Mechanisms of action of antibacterial biocides

Antibacterial biocides are represented by a wide range of chemical agents. This chemical diversity offers a multiplicity of potentially damaging interactions with the bacterial cell. Only rarely, however, are these interactions non-specific in nature; more frequently, the morphology and physiology of the cell, when combined with the physicochemical properties of the biocide, will dictate specific targets or target regions. A knowledge and understanding of these lesions offers a powerful tool in the search for novel chemistries and improved biocidal capabilities.     

Mechanisms of bacterial biocide and antibiotic resistance

Resistance to antibiotics is increasingly commonplace amongst important human pathogens. Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux. While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems. Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes. Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms. Still, target site mutations leading to biocide resistance, though rare, are known. Intriguingly, many multidrug efflux systems also accommodate biocides (e.g. triclosan) such that strains expressing these are both antibiotic- and biocide-resistant. Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics. Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan. These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two. Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems. Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.     

Cellular impermeability and uptake of biocides and antibiotics in gram-negative bacteria

The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.     

Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?

Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.     

Biochemical significance of the hard and soft acids and bases principle.

The hard and soft acids and bases (HSAB) principle, which states that hard acids bind preferentially to hard bases and soft acids to soft bases, may serve to assess specific chemico-biological interactions. As living systems are composed mainly of "hard" elements, molecular events taking place within the cell are dominated by "hard-hard interactions". On this premise, it becomes likely that extraneous "soft" agents are particularly injurious to life. In the HSAB context a selected number of variegated phenomena are briefly discussed qualitatively; these include biocidal actions, heavy metal poisoning, chemical carcinogenesis, some enzymic reactions, and nucleic acid complexations. Although the HSAB principle cannot be used as a tool for mechanistic explanations of biochemical processes, it may provide clues to likely target molecules and the loci of action.     

On the possible use of exogenous histones in cell technology

The prospect of developing transport systems using histones for site-specific delivery of therapeutic agents that have poor penetration characteristics through cellular membranes and tissue barriers has been investigated. Histones immobilized on microspheres can also be used to modify surfaces intended for cell cultivation, facilitating adhesion, proliferation and network formation by interactions of cells through contacts with several microspheres. They can be applied to three-dimensional pore matrices that are designed for producing tissue-like structures in vitro.     

Modifications of nucleosides by endogenous mutagens-DNA adducts arising from cellular processes

DNA damage plays a significant role in mutagenesis, carcinogenesis and ageing. Chemical transformations leading to DNA damage include reactions of the base units with agents of endogenous and exogenous origin. The vast majority of damage arising from cellular processes such as metabolism and lipid peroxidation are identical or very similar to those induced by exposure to environmental agents. A detailed knowledge of the types and prevalence of endogenous DNA damage provides insight into the chemical nature of species involved in these modifications and may be of help in understanding their influence on the induction of cancer or other diseases. This knowledge may also be essential to the development of rational chemopreventive strategies directed against the initiation of oxidative stress- and lipid peroxidation-associated pathology.The present work reviews findings regarding the interaction between DNA bases and various reactive species arising from lipid peroxidation and other cellular processes, drawing attention to the mechanism responsible for the formation of the resulted modifications. The biological consequences of these interactions are also briefly discussed.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Summary Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress within vivo gene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissuesin vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages forin vivo applications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumventedin vivo, which is possible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCMin vivo. When labeled with a radioisotope (e.g.,¹²⁵I or¹¹¹In), the antisense chimeric peptide provides imaging of gene expression in the brainin vivo in a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow forin vivo imaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.     

Targeted therapy for cancer using pH-responsive nanocarrier systems

Most of the conventional chemotherapeutic agents used against cancer have poor efficacy. An approach to improve the efficacy of cancer chemotherapy is the development of carrier systems that can be triggered to release the anticancer drug in response to extracellular or intracellular chemical stimuli. To this end, pH-responsive nanocarriers have been developed to target drugs either to the slightly acidic extracellular fluids of tumor tissue or, after endocytosis, to the endosomes or lysosomes within cancer cells. These systems can release the drug by specific processes after accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect or they can release the drugs in endosomes or lysosomes by pH-controlled hydrolysis after they are taken up by the cell via the endocytic pathway. This strategy facilitates the specific delivery of the drug while reducing systemic side-effects with high potential for improving the efficacy of cancer chemotherapy.     

Signal transmission using second messengers

Intercellular communication needs signal molecules (hormones, neurotransmitters, growth factors) emitted by a cell and recognized by targets. A widespread mechanism of cellular action of these molecules involves a membrane receptor, a GTP-dependent coupling protein, an effector which modulates the intracellular concentration of a second messenger (cAMP, cGMP, inositol triphosphate, Ca++), and a target for final effect of this second messenger (kinases, channel). Hormonal systems and visual processes in vertebrates rod outer segments are the best known examples of this mechanism; they are taken here to illustrate the state of knowledge of the interactions between the actors of the transductional enzymatic cascades.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress with in vivogene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissues in vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages for in vivoapplications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumvented in vivo, which ispossible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCM in vivo. When labeled with a radioisotope (e.g., ¹²⁵I or ¹¹¹In), the antisense chimeric peptide provides imaging of gene expressionin the brain in vivoin a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow for in vivoimaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Physical properties of compounds promoting oral delivery of macromolecular drugs

