Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial 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.     

Antibiotic and biocide resistance in bacteria: introduction

Drug resistance in bacteria is increasing and the pace at which new antibiotics are being produced is slowing. It is now almost commonplace to hear about methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistance in Mycobacterium tuberculosis (MDRTB) strains and multi-drug-resistant (MDR) gram-negative bacteria. So-called new and emerging pathogens add to the gravity of the situation. Reduced susceptibility to biocides is also apparently increasing, but is more likely to be low level in nature and to concentrations well below those used in hospital, domestic an industrial practice. A particular problem, however, is found with bacteria and other micro-organisms present in biofilms, where a variety of factors can contribute to greater insusceptibility compared with cells in planktonic culture. Also of potential concern is the possibility that widespread usage of biocides is responsible for the selection and maintenance of antibiotic-resistant bacteria. The basic mechanisms of action of, and bacterial resistance to, antibiotics are generally well documented, although data continue to accumulate about the nature and importance of efflux systems. In contrast, the modes of action of most biocides are poorly understood and consequently, detailed evaluation of bacterial resistance mechanisms is often disappointing. During this Symposium, the mechanisms of bacterial resistance to antibiotics and biocides are discussed at length. It is hoped that this knowledge will be used to develop newer, more effective drugs and biocides that can be better and perhaps, on occasion, more logically used to combat the increasing problem of bacterial resistance.     

Resistance mechanisms of bacteria to antimicrobial compounds

A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood.Three mechanisms of resistance that have been reported include:

• limited diffusion of antimicrobial agents through the biofilm matrix,

• interaction of the antimicrobial agents with the biofilm matrix (cells and polymer),

• enzyme mediated resistance,

• level of metabolic activity within the biofilm

• genetic adaptation

• efflux pumps and

• outer membrane structure.

     

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.     

Mechanisms of action of biocides

Detailed studies have clearly demonstrated that few biocides can be considered now as general cell poisons. Biocidal action may result through physicochemical interaction with microbial target structures, specific reactions with biological molecules, or disturbance of selected metabolic or energetic processes. Mechanism of action studies, if intelligently applied, can provide direction to the development of novel biocides and biocidal systems.     

The biochemical basis of anthelmintic action and resistance

The most commonly used modern anthelmintics include the benzimidazoles, the nicotinic agonists. praziquantel, triclabendazole and the macrocyclic lactones. These drugs interfere with target sites that are either unique to the parasite or differ in their structural features from those of the homologous counterpart present in the vertebrate host. The benzimidazoles exert their effect by binding selectively and with high affinity to the beta-subunit of helminth microtubule protein. The target site of the nicotinic agonists (e.g. levamisole, tetrahydropyrimidines) is a pharmacologically distinct nicotinic acetylcholine receptor channel in nematodes. The macrocyclic lactones (e.g. ivermectin, moxidectin) act as agonists of a family of invertebrate-specific inhibitory chloride channels that are activated by glutamic acid. The primary mode of action of other important anthelmintics (e.g. praziquantel, triclabendazole) is unknown. Anthelmintic resistance is wide-spread and a serious threat to effective control of helminth infections, especially in the veterinary area. The biochemical and genetic mechanisms underlying anthelmintic resistance are not well understood, but appear to be complex and vary among different helminth species and even isolates. The major mechanisms helminths use to acquire drug resistance appear to be through receptor loss or decrease of the target site affinity for the drug. Knowledge on the mechanisms of drug action and resistance may be exploitable for the development of new drugs and may provide information on ways to overcome parasite resistance, respectively.     

Introduction of biocides into clinical practice and the impact on antibiotic-resistant bacteria

Biocides and other antimicrobial agents have been employed for centuries. Much later, iodine found use as a wound disinfectant, chlorine water in obstetrics, alcohol as a hand disinfectant and phenol as a wound dressing and in antiseptic surgery. In the early part of the twentieth century, other chlorine-releasing agents (CRAs), and acridine and other dyes were introduced, as were some quaternary ammonium compounds (QACs, although these were only used as biocides from the 1930s). Later still, various phenolics and alcohols, formaldehyde and hydrogen peroxide were introduced and subsequently (although some had actually been produced at an earlier date) biguanides, iodophors, bisphenols, aldehydes, diamidines, isocyanurates, isothiazolones and peracetic acid. Antibiotics were introduced clinically in the 1940s, although sulphonamides had been synthesized and used previously. After penicillin came streptomycin and other aminoglycosides-aminocyclitols, tetracyclines, chloramphenicol, macrolides, semi-synthetic beta-lactams, glycopeptides, lincosamides, 4-quinolones and diaminopyrimidines. Bacterial resistance to antibiotics is causing great concern. Mechanisms of such resistance include cell impermeability, target site mutation, drug inactivation and drug efflux. Bacterial resistance to biocides was described in the 1950s and 1960s and is also apparently increasing. Of the biocides listed above, cationic agents (QACs, chlorhexidine, diamidines, acridines) and triclosan have been implicated as possible causes for the selection and persistence of bacterial strains with low-level antibiotic resistance. It has been claimed that the chronological emergence of qacA and qacB determinants in clinical isolates of Staphylococcus aureus mirrors the introduction and usage of cationic biocides.     

