Effect of 2, 4-di-tert-butylphenol on growth and biofilm formation by an opportunistic fungus Candida albicans

Candida albicans, an opportunistic pathogen, has been known to form hypoxic biofilms on medical devices which in turn confers resistance towards antifungals, resulting in subsequent therapeutic failures. Inclusion of anti-biofilm agents in the control of infections is a topic of current interest in developing potential anti-infectives. The in vitro anti-fungal and anti-biofilm efficacy of 2,4-di-tert-butyl phenol [DTBP] was evaluated in this study, which revealed the potential fungicidal action of DTBP at higher concentrations where fluconazole failed to act completely. DTBP also inhibited the production of hemolysins, phospholipases and secreted aspartyl proteinase which are the crucial virulence factors required for the invasion of C. albicans. Various anti-biofilm assays and morphological observations revealed the efficacy of DTBP in both inhibiting and disrupting biofilms of C. albicans. Inhibition of hyphal development, a key process that aids in initial adhesion of C. albicans, was observed, and this could be a mechanism for the anti-biofilm activity of DTBP.     

Riboflavin analogs and inhibitors of riboflavin biosynthesis

Flavins are active components of many enzymes. In most cases, riboflavin (vitamin B₂) as a coenzyme represents the catalytic part of the holoenzyme. Riboflavin is an amphiphatic molecule and allows a large variety of different interactions with the enzyme itself and also with the substrate. A great number of active riboflavin analogs can readily be synthesized by chemical methods and, thus, a large number of possible inhibitors for many different enzyme targets is conceivable. As mammalian and especially human biochemistry depends on flavins as well, the target of the inhibiting flavin analog has to be carefully selected to avoid unwanted effects. In addition to flavoproteins, enzymes, which are involved in the biosynthesis of flavins, are possible targets for anti-infectives. Only a few flavin analogs or inhibitors of flavin biosynthesis have been subjected to detailed studies to evaluate their biological activity. Nevertheless, flavin analogs certainly have the potential to serve as basic structures for the development of novel anti-infectives and it is possible that, in the future, the urgent need for new molecules to fight multiresistant microorganisms will be met.     

Anti-infective therapy at the end of life: ethical decision-making in hospice-eligible patients

Clear guidelines addressing the ethically appropriate use of anti-infectives in the setting of hospice care do not exist. There is lack of understanding about key treatment decisions related to infection treatment for patients who are eligible for hospice care. Ethical concerns about anti-infective use at the end of life include: (1) delaying transition to hospice, (2) prolonging a dying process, (3) prescribing regimens incongruent with a short life expectancy and goals of care, (4) increasing the reservoir of potential resistant pathogens, (5) placing unreasonable costs on a capitated hospice system. Although anti-infectives are thought to be relatively safe, they can place a burden on patients and be inconsistent to particular care plans. The current complex, and at times fragmented, medical care often fails to address these issues in decision-making. In many ways, the ethics governing the end of life decisions related to dialysis, hydration/nutrition, and hypercalcemia parallel those of anti-infectives. In this article we articulate important elements in ethical decision-making in the application of anti-infectives for patients who are eligible for hospice care, and we point to the need for prospective studies to help refine particular guidelines in these cases.     

Inhibitors of pathogen intercellular signals as selective anti-infective compounds

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.     

Surpassing nature: rational design of sterile-surface materials

The rise of multidrug-resistant pathogens and recalcitrance of biofilm infections present a formidable challenge to combating infectious diseases. There are numerous disinfectants and antiseptics for treating materials in hospitals and community settings, and devices such as catheters impregnated with anti-infectives have been introduced into practice. However, there are many limitations of materials impregnated with a leaching antibacterial agent. Recently, non-leaching, permanent, sterile-surface materials have been developed in which one end of a long-chained hydrophobic polycation containing antimicrobial monomers is attached covalently to the surface of a material, for example, cotton or plastic. The polymeric chain allows the antimicrobial moieties to permeate into, and kill, the cells of the pathogen. These sterile-surface materials kill both air- and waterborne pathogens and are not susceptible to existing resistance mechanisms.     

Expression of the carbohydrate recognition domain of FimH and development of a competitive binding assay

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays.     

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.     

