Symposium series (Society for Applied Microbiology)最新文献

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.

The Centers for Disease Control and Prevention have estimated that there are 3,713,000 cases of infectious disease associated with day care facilities each year. The objective of this study was to examine the risk reduction achieved from using different soap formulations after diaper changing using a microbial quantitative risk assessment approach. To achieve this, a probability of infection model and an exposure assessment based on micro-organism transfer were used to evaluate the efficacy of different soap formulations in reducing the probability of disease following hand contact with an enteric pathogen. Based on this model, it was determined that the probability of infection ranged from 24/100 to 91/100 for those changing diapers of babies with symptomatic shigellosis who used a control product (soap without an antibacterial ingredient), 22/100 to 91/100 for those who used an antibacterial soap (chlorohexadine 4%), and 15/100 to 90/100 for those who used a triclosan (1.5%) antibacterial soap. Those with asymptomatic shigellosis who used a non-antibacterial control soap had a risk between 49/100,000 and 53/100, those who used the 4% chlorohexadine-containing soap had a risk between 43/100,000 and 51/100, and for those who used a 1.5% triclosan soap had a risk between 21/100,000 and 43/100. The adequate washing of hands after diapering reduces risk and can be further reduced by a factor of 20% by the use of an antibacterial soap. Quantitative risk assessment is a valuable tool in the evaluation of household sanitizing agents and low risk outcomes.

Aims: The aims of the project were threefold: to survey the use of disinfectants in the UK food industry; to assess the product and environmental microflora of selected food factories for the persistence of Listeria monocytogenes and Escherichia coli; and to determine the disinfectant resistance of any persistent strains.

Methods and results: A survey of the use of disinfectants in the UK food industry was undertaken in which a total of 40 sites were visited and a further 77 postal questionnaires were returned from farms, food manufacture, food transport and food retail sites. Quaternary ammonium compounds (QACs) were predominantly used, applied in small volumes as a mist. Approximately 30,000 samples from the product and environment of five chilled food factories were examined for L. monocytogenes and E. coli over a 3 year period. A total of 181 L. monocytogenes and 176 E. coli isolates were ribotyped to yield 19 and 34 ribogroups, respectively. Some strains were isolated only from the product, a number only from the environment and others from both niches. Some strains were seen to be persistent for the duration of the sampling exercise (2-3 years). The most common L. monocytogenes and E. coli strains, together with two environmental L. monocytogenes strains, were assessed for any resistance to commercial disinfectants as compared with a laboratory L. monocytogenes disinfectant testing strain. The resistance of the L. monocytogenes and E. coli strains isolated from the factory were not significantly different from the laboratory control strain.

Conclusions: Persistent strains of L. monocytogenes and E. coli are found in the UK food industry, though this persistence is not related to their increased susceptibility to the most commonly used disinfectants.

Significance and impact of the study: The concept of a persistent microflora in food factories will have an impact on the future selection of suitable control options, including the use of biocides.

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

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.

The mechanisms of action for chemical germicides and antibiotics for inactivating microorganisms are significantly different and methods for determining resistance by microorganisms to these agents are also different. Chemical germicides usually have multiple targets and the mechanisms for inactivation and resistance are not measured in absolute terms but rather in the rapidity with which they reduce levels of microorganisms. The term tolerance is much more suited for germicides than the term resistance. The mechanism of resistance to chemical germicides is often dependent on the concentration of the germicide. At high concentrations multiple cellular and metabolic targets are involved, and at low concentrations fewer cellular targets. In contrast antibiotics usually have a singular cellular or metabolic target and resistance implies the ability of the microorganism to grow in the presence of the antibiotic, and in a clinical sense, to initiate or continue infection in the presence of the antibiotic. When methods used to assess resistance to antibiotics are applied to chemical germicides, inappropriate interpretations can be made regarding the ability of microorganisms to develop resistance to antibiotics as a result of developing resistance to chemical germicides. The use of chemical germicides in health-care institutions and especially the home setting has increased in recent years. Although there may be an overuse of germicides in these settings the consequence is a cost issue and not one that involves the development of antibiotic resistant microorganisms.

The introduction of antibiotics for the chemotherapy of bacterial infections has been one of the most important medical achievements of the past 50 years. However, the emergence of bacterial resistance to antibiotics undermines the therapeutic utility of existing agents, creating a requirement for the discovery of new antibacterial drugs. Several drug discovery strategies have emerged, including incremental improvements to existing antibiotics by chemical manipulation and the search for novel drug targets based on genomic approaches. An alternative strategy seeks to exploit opportunities for drug discovery arising from an understanding of the mode of action of existing antibiotics. Thus biochemical pathways or processes inhibited by antibiotics already in clinical use may nevertheless contain key functions that represent unexploited targets for further drug discovery. A major benefit of employing pathways or processes that are already known to contain drug targets is that proof of principle for drug intervention is already established. This approach to drug discovery is illustrated by reviewing target sites for existing antibiotics and considering how this information might be applied for the discovery of new agents inhibiting peptidoglycan synthesis, tRNA synthesis, transcription and DNA replication.

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.

Antibiotic resistance is an increasing threat in hospitals and both morbidity and mortality from infections are greater when caused by drug-resistant organisms. Whilst hospitals are universally blamed for this increase, there is an insufficient appreciation of external sources of resistance, such as when patients are admitted to hospitals from long-term care facilities in the community. The use of antibiotics in family practice and animal husbandry has also been linked to drug resistance being encountered in the hospital setting. Justifiable hospital antibiotic use, which can be life saving, may lead to 'collateral damage' with the emergence of resistance in non-target bacteria in the bowel, for example, with subsequent spread by cross-infection. At a management level, antibiotic resistance can have a significant impact on the ability of hospitals to maintain services since cohorting of patients and ward closures from outbreaks add to continuing bed shortages and waiting lists. Hospital laboratories must review their standard operating procedures since some resistance mechanisms may be missed by current methods of antibiotic susceptibility testing. With increasing public concern from press reports of 'multiresistant Staphylococcus aureus killer virus' and other drug-resistant organisms, there will inevitably be a push by national authorities for more surveillance data on antibiotic resistance; however, the cost-effectiveness of different surveillance strategies should be considered. Clinical governance and risk management are dominant themes in the National Health Service and hospital hygiene and antibiotic resistance are likely to feature prominently in audits related to these themes in the near future.