Mechanisms of bacterial resistance to biocides

Bacterial resistance to biocides is basically of two types: (i) intrinsic, a natural chromosomally-controlled property of an organism, (ii) acquired, resulting from genetic changes in a cell and arising either by mutation or by the acquisition of genetic material. Both types of resistance are discussed together with the underlying biochemical mechanisms where known. Specific examples of organisms are provided by reference to bacterial spores, mycobacteria, other Gram-positive bacteria and Gram-negative bacteria. The stability of resistance to biocides is considered, as is the possible linkage between biocide and antibiotic resistance.     

The effect of fifteen biocides on formaldehyde-resistant strains ofPseudomonas aeruginosa

Summary Evaluation of formaldehyde and fifteen biocides in formaldehyde sensitive (S) and resistant (R) strains ofPseudomonas aeruginosa revealed a pattern of response that allowed a comparison of the mode of action of these biocides. The response of these strains to the various biocides, as well as the induction of transient resistance or cross-resistance in the (S) strain, allowed a grouping of biocides based on this pattern of response. Group 1 biocides acted in a manner indistinguishable from formaldehyde for both the (S) and (R) strains. Group 2 biocides were not effective against either the (S) or (R) strains at concentrations calculated to release equimolar concentrations of formaldehyde. However, treatment of the (S) strain with formaldehyde or Group 2 biocides resulted in the development of cross-resistance. Group 3 biocides were equally effective against the (S) and (R) strain, but the (S) strain survivors of treatment with Group 3 biocides were resistant to formaldehyde. Group 4 biocides (controls) had no presumed connection to formaldehyde mode of action. These four groupings, based on pattern of response, also resulted in groupings of biocides based on chemical structure.     

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates.     

Possible implications of biocide accumulation in the environment on the prevalence of bacterial antibiotic resistance

The lethality of biocides depends upon their interaction with a number of distinct biochemical targets. This often reflects reactive chemistry for any given agent, such as thiol oxidation. Susceptibility may vary markedly between different target organisms, and changes within the more sensitive targets can alter the inhibitory effect. The multiplicity of potential targets, however, usually dictates against the development of overt resistance to concentrations used for hygienic applications. Similarly, although changes in cellular permeability toward such agents, mediated either by envelope modification or the induction of efflux-pumps may reduce susceptibility, they rarely influence the outcome of treatments at use-concentration. It has recently been proposed that chronic exposure of the environment to biocides used in a variety of commercial products might expose some microbial communities to subeffective concentrations causing emergence of resistant clones. Such resistance might relate to mutational changes in the most susceptible target or to regulatory mutants that cause the constitutive expression of certain efflux pumps. Although selection of organisms with such modifications is unlikely to influence the effectiveness of the biocides, changes in their susceptibility to third-party antibiotics can be postulated. This is particularly the case where a cellular target is shared between a biocide and an antibiotic, or where induction of efflux is sufficient to confer antibiotic resistance in the clinic. Although such linkage has been demonstrated in the laboratory in pure culture, it has not been documented in environments commonly exposed to biocides. In nature, the effects of chronic stressing with biocides are complicated by competition between microbial community members that may result in clonal expansion of naturally insusceptible clones. Received 11 March 2002/ Accepted in revised form 16 August 2002     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.     

Factors involved in bactericidal activities of formaldehyde and formaldehyde and formaldehyde condensate/isothiazolone mixtures

Enhanced antimicrobial activities of formaldehyde (FA)_and 5-choloro-2-methyl-4-isothiazolin-3-one (IT) mixtures were determined in tryptic soy broth (TSB), minimal salt-based medium, saline, and soluble oil emulsion using a Pseudomonas aeruginosa strain as a test organism. The apparent higher activity of mixture was the result of simultaneous and combined action of the biocides. The involvement of sulfhydryl groups as targets in the mode of action of both biocides was investigated using S-nitroso thioglycollic acid as a covalent modifier of exterior sulfhydryl groups of the cells. The results suggest their involvement in the mode of action of IT. These studies also indicate a considerable independent action by both biocides when the mixture is used with absence of cross-resistance in resistant isolates and independent induction of resistance in Pseudomonas aeruginosa to these biocides. Replacement of FA in the mixture with 1, 3,5-tris (2-hydroxyethyl)-hexahydrotriazine at equimolar concentrations of FA in TSB showed essentially the same levels of increased activity with no apparent adverse effects on the antimicrobial activity of IT from the amine portion of the molecule. Furthermore, in-vitro studies also indicated direct protection of IT by FA and FA condensate biocides in the presence of nucleophiles.     

