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.     

A Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to the global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reducing ATP production and inhibiting the activity of efflux pumps), impairs the respiration chain, promotes the production of reactive oxygen species (ROS) in bacterial cells and induces intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and the elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa.     

Breaking membrane barriers to neutralize E. coli and K. pneumoniae virulence with PEGylated branched polyethylenimine

Bacterial infections caused by Gram-negative pathogens, such as those in the family Enterobacteriaceae, are among the most difficult to treat because effective therapeutic options are either very limited or non-existent. This raises serious concern regarding the emergence and spread of multi-drug resistant (MDR) pathogens in the community setting; and thus, creates the need for discovery efforts and/or early-stage development of novel therapies for infections. Our work is directed towards branched polyethylenimine (BPEI) modified with polyethylene glycol (PEG) as a strategy for targeting virulence from Gram-negative bacterial pathogens. Here, we neutralize lipopolysaccharide (LPS) as a barrier to the influx of antibiotics. Data demonstrate that the β-lactam antibiotic oxacillin, generally regarded as ineffective against Gram-negative bacteria, can be potentiated by 600 Da BPEI to kill some Escherichia coli and some Klebsiella pneumoniae. Modification of 600 Da BPEI with polyethylene glycol (PEG) could increase drug safety and improves potentiation activity. The ability to use the Gram-positive agent, oxacillin, against Gram-negative pathogens could expand the capability to deliver effective treatments that simplify, reduce, or eliminate some complicated treatment regimens.     

The natural plant compound carvacrol as an antimicrobial and anti-biofilm agent: mechanisms,synergies and bio-inspired anti-infective materials

Abstract

Carvacrol (5-isopropyl-2-methyl phenol) is a natural compound that occurs in the leaves of a number of plants and herbs including wild bergamot, thyme and pepperwort, but which is most abundant in oregano. The aim of this review is to analyse the scientific data from the last five years (2012-2017) on the antimicrobial and anti-biofilm activities of carvacrol, targeting different bacteria and fungi responsible for human infectious diseases. The antimicrobial and anti-biofilm mechanisms of carvacrol and its synergies with antibiotics are illustrated. The potential of carvacrol-loaded anti-infective nanomaterials is underlined. Carvacrol shows excellent antimicrobial and anti-biofilm activities, and is a very interesting bioactive compound against fungi and a wide range of Gram-positive and Gram-negative bacteria, and being active against both planktonic and sessile human pathogens. Moreover, carvacrol lends itself to being combined with nanomaterials, thus providing an opportunity for preventing biofilm-associated infections by new bio-inspired, anti-infective materials.     

Synthesis of linear and cyclic guazatine derivatives endowed with antibacterial activity

Antibiotic resistance has reached alarming levels in many clinically-relevant human pathogens, and there is an increasing clinical need for new antibiotics active on drug-resistant Gram-negative pathogens who rapidly evolve towards pandrug resistance phenotypes. Here, we report on two related classes of guanidinic compounds endowed with antibacterial activity. The two best compounds (9a and 13d) exhibited the most potent antibacterial activity with MIC values ranging 0.12–8 μg/ml with most tested pathogens, including both Gram-positive and Gram-negative bacteria. Interestingly, MIC values were not affected (1–8 μg/ml) when measured using recent clinical isolates with various antibiotic resistance determinants. The results reported herein identify guazatine derivatives as an interesting starting point for the optimization of a potentially novel class of antibacterial agents.     

Susceptibility of Human Pathogenic Bacteria to Antimicrobial Peptides from Sesame Kernels

Hospital infection caused by Gram-negative bacteria is a serious and common problem, especially in developing countries. Aiming to reduce these infections, this report focuses on the identification and characterization of novel antimicrobial peptides from sesame (Sesamum indicum) kernel meals. Thus, sesame flour was extracted and precipitated with ammonium sulfate (100%). After dialysis, a rich fraction was applied to affinity red-Sepharose CL-6B chromatography, followed by reversed-phase high-performance liquid chromatography. Mass spectrometry analysis indicated the presence of a major peptide with molecular mass of ∼5.8 kDa in both cultivars. The bactericidal activities of antimicrobial peptides were evaluated against several human pathogens that had been effective only against Klebsiella sp., a Gram-negative bacterium responsible for human urinary infection. These data indicate the biotechnological potential of sesame peptides as an alternative method for hospital infection control and also the decrease of bacterial resistance to synthetic antibiotics.     

Inhibition of Quorum Sensing–Regulated Behaviors by <Emphasis Type="Italic">Scorzonera sandrasica</Emphasis>

Many Gram-negative bacteria use N-acyl homoserine lactone signal molecules to monitor their own population density and coordinate gene regulation in a process called quorum sensing (QS). Increasing evidence implies that certain eukaryotes produce QS-inhibitory compounds. In this work, we tested 46 terrestrial plants materials for their ability to inhibit QS-regulated behaviors in different bacterial species. Plant materials were dried and extracted using different solvents. The chloroform-soluble compounds extracted from Scorzonera sandrasica were found to inhibit violacein production, a QS-regulated behavior in Chromobacterium violaceum. In addition, the chloroform extract was also able to inhibit QS-regulated carbapenem antibiotic production in Erwinia carotovora. Because the regulation of many bacterial processes is controlled by QS systems, the finding of natural compounds acting as QS inhibitors suggests an attractive tool to control and handle detrimental infections caused by human, animal, and plant pathogens.     

