Silver nanoparticles as a new generation of antimicrobials.

The Infectious Diseases Society of America (IDSA) continues to view with concern the lean pipeline for novel therapeutics to treat drug-resistant infections, especially those caused by gram-negative pathogens. Infections now occur that are resistant to all current antibacterial options. Although the IDSA is encouraged by the prospect of success for some agents currently in preclinical development, there is an urgent, immediate need for new agents with activity against these panresistant organisms. There is no evidence that this need will be met in the foreseeable future. Furthermore, we remain concerned that the infrastructure for discovering and developing new antibacterials continues to stagnate, thereby risking the future pipeline of antibacterial drugs. The IDSA proposed solutions in its 2004 policy report, “Bad Bugs, No Drugs: As Antibiotic R&D Stagnates, a Public Health Crisis Brews,” and recently issued a “Call to Action” to provide an update on the scope of the problem and the proposed solutions. A primary objective of these periodic reports is to encourage a community and legislative response to establish greater financial parity between the antimicrobial development and the development of other drugs. Although recent actions of the Food and Drug Administration and the 110th US Congress present a glimmer of hope, significant uncertainly remains. Now, more than ever, it is essential to create a robust and sustainable antibacterial research and development infrastructure—one that can respond to current antibacterial resistance now and anticipate evolving resistance. This challenge requires that industry, academia, the National Institutes of Health, the Food and Drug Administration, the Centers for Disease Control and Prevention, the US Department of Defense, and the new Biomedical Advanced Research and Development Authority at the Department of Health and Human Services work productively together. This report provides an update on potentially effective antibacterial drugs in the late-stage development pipeline, in the hope of encouraging such collaborative action.

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Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America

Antiseptics and disinfectants are extensively used in hospitals and other health care settings for a variety of topical and hard-surface applications A wide variety of active chemical agents (biocides) are found in these products, many of which have been used for hundreds of years, including alcohols, phenols, iodine, and chlorine Most of these active agents demonstrate broad-spectrum antimicrobial activity; however, little is known about the mode of action of these agents in comparison to antibiotics This review considers what is known about the mode of action and spectrum of activity of antiseptics and disinfectants The widespread use of these products has prompted some speculation on the development of microbial resistance, in particular whether antibiotic resistance is induced by antiseptics or disinfectants Known mechanisms of microbial resistance (both intrinsic and acquired) to biocides are reviewed, with emphasis on the clinical implications of these reports

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Antiseptics and Disinfectants: Activity, Action, and Resistance

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecological perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecological success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.

Biofilms: an emergent form of bacterial life.

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme mur D

The basis of tetracycline resistance mediated by TetA determinants and joint resistance to tetracycline and minocycline coded by TetB determinants was investigated. The TetA class of determinants was represented by those carried on plasmids pSC101, RP1 and pIP7 and TetB by Tn10. The relationships between expression of tetracycline and minocycline resistance and accumulation of these antibiotics suggest that there are three mechanisms of plasmid-determined resistance conferring (1) about a 10- to 20-fold increase in resistance to tetracycline that is not associated with decreased antibiotic accumulation, (2) a 4- to 7-fold increase in resistance to tetracycline that is associated with decreased drug accumulation, and (3) about a 2- to 3-fold increase in resistance to both tetracycline and minocycline that is not associated with decreased accumulation of either antibiotic. Mechanism 1 was coded by the tetracycline resistance determinant of pSC101 (TetA), mechanisms 1 and 2 by the determinants in RP1 and pIP7 (TetA) and all three mechanisms by Tn10 (TetB).

Evidence for more than one mechanism of plasmid-determined tetracycline resistance in Escherichia coli.

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together (Shales et al., 1980). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal BglII fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.

Reduced expression of Tn 10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon.

Objectives The antioxidant tert-butylhydroquinone (TBHQ) is a food additive reported to have antibacterial activity, and may therefore have application in the healthcare setting. This study sought to characterize the antibacterial activity and mode of action of TBHQ and its oxidation product, tert-butylbenzoquinone (TBBQ). Methods The stability of TBHQ/TBBQ was studied in buffer. Susceptibility testing was performed by broth microdilution, and killing and lytic activity were evaluated by viable counting and culture turbidity measurements. Mode of action studies included following the incorporation of radiolabelled precursors into macromolecules. The effect of TBHQ/TBBQ upon bacterial and mammalian membranes was assessed using the BacLight(TM) assay and by monitoring the haemolysis of equine erythrocytes. Results TBHQ underwent oxidation in solution to form TBBQ. When oxidation was prevented, TBHQ lacked useful antibacterial activity, indicating that TBBQ is responsible for the antibacterial activity attributed to TBHQ. TBBQ demonstrated activity against Staphylococcus aureus SH1000 (MIC 8 mg/L) and against a panel of clinical S. aureus isolates (MIC90 16 mg/L). TBBQ at 4× MIC caused a >4 log10 drop in cell viability within 6 h without lysis, and eradicated staphylococcal biofilms at 8× MIC. TBBQ did not display preferential inhibition of any single macromolecular synthetic pathway, but caused loss of staphylococcal membrane integrity without haemolytic activity. Conclusions TBBQ is responsible for the antibacterial activity previously ascribed to TBHQ. TBBQ prompts loss of staphylococcal membrane integrity; it is rapidly and extensively bactericidal, but is non-lytic. In view of the potent and selective bactericidal activity of TBBQ, this compound warrants further investigation as a candidate antistaphylococcal agent.

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Antibacterial activity and mode of action of tert-butylhydroquinone (TBHQ) and its oxidation product, tert-butylbenzoquinone (TBBQ)

The biofilm mode of growth can lead to diversification of the bacterial population by promoting the emergence of variants. Here we report the identification and characterization of two major subpopulations of morphological variants arising in biofilms of S. aureus. One of these lacked pigmentation (termed white variants; WVs), whilst the other formed colonies on agar that were larger and paler than the parental strain (termed large pale variants; LPVs). WVs were unable to form biofilms, and exhibited increased proteolysis and haemolysis; all phenotypes attributable to loss-of-function mutations identified in the gene encoding the alternative sigma factor, sigB. For LPVs, no differences in biofilm forming capacity or proteolysis were observed compared with the parental strain. Genetic analysis of LPVs revealed that they had undergone mutation in the accessory gene regulator system (agrA), and deficiency in agr was confirmed by demonstrating loss of both colony spreading and haemolytic activity. The observation that S. aureus biofilms elaborate large subpopulations of sigB and agr mutants, both genotypes that have independently been shown to be of importance in staphylococcal disease, has implications for our understanding of staphylococcal infections involving a biofilm component.

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Ian Chopra

Papers

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme mur D

Evidence for more than one mechanism of plasmid-determined tetracycline resistance in Escherichia coli.

Reduced expression of Tn 10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon.

Antibacterial activity and mode of action of tert-butylhydroquinone (TBHQ) and its oxidation product, tert-butylbenzoquinone (TBBQ)

Population diversification in Staphylococcus aureus biofilms may promote dissemination and persistence.