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria

Studies of lactic acid bacteria (LAB) as delivery vehicles have focused mainly on the development of mucosal vaccines, with much effort being devoted to the generation of genetic tools for antigen expression in different bacterial locations. Subsequently, interleukins have been co-expressed with antigens in LAB to enhance the immune response that is raised against the antigen. LAB have also been used as a delivery system for a range of molecules that have different applications, including anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. Now that the first human trial with a Lactococcus strain that expresses recombinant interleukin-10 has been completed, we discuss what we have learnt, what we do not yet understand and what the future holds for therapy and prophylaxis with LAB.     

Bacterial cell wall assembly: still an attractive antibacterial target

The development of new antibacterial agents to combat worsening antibiotic resistance is still a priority area in anti-infectives research, but in the post-genomic era it has been more difficult than expected to identify new lead compounds from high-throughput screening, and very challenging to obtain antibacterial activity for lead compounds. Bacterial cell-wall peptidoglycan biosynthesis is a well-established target for antibacterial chemotherapy, and recent developments enable the entire biosynthetic pathway to be reconstituted for detailed biochemical study and high-throughput inhibitor screening. This review article discusses recent developments in the availability of peptidoglycan biosynthetic intermediates, the identification of lead compounds for both the earlier cytoplasmic steps and the later lipid-linked steps, and the application of new methods such as structure-based drug design, phage display and surface science.     

Bioengineered bugs expressing oligosaccharide receptor mimics: toxin-binding probiotics for treatment and prevention of enteric infections

Many microbial pathogens recognize oligosaccharides displayed on the surface of host cells as receptors for toxins and adhesins. These ligand-receptor interactions are critical for disease pathogenesis, making them promising targets for novel anti-infectives. One strategy with particular utility against enteric infections involves expression of molecular mimics of host oligosaccharides on the surface of harmless bacteria capable of surviving in the gut. This can be achieved in Gram-negative bacteria by manipulating the outer core region of the lipopolysaccharide (LPS) through expression of cloned heterologous glycosyltransferases. The resultant chimeric LPS molecules are incorporated into the outer membrane by the normal assembly route and presented as a closely packed 2-D array of receptor mimics. Several such "designer probiotics" have been constructed, and these bind bacterial toxins in the gut lumen with very high avidity, blocking their uptake by host cells and thereby preventing disease.     

Seeing is believing: the impact of structural genomics on antimicrobial drug discovery

Over the past decade, the availability of complete microbial genome sequences has led to changes in the strategies that are used to search for novel anti-infectives. However, despite the identification of many new potential drug targets, novel antimicrobial agents have been slow to emerge from these efforts. In part, this reflects the long discovery and development times that are needed to bring new drugs to market and the bottlenecks at the stages of identifying good lead compounds and optimizing these leads into drug candidates. Structural genomics will hopefully provide opportunities to overcome these bottlenecks and populate the antimicrobial pipeline.     

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii

The resistance of Gram-negative bacteria to β-lactam antibiotics stems mainly from β-lactamase proteins that hydrolytically deactivate the β-lactams. Of particular concern are the β-lactamases that can deactivate a class of β-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by β-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D β-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the “real-time” deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a β-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or β-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling β-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.     

Staphylococcus and biofilms

The genetic and molecular basis of biofilm formation in staphylococci is multifaceted. The ability to form a biofilm affords at least two properties: the adherence of cells to a surface and accumulation to form multilayered cell clusters. A trademark is the production of the slime substance PIA, a polysaccharide composed of beta-1,6-linked N-acetylglucosamines with partly deacetylated residues, in which the cells are embedded and protected against the host's immune defence and antibiotic treatment. Mutations in the corresponding biosynthesis genes (ica operon) lead to a pleiotropic phenotype; the cells are biofilm and haemagglutination negative, less virulent and less adhesive on hydrophilic surfaces. ica expression is modulated by various environmental conditions, appears to be controlled by SigB and can be turned on and off by insertion sequence (IS) elements. A number of biofilm-negative mutants have been isolated in which polysaccharide intercellular adhesin (PIA) production appears to be unaffected. Two of the characterized mutants are affected in the major autolysin (atlE) and in D-alanine esterification of teichoic acids (dltA). Proteins have been identified that are also involved in biofilm formation, such as the accumulation-associated protein (AAP), the clumping factor A (ClfA), the staphylococcal surface protein (SSP1) and the biofilm-associated protein (Bap). Concepts for the prevention of obstinate polymer-associated infections include the search for new anti-infectives active in biofilms and new biocompatible materials that complicate biofilm formation and the development of vaccines.     