Comparative proteomic analysis of Salmonella tolerance to the biocide active agent triclosan

Concern has been expressed about the overuse of biocides in farm animal production and food industries. Biocide application can create selective pressures that lead to increased tolerance to one or more of these compounds and are concomitant with the emergence of cross-resistance to antibiotics. A triclosan sensitive Salmonella enterica serovar Typhimurium and the isogenic triclosan tolerant mutant were studied at the proteomic level in order to elucidate cellular mechanisms that facilitate biocide tolerance. 2-D differential fluorescent gel electrophoresis (DIGE) compared protein profiles of parent and mutant Salmonella, in the presence and absence of triclosan. Differentially expressed proteins were identified by mass spectrometry and divided into two groups: Group A describes proteins differentially expressed between susceptible and triclosan tolerant Salmonella and includes the known triclosan target FabI which contained a mutation at the triclosan target binding site. Group B identified proteins differentially expressed in response to triclosan exposure and defines a general cell defence network. Only four proteins were common to both groups highlighting the diverse range of pathways employed by Salmonella to counteract biocides. These data suggest that sub-lethal concentrations of triclosan induce discernible changes in the proteome of exposed Salmonella and provide insights into mechanisms of response and tolerance.     

Susceptibility of antibiotic-resistant cocci to biocides

Biocide resistance has hitherto been a poorly studied subject, possibly due to the belief that such resistance was rare and clinically insignificant. Various recent findings, however, have underlined the importance of biocide resistance as a clinically relevant phenomenon. Outbreaks of biocide-resistant organisms in hospitals have been described and the genetic mechanism for resistance to quaternary ammonium compounds (QACs) in Staphylococcus aureus has now been elucidated. Mycobacteria resistant to commonly used endoscope disinfectants are now commonly reported and have caused numerous adverse clinical events. Cross-resistance between triclosan and antituberculous drugs has been demonstrated in other strains of mycobacteria. This is related to a common mechanism of action. The work presented here describes studies into the biocide resistance of antibiotic-resistant cocci and attempts to create biocide-resistant strains in vitro. Strains of staphylococci (including methicillin-resistant Staph. aureus (MRSA)) and enterococci (including vancomycin-resistant enterococci (VRE)) had their susceptibility to biocides assayed using broth macro dilution methods and resistant strains were selected by serial subculture on biocide-containing media. Mutants were created with relative ease; for instance, triclosan minimal bactericidal concentrations (MBCs) increased from 0.002 to 3.12 mg l(-1). Some strains of MRSA which have intermediate resistance to glycopeptides were demonstrated to have decreased susceptibility to some biocides. Biocide resistance amongst enterococci was demonstrated although there was no clear correlation between biocide and antibiotic resistance. The exact mechanisms of resistance in these strains are still being studied but it is clear that biocide resistance is an important clinical phenomenon.     

Biocides, drug resistance and microbial evolution

Antimicrobial biocides are widely used in critical human health situations in which rigorous infection control is needed. Increasingly, biocidal agents are being marketed for home use, although there is little evidence that they significantly improve home hygiene. Biocide resistance mechanisms share many themes with antibiotic resistance mechanisms.     

Theoretical aspects of enzyme induction and inhibition leading to the reversal of resistance to biocides

The well-known phenomena of enzyme induction and inhibition have been applied in the enunciation of two mechanisms which could be used in the reversal of resistance which organisms develop towards biocides (drugs and pesticides) in many cases. For those biocides active per se which are metabolized by inducible enzymes to non-toxic metabolites, resistant organisms would be those possessing high levels of drug-metabolizing enzymes (Mechanism 1). For those biocides inactive per se but requiring metabolic activation for activity, resistant organisms would be those possessing low levels of drug metabolizing enzymes (Mechanism 2). In mechanism 1, the addition of enzyme inhibitors to the biocide would be effective in reversing resistance. In mechanism 2 the addition of an enzyme inducer to the biocide would increase the susceptibility of the resistant organisms. An ectoparasite insecticide 2-chloro-1-(2,4 dichlorophenyl) vinyl diethylphosphate (chlorfenvinphos or supona) is used as an example for mechanism 1. The malarial drugs primaquine and chloroquine are used as examples of mechanism 2.     

Aminoglycosides versus bacteria – a description of the action, resistance mechanism, and nosocomial battleground

Since 1944, we have come a long way using aminoglycosides as antibiotics. Bacteria also have got them selected with hardier resistance mechanisms. Aminoglycosides are aminocyclitols that kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane. Aminoglycoside resistance mechanisms include: (a) the deactivation of aminoglycosides by N-acetylation, adenylylation or O-phosphorylation, (b) the reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport, active efflux, and drug trapping, (c) the alteration of the 30S ribosomal subunit target by mutation, and (d) methylation of the aminoglycoside binding site. There is an alarming increase in resistance outbreaks in hospital setting. Our review explores the molecular understanding of aminoglycoside action and resistance with an aim to minimize the spread of resistance.