为迫切,而现今的研究已经发现细菌的生物被膜是其耐药作用的一个独特机制。许多中药被发现对于细菌的生物被膜具有抑制作用,是细菌耐药性研究中一个独特的研究领域。通过查阅分析国内外有关中药对细菌生物被膜抗菌作用的文献,从细菌生物被膜形成过程、细菌生物被膜的耐药机制以及中药对于细菌生物被膜的作用等方面进行综述,为进一步研究中药的抗菌作用提供参考。     

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.     

Prospects for the prevention and control of pseudomonal infection in children with cystic fibrosis

Most patients with cystic fibrosis (CF) experience recurrent and chronic endobronchial Pseudomonas aeruginosa infections. It is possible to prevent or delay the onset of these chronic infections in most patients with CF by eliminating cross-infection and by early aggressive antibiotic treatment of the first positive sputum culture and of subsequent intermittent colonisation. Lung tissue damage is caused by activation of the immunologically specific inflammatory defence mechanisms of the lungs, which are initiated by the antibody response and dominated by polymorphonuclear neutrophil leucocytes and their proteolytic and oxidative products. This inflammation induces a phenotypic shift from nonmucoid to mucoid, alginate-producing phenotypes of P. aeruginosa which then grow, endobronchially, as a biofilm. Such biofilms are impossible to eradicate with antibiotics. By using chronic suppressive antibiotic maintenance therapy and anti-inflammatory drugs it is however, possible to maintain the lung function of these patients for a number of years.     

Synergistic antimicrobial effect against early biofilm formation: micropatterned surface plus antibiotic treatment

The detrimental effects of biofilms are a cause of great concern in medical, industrial and environmental areas. In this study, we proposed a novel eradication strategy consisting of the combined use of micropatterned surfaces and antibiotics on biofilms to reduce the rate of bacterial colonisation. Pseudomonas fluorescens biofilms were used to perform a comparative evaluation of possible strategies to eradicate these biological layers. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of planktonic cultures were determined. Subsequently, adhesion of bacteria on microstructured gold surfaces (MS) with patterned features that were similar to the bacterial diameter as well as on smooth nanostructured gold (NS) was assessed. As expected, lower bacterial attachment as well as inhibition of bacterial aggregation were observed on MS. The effect of streptomycin treatment (ST) in the concentration range 1-4mg/L (0.25-1× MIC) on biofilms grown on MS and NS was also evaluated. The combined strategy involving the use of micropatterned surfaces and antibiotic treatment (MS+ST) to eradicate Pseudomonas biofilms was then investigated. Results showed a synergistic effect of MS+ST that yielded a reduction of ≥1000-fold in the number of surviving biofilm bacteria with respect to those obtained with single ST or MS. The combined strategy may be a significant contribution to the eradication of biofilms from different environments. In addition, the important role of early monolayer bacterial aggregates in increasing resistance to antimicrobial agents was demonstrated.     

In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xylitol

The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.     

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.     

Anaerobic Pseudomonas aeruginosa and other obligately anaerobic bacterial biofilms growing in the thick airway mucus of chronically infected cystic fibrosis patients: an emerging paradigm or "Old Hat"?

Introduction: The cystic fibrosis (CF) airway mucus is an ideal niche in which many bacteria can develop antibiotic- and phagocyte-resistance in unique structures known as "mode II biofilms" where bacteria are embedded within the mucus, yet unattached to airway epithelial cells. Pseudomonas aeruginosa is the dominant CF pathogen, yet herein the authors provide burgeoning evidence that obligate anaerobic bacteria (e.g., Prevotella) actually thrive within the CF mucus, a paradigmatic shift that chronic CF is an "aerobic" disease. Interestingly, CF organisms repress virulence factor production (e.g., P. aeruginosa) while others (e.g., S. aureus) increase them under anaerobic conditions. Areas covered: The authors shed additional light on (i) the anoxic nature of the CF airway mucus, (ii) the relative commonality of anaerobic bacteria isolated from CF sputum, (iii) virulence factor production and cross-talk between obligate anaerobes and P. aeruginosa relative to disease progression/remission, (iv) the role of mucoidy in CF, and (v) the role of nitrosative stress in activation of bacteriophage and pyocins within biofilms. Expert opinion: The authors conclude with insight as to how we might treat some CF bacteria during mode II biofilm infections that utilizes a metabolite of bacterial anaerobic respiration and an aerobic oxidation product of airway-generated NO, acidified NO(2) (-).     

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.