Aedesin: Structure and Antimicrobial Activity against Multidrug Resistant Bacterial Strains

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4^th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.     

A Fatty Acid from the Diatom Phaeodactylum tricornutum is Antibacterial Against Diverse Bacteria Including Multi-resistant Staphylococcus aureus (MRSA)

Pathogenic bacteria, such as multidrug-resistant Staphylococcus aureus (MRSA), which are not susceptible to most conventional antibiotics, are causing increased concern in healthcare institutions worldwide. The discovery of novel antibacterial compounds for biomedical exploitation is one avenue that is being pursued to combat these problematic bacteria. Marine eukaryotic microalgae are known to produce numerous useful products but have attracted little attention in the search for novel antibiotic compounds. Cell lysates of the marine diatom, Phaeodactylum tricornutum Bohlin, have been reported to display antibacterial activity in vitro, but the compounds responsible have not been fully identified. In this paper, using column chromatography and reversed-phase high-performance liquid chromatography, we report the isolation of an antibacterial fatty acid. Mass spectrometry and ¹H-nuclear magnetic resonance spectroscopy revealed it to be the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). We show that EPA is active against a range of both Gram-positive and Gram-negative bacteria, including MRSA, at micromolar concentrations. These data indicate that it could find application in the topical and systemic treatment of drug-resistant bacterial infections.     

Homology modeling,docking and structure-based virtual screening for new inhibitor identification of Klebsiella pneumoniae heptosyltransferase-III

Abstract

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative opportunistic pathogen commonly associated with hospital-acquired infections that are often resistant even to antibiotics. Heptosyltransferase (HEP) belongs to the family of glycosyltransferase-B (GT-B) and plays an important in the synthesis of lipopolysaccharides (LPS) essential for the formation of bacterial cell membrane. HEP-III participates in the transfer of heptose sugar to the outer surface of bacteria to synthesize LPS. LPS truncation increases the bacterial sensitivity to hydrophobic antibiotics and detergents, making the HEP as a novel drug target. In the present study, we report the 3D homology model of K. pneumoniae HEP-III and its structure validation. Active site was identified based on similarities with known structures using Dali server, and structure-based pharmacophore model was developed for the active site substrate ADP. The generated pharmacophore model was used as a 3D search query for virtual screening of the ASINEX database. The hit compounds were further filtered based on fit value, molecular docking, docking scores, molecular dynamics (MD) simulations of HEP-III complexed with hit molecules, followed by binding free energy calculations using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA). The insights obtained in this work provide the rationale for design of novel inhibitors targeting K. pneumoniae HEP-III and the mechanistic aspects of their binding.

Communicated by Ramaswamy H. Sarma     

Modulation of antibiotic resistance in bacterial pathogens by oleanolic acid and ursolic acid

Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. Some plant-derived compounds, e.g. pentacyclic triterpenoids such as oleanolic acid (OA) and ursolic acid (UA), have potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and specifically target the cell envelope. The aim of the present study was to investigate the influence of OA and UA on the susceptibility of four bacterial pathogens (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidis) to the β-lactam antibiotics ampicillin (Ap) and oxacillin (Ox). Antimicrobial assays were conducted with bacteria growing in liquid suspension cultures (planktonic cells) or as biofilms. Using FICI value estimation and the time-kill method it was demonstrated that in some combinations, the tested compounds acted in synergy to lower the susceptibility of S. aureus, S. epidermidis and L. monocytogenes to ampicillin and oxacillin, but no synergy was observed for P. aeruginosa. These results indicate that OA and UA may be useful when administered in combination with β-lactam antibiotics to combat bacterial infections caused by some Gram-positive pathogens.     

铜绿假单胞菌生物被膜组成及其受群体感应系统和c-di-GMP调控的研究进展

作为人类条件性感染的前三大病原菌之一的铜绿假单胞菌,是一种革兰氏阴性细菌,对免疫功能低下和囊性纤维化患者可以造成严重和持续性感染。造成这种持续感染的原因主要是由于细菌接收外界信号后,在自身调控网络的协同作用下,会依附于固体表面,并产生胞外多糖、基质蛋白和胞外DNA等大分子物质形成高度结构化的膜状复合物将自身包裹形成生物被膜群体结构。生物被膜可以有效帮助细菌定殖、提高细菌对抗菌物质和宿主免疫反应的抵抗能力、促进群落细菌的细胞-细胞之间的信号交流等,是临床治疗中病原菌慢性感染和反复感染最重要的原因之一。本篇综述重点介绍了铜绿假单胞菌生物被膜的各组成成分及其在生物被膜形成中的重要功能,并进一步阐述了群体感应系统(las、rhl、pqs与iqs)和c-di-GMP对铜绿假单胞菌生物被膜形成的调控作用。通过本篇综述可以更清晰地了解细菌生物被膜形成和调控的过程,为开发新的治疗生物被膜感染策略提供帮助。     

Antifungal-activity-producing lactic acid bacteria as biocontrol agents in plants

Fungal infection represents a severe problem that decreases the yield and market value of fruit crops. The use of fungicides is a conventional method to control infections but it is associated with disadvantages, such as hazardous impact on public health, environmental contamination, resistance development among pathogens and high cost of agrochemicals. Biological control is an alternative approach for the treatment of fungal infections. The species of Bacillus, Pseudomonas, Enterobacter, Pantoea, Burkholderia, Lysobacter and Serratia have been successfully used in the control of fungal infections. The mechanisms involved in biocontrol are hyperparasitism or predation, production of antibiotics, lytic enzymes and induction of host resistance. Lactic acid bacteria have been used as biopreservative organisms in food and feed systems. They are a cluster of Gram-positive bacteria and include species of the genera Enterococcus, Lactobacillus, Leuconostoc, Lactococcus and Pediococcus. The ability to produce several antibacterial and antifungal substances confers biopreservation potential to lactic acid bacteria. Many have ‘generally regarded as safe’ status and are considered as safe from both human and environmental points of view. Their isolation is reported from vegetables, aerial plant surfaces, pickled cabbage, grass silage, malted cereals and also from soil. They produce antifungal substances, such as cyclic dipeptides, proteinaceous compounds, organic acids, fatty acids and reuterin. The biocontrol potential of lactic acid bacteria is demonstrated in the prevention of fungal infections of fruits, such as apples and grapes. Thus, living cells or product formulations of antifungal lactic acid bacteria may be prepared and used as an alternative biocontrol technology.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.     

Characteristic of Bacteriocins of Lactobacillus rhamnosus BTK 20-12 Potential Probiotic Strain

Multidrug resistance (MDR) is a serious health threat throughout the world resulting in reduced efficacy of antibacterial, antiparasitic, antiviral, and antifungal drugs. One of the most promising concepts that may represent a good alternative to antibiotics can be the use of bacteriocins obtained from lactic acid bacteria. The L. rhamnosus BTK 20-12 strain was isolated from traditional Armenian naturally fermented salted cheese. The probiotic potential of the strain was approved. It was shown that strain produced at less two bacteriocins (BCN 1 and BCN 2) with different molecular weight (1427 Da and 602.6 Da, respectively). Bacteriocins inhibited the growth of multidrug-resistant bacteria of different etiologies and belong to different taxonomic groups with diverse efficiency and it depends on properties of bacteriocins, as well as from isolation sources of pathogens. Thus, bacteriocins of L. rhamnosus BTK 20-12 have protein-like nature and a broad range of activity and are excellent candidates for the development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics.

     

Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics

The research reported here deals with co-action of the hop (Humulus lupulus)-derived anti-bacterial compounds, lupulone and xanthohumol, with several antibiotics. Among the antibiotics investigated for their co-action, polymyxin B sulfate, tobramycin and ciprofloxacin had a positive co-action in inhibiting selected test bacteria. The disc/well-diffusion assay and the minimum inhibitory concentration test (MIC) were employed to determine co-action. Both Gram-positive and Gram-negative bacteria were used in the evaluation. There was some co-action against all Gram-positive bacteria tested. Surprisingly, there was some positive co-action even against certain Gram-negative bacteria but not against others. Particularly, there was no co-action against E.coli. An antibacterial cream with lupulone, neomycin and polymyxin B sulfate was prepared and showed co-action. Ideas for other practical applications of this effect are put forth. The mechanism of the synergistic effect is briefly discussed but no attempt was made to prove it experimentally.     

Lipopolysaccharide as an antibiotic target

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.     

某院感染科病房住院患者常见病原菌分布及耐药性分析

目的 对感染科病房住院患者临床常见病原菌的分布及其耐药性进行分析,为临床预防和治疗感染性疾病提供依据。方法 回顾性分析中国医科大学附属第一医院感染科病房2012年1月至2016年12月住院患者体液及组织样本中分离的病原菌,对其耐药情况进行分析。结果 5年中感染科共分离出非重复病原菌1 266株,其中革兰阴性菌786株,占62.09%,分离率居前3位的是大肠埃希菌、肺炎克雷伯菌以及铜绿假单胞菌,分别占17.22%、15.24%和10.58%;革兰阳性菌480株,占37.91%,分离率居前3位的是屎肠球菌、草绿色链球菌以及金黄色葡萄球菌,分别占9.79%、7.50%和6.00%。产超广谱β-内酰胺酶（ESBLs）大肠埃希菌和肺炎克雷伯菌检出率分别为66.7%和28.8%。大肠埃希菌对亚胺培南和美罗培南的耐药率分别为0.95%和3.79%,肺炎克雷伯菌对亚胺培南和美罗培南的耐药率分别为2.80%和2.80%。结论 我院感染科病房住院患者感染病原菌以革兰阴性菌为主,为有效的控制和避免耐药菌感染的发生,临床应根据药敏试验结果合理应用抗菌药物。