Inhibition of β-carbonic anhydrases from Brucella suis with C-cinnamoyl glycosides incorporating the phenol moiety

Abstract

A small series of C-glycosides containing the phenol moiety was tested for the inhibition of the β-class carbonic anhydrases (βCAs, EC 4.2.1.1) from Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. Glycosides incorporating the 3-hydroxyphenyl moiety showed the best inhibition profile, and therefore this functionality represents lead for the development of novel anti-infectives with a new mechanism of action.     

Effects of perceived patient demand on prescribing anti-infective drugs

BACKGROUND: Although patient demand is frequently cited by physicians as a reason for inappropriate prescribing, the phenomenon has not been adequately studied. The objectives of this study were to determine the prevalence of perceived patient demand in physician-patient encounters; to identify characteristics of the patient, physician and prescribing situation that are associated with perceived demand; and to determine the influence of perceived demand on physicians'' prescribing behaviour. METHODS: An observational study using 2 survey approaches was conducted in February and March 1996. Over a 2-day period 20 family physicians in the Toronto area completed a brief questionnaire for each patient encounter related to suspected infectious disease. Physicians were later asked in an interview to select and describe 1 or 2 incidents from these encounters during which perceived patient demand influenced their prescribing (critical incident technique). RESULTS: Perceived patient demand was reported in 124 (48%) of the 260 physician-patient encounters; however, in almost 80% of these encounters physicians did not think that the demand had much influence on their decision to prescribe an anti-infective. When clinical need was uncertain, 28 (82%) of 34 patients seeking an anti-infective were prescribed one, and physicians reported that they were influenced either "moderately" or "quite a bit" by perceived patient demand in over 50% of these cases. Of the 35 critical prescribing incidents identified during the interviews, anti-infectives were prescribed in 17 (49%); the reasons for prescribing in these situations were categorized. INTERPRETATION: This study provides preliminary data on the prevalence and influence of perceived patient demand in prescribing anti-infectives. Patient demand had more influence on prescribing when physicians were uncertain of the need for an anti-infective.     

C-glycosides incorporating the 6-methoxy-2-naphthyl moiety are selective inhibitors of fungal and bacterial carbonic anhydrases

Abstract

A small series of C-glycosides containing the methoxyaryl moieties was tested for the inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from Cryptococcus neoformans and Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. The deprotected glycosides incorporating the 6-methoxy-2-naphthyl moiety showed the best inhibition profile and therefore represent leads for the development of novel anti-infectives with a new mechanism of action.     

Host-defense peptides of the skin with therapeutic potential: From hagfish to human

It is now well established that peptides that were first identified on the basis of their ability to inhibit growth of bacteria and fungi are multifunctional and so are more informatively described as host-defense peptides. In some cases, their role in protecting the organism against pathogenic microorganisms, although of importance, may be secondary. A previous article in the journal (Peptides 2014; 57:67–77) assessed the potential of peptides present in the skin secretions of frogs for development into anticancer, antiviral, immunomodulatory and antidiabetic drugs. This review aims to extend the scope of this earlier article by focusing upon therapeutic applications of host-defense peptides present in skin secretions and/or skin extracts of species belonging to other vertebrate classes (Agnatha, Elasmobranchii, Teleostei, Reptilia, and Mammalia as represented by the human) that supplement their potential role as anti-infectives for use against multidrug-resistant microorganisms.     

Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and SRC family tyrosine kinases

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.     

State-of-the-art therapeutic medical countermeasures for viral threat agents

In recent years, there has been an increase in the perceived threat of biological agents being used against civilian populations. This has prompted an urgent need for the development and procurement of medical countermeasures (MCMs) against highly pathogenic viruses that can prevent morbidity and mortality from infections caused by these agents. To date, antiviral drug development has been largely focused on clinically prevalent chronic infections due to their commercial viability. This has left a huge gap in the drug development path for acute infections of biodefense importance. In this review, we discuss the antiviral research and development initiatives focusing specifically on poxviruses, filoviruses, and equine encephalitis viruses (EEV). We discuss the benefits and technical challenges in the current development strategies and the hurdles in the licensure path for MCMs against these highly pathogenic viruses under the FDA Animal Rule, and we provide recommendations for the path forward.     

Making space for anti-infective drug discovery

The dwindling supply of effective treatments for infectious disease is cause for alarm. Searches for anti-infectives yielding fewer and fewer novel discoveries have been concentrated in overly restricted regions of target space, screening space, chemical space, and competition space. Appreciating the diverse axes of these spaces may encourage wider exploration.