Neil Woodford

The rising prevalence of the members of the Enterobacteriaceae, Pseudomonas spp. and Acinetobacter baumannii that produce extended-spectrum b-lactamases (ESBLs) and carbapenem-hydrolysing b-lactamases (carbapenemases) represents one of the largest new resistance problems faced by clinicians and microbiologists during the last 10 years. These diverse enzymes have emerged globally and represent serious health challenges, compromising therapeutic choice and complicating patient management. The...

Virve I Enne and Peter M Bennett

The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly...

Jonathan Pratten and Derren Ready Abstract

There are many laboratory biofilm models available which can be used to assess the susceptibility of these distinctive resistant phenotypes. The complexities of these models vary considerably and indeed, the antimicrobial susceptibility of biofilms grown in these different models are also not standardised. It is clear that such methods are necessary for the testing of antibiotics and antimicrobial agents since these persistent communities are far more resistant than their planktonic...

Miguel Viveiros Liliana Rodrigues Marta Martins Isabel Couto Gabriella Spengler Ana Martins and Leonard Amaral

A semi-automated method that uses the common efflux pump (EP) substrate ethidium bromide (EB) is described for the assessment of EP systems of bacteria. The method employs the Rotor-GeneTM 3000 thermocycler (Corbett Research) for the real-time assessment of accumulation and efflux of EB in Phosphate-Buffered Solution (PBS) under varying physiological conditions, such as temperature, pH, presence and absence ofthe energy source, and presence of efflux pumps inhibitors (EPIs). The method is...

Marta Martins Isabel Couto Miguel Viveiros and Leonard Amaral

Two simple, instrument-free, user-friendly methods that can readily be implemented by a routine microbiology laboratory are described for the detection of multi-drug-resistant (MDR) isolates that overexpress efflux pump (EP) systems responsible for the MDR phenotype. The first method employs the universal EP substrate ethidium bromide (EB) at varying concentrations in agar-containing plates upon which the contents of an overnight culture are swabbed as spokes of a wheel. In this method, named...

Hee Jeon Hong Abstract

Gene swapping is a simple but effective genetic tool for characterizing the functioning of a gene, where the gene in question is known to fulfil a distinctive biological role in the cell. VanS is a sensor kinase which, in conjunction with its cognate response regulator VanR, triggers resistance to vancomycin. One of the most important questions yet to be answered in the study of vancomycin resistance is the nature of the specific ligand recognized by the VanS sensor. A VanRS-swap experiment...

Jodi A Lindsay and Julia ML Sung Abstract

Staphylococcus aureus lineages evolve independently and differ in hundreds of genes. Identification of lineages can be useful for epidemiological typing and infection control at the local or global level, and can also be useful when investigating differences in pathogenesis between strains. MLST (multilocus sequence typing) and spa typing (polymorphisms in the protein A gene) are useful methods for identifying lineages but can be time-consuming and expensive. Here, we describe a method for...

Joanna Bacon Kim A Hatch and Jon Allnutt

The ability of all pathogens to survive within the host is key to their success in establishing disease. Environmental conditions that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying the response of Mycobacterium tuberculosis (M. tuberculosis) exposed to these relevant host conditions in vitro will further increase our understanding of how these environments have an impact on the molecular mechanisms M....

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Fig. 1. (a) Cartwheel pattern for the swabbing of the strains to be tested by the EB-agar method. The pattern may be made on the bottom side of the agar plate with a water-removable marking pen. One or more control strains can be used, according to the specific design. If possible, we recommend the use of two control strains one that shows no efflux activity (to be used as the reference strain) and one that has an overex-pressed efflux system. The reference strain will begin to fluoresce at low...