Symposium series (Society for Applied Microbiology)最新文献

Recent events have raised awareness of the need for effective hygiene in the home. Not least is the requirement to reduce antibiotic resistance by reducing the need for antibiotic prescribing. Current evidence suggests that improved hygiene in the domestic setting could have a significant impact. Recently, it has been suggested that widespread biocide usage, particularly in consumer products, may be a contributory factor in antibiotic resistance. In developing home hygiene policies, however, it is important that biocide use as an integral part of good hygiene practice is not discouraged in situations where there is real benefit. Although laboratory data indicate possible links, it is necessary to assess whether and to what extent biocide exposure could contribute to antibiotic resistance in clinical practice. The extent to which reduced susceptibility to biocides resulting from biocide exposure could compromise their 'in-use' effectiveness must also be considered. Equally, it is important that changes in susceptibility induced by biocide exposure are assessed relative to those induced by antibiotic exposure or the phenotypic changes induced by 'normal' environmental 'stresses'. It is proposed that to be effective, home hygiene policy should be based on the concept of risk assessment and risk prevention. Using this approach, critical risk situations are identified and appropriate hygiene procedures applied to reduce risks. This may involve either soap and water cleaning, or cleaning combined with a disinfection process. A 'targeted' hygiene approach not only provides the most effective means of preventing infectious disease, it also offers a means of addressing concerns about 'too much hygiene' and 'too many antibacterials' amongst a public who have lost confidence regarding appropriate hygiene for their home environment.

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.

Bacteria resistant to both the agents deployed to prevent infections and those used to treat infections would be formidable nosocomial pathogens. The aim of this paper is to review the evidence that gram-negative bacteria resistant to antibiotics and biocides have emerged and been responsible for catheter-associated urinary tract infection. A study of patients undergoing intermittent bladder catheterization revealed that the frequent application of the antiseptic chlorhexidine to the perineal skin prior to the insertion of the catheter was effective against the normal gram-positive skin flora but not against the gram-negative organisms that subsequently colonized this site. Organisms such as Providencia stuartii, Pseudomonas aeruginosa and Proteus mirabilis were repeatedly isolated from the skin of these patients and inevitably went on to cause urinary infections. The minimum inhibitory concentration (MIC) of chlorhexidine for many of these strains proved to be 200-800 microg ml(-1) compared with the 10-50 microg ml(-1) recorded for reference strains of gram-negative species. A subsequent survey of over 800 gram-negative isolates from urinary tract infections in patients from both hospitals and the community revealed that chlorhexidine resistance was not a widespread phenomenon, but was restricted to these species and to units where the care of catheterized patients involved the extensive use of chlorhexidine. Analysis of the antibiotic resistance patterns revealed that the chlorhexidine-resistant strains were also multidrug resistant. Other clinical studies also reported catheter-associated infections with chlorhexidine- and multidrug-resistant strains of Pr. mirabilis when chlorhexidine was being used extensively. This species poses particular problems to the catheterized patient. Chlorhexidine thus proved counterproductive in the care of catheters and its use in this context has been largely abandoned. Suggestions of reintroducing this agent in the form of biocide-impregnated catheters should be resisted.

Recent advances in DNA sequencing technology have made it possible to elucidate the entire genomes of pathogenic bacteria, and advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are dramatically increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene knockout technologies amenable to pathogenic organisms have enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. Specific target examples in the areas of cell wall biosynthesis, aromatic amino acid biosynthesis, cell division, two component signal transduction, fatty acid biosynthesis, isopreniod biosynthesis and tRNA synthetases illustrate how aspects of the above capabilities are impacting on the discovery and characterization of novel antibacterial targets. An example of a novel inhibitor of bacterial fatty acid biosynthesis discovered from high throughput screening processes is described, along with its subsequent chemical optimization. Furthermore, the application and importance of technologies for tracking the mode of antibacterial action of these novel inhibitors is discussed.

Evolution of antibiotic resistance (AR) is increasingly perceived as a major clinical problem. The use of bactericidal antibiotics may protect against this, to some extent, by eradication of the pathogen, but the borders between cidal and inhibitory activity in the patient are often blurred. In addition, there are clinical reasons why eradication of the pathogen may not always be desirable. Antibiotic dosing schedules are currently driven by the perception that T > MIC and AUIC are the main predictors of outcome for time-dependent and concentration-dependent antibiotics, respectively. In the context of protecting against development of resistance in the pathogen however, peak antibiotic concentration and the concept of mutant prevention concentrations may be more important. The role of post-antibiotic and sub-MIC effects is more conjectural. Considerations of mechanisms of resistance and their relationship to antibiotic dosing schedules will also be highlighted. Lastly, the relevance of all this to the development of resistance in the normal bacterial flora will be discussed.

The extent to which antibiotics given to animals contribute to the overall problem of antibiotic resistance in man is still uncertain. The development of resistance in some human pathogens, such as methicillin-resistant Staphylococcus aureus and multi-drug resistant Mycobacterium tuberculosis, is linked to the use of antimicrobials in man and there is no evidence for animal involvement. However, there are several good examples of transfer of resistant bacteria or bacterial resistance genes from animals to man via the food chain. A bacterial ecosystem exists with simple and complex routes of transfer of resistance genes between the bacterial populations; in addition to transfer of organisms from animals to man, there is also evidence of resistance genes spilling back from humans into the animal population. This is important because of the amplification that can occur in animal populations. The most important factor in the selection of resistant bacteria is generally agreed to be usage of antimicrobial agents and in general, there is a close association between the quantities of antimicrobials used and the rate of development of resistance. The use of antimicrobials is not restricted to animal husbandry but also occurs in horticulture (for example, aminoglycosides in apple growing) and in some other industrial processes such as oil production.

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.

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.

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.

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.