The spectroscopic and solution properties of a series of amidated acids (delivery agents), which promote the gastrointestinal absorption of USP heparin and other drugs that show poor oral bioavailability, are investigated using Raman and NMR spectroscopy. The results show evidence for self-association at low concentrations of delivery agents that increases as the concentration of the delivery agent is increased. The self-associate is characterized by ring-ring stacking interactions, and the best geometrical arrangement for the stacking is the parallel-shifted arrangement of the rings. In addition, the amide group participates in the formation of intermolecular hydrogen bonds in the self-associate. Unlike the rigid ring, the tails of these delivery agents remain relatively flexible in the self-associate. It is suggested that the limited solubility of the delivery agents at physiological pH arises from a percentage of protonated carboxyls. Their presence promotes the formation of intermolecular hydrophobic and ring stacking interactions, which are otherwise weakened by an ionized carboxyl group.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Summary Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress with in vivo gene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissues in vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages for in vivo applications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumvented in vivo, which is possible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCM in vivo. When labeled with a radioisotope (e.g., ¹²⁵I or ¹¹¹In), the antisense chimeric peptide provides imaging of gene expression in the brain in vivo in a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow for in vivo imaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations

AIMS: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants. METHODS AND RESULTS: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients. CONCLUSIONS: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.     

New directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids

This review discusses new directions in the emerging field of carbohydrate engineering. Specifically, it describes substrate-based methodologies that are complementary to the recombinant DNA techniques that now dominate metabolic and cellular engineering endeavors. A substrate-based approach consists of intercepting a biosynthetic pathway with an unnatural analog of a metabolic intermediate. The unnatural compound competes with the endogenous substrate for biosynthetic incorporation into a cellular component by action of the natural enzymes of the cell. The incorporation of the unnatural compound into the cellular architecture can directly modulate cellular properties and biological processes. Alternatively, a molecular handle can be included in the design of the unnatural substrate that allows further elaboration upon reaction with an externally delivered reagent. The sialic acid biosynthetic pathway is presented as a model system to illustrate both the practical aspects and theoretical considerations of a substrate-based cellular engineering approach. Specific applications of carbohydrate-based cell surface engineering include chemical construction of new glycosylation patterns on cells, new approaches to targeting tumor cell with either diagnostic or therapeutic agents, and installation of novel receptors on cells for facilitating viral-mediated gene delivery.     

The human LT system. VII. Release of soluble forms and delivery to target L-929 cells by lectin-preactivated human lymphocytes in the absence of protein synthesis and secretory processes.

We have investigated the effects of inhibitors of cellular protein synthesis (emetine, cycloheximide) and secretion (colchicine, cytochalasin B) on the capacity of primary or secondary lectin-activated human lymphocytes to release LT molecules or to cause lectin-induced destruction (LICC) of murine L-929 cells in vitro. Our findings reveal: (a) agents which inhibit protein synthesis or secretion block the release of LT activity into the supernatant and LICC when primary lectin-stimulated human adenoid lymphocytes are employed as effector cells; (b) these same agents are ineffective at blocking LT release or LICC when 3- or 5-day lectin-prestimulated lymphocytes are employed; and (c) anti-human α-LT serum blocks LICC of L-929 cells mediated by primary or secondary lectin-activated human lymphocytes. The difference in participation of effector cellular processes in LICC between primary and secondary lectin-stimulated cells correlates with the findings that preactivated lymphoid cells possess high levels of preformed intracellular, as well as membrane associated, LT molecules, and that release of these materials into the supernatant or delivery to the target cell can occur independently of active protein biosynthesis or classical secretory systems.     

Mechanistically comparing reproductive manipulations caused by selfish chromosomes and bacterial symbionts

Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.Subject terms: Development, Genomic instability     

Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems: an exploration of universality

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan ontogenesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifying principles. Requirements for multicellularity are (a) the construction of three‐dimensional structures involving a crucial balance between adhesiveness and motility; and (b) the establishment of integration at molecular, cellular, tissue, and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (if) cell movement, (Hi) cell‐cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration with patterning, growth, and other developmental processes are all crucial to metazoan development, and must have been present for the emergence and radiation of Metazoa. The principal unifying themes of this review are the dynamics and regulation of cell contact phenomena. Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains fragmentary. Here we examine the molecular bases of cell contact phenomena using extant model developmental systems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protist Dictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contact phenomena in a broad developmental context. The molecular language of cell contact phenomena is complex; it involves a plethora of structurally and functionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/or carbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter‐molecular interactions, the requirement for a multitude of different signals, and the apparent requirement for an increasingly large repertoire of cell contact molecules in more complex developmental systems, such as the developing vertebrate nervous system. However, comparison of molecular models for dynamic adhesion in sponges and in vertebrates indicates that, in spite of significant differences in the details of the way specific cell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members of disparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions; (b) low‐affinity intermolecular interactions with fast on–off rates; (c) the compounding of multiple intermolecular interactions; (d) associated regulatory signalling systems. The apparent widespread employment of molecular mechanisms involving cadherin‐like cell adhesion molecules suggests the fundamental importance of cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregation of cells.