Application of green fluorescent protein to measure antimicrobial efficacy and the kinetics of cell death against Escherichia coli.

Industrial antimicrobials have been extensively used to control unwanted microbial growth by incorporation into a variety of products such as plastics and paints, reducing biodeterioration and biofouling and extending the lifespan of the product. Industrial antimicrobials generally have broad sites of action affecting core cellular functions such as central metabolism, enzyme function, cell wall or DNA synthesis and can either be biocidal or biostatic. In addition, susceptibility can be affected by the metabolic state of the microbe, with metabolically inactive cells generally more resistant than metabolically active cells. Previously it was demonstrated that cytosolically expressed green fluorescent protein could be used as a real-time viability indicator in the yeast Aureobasidium pullulans based on the pH dependent fluorescence of GFP and the collapse of the proton gradient across the cell membrane during cell death. In this study we report on the development and validation of an equivalent GFP fluorescence viability assay in Escherichia coli and used this assay to study the effect of five antimicrobials commonly used in plastics; 4,5-dichloro-2-octyl-isothiazol-3-one (DCOIT), sodium pyrithione, 1,2-benzisothiazol-3-one (BIT), 2-octyl-isothiazol-3-one (OIT) and n-butyl-1,2-benzisothiazol-3-one (BBIT). The results demonstrate broad differences amongst the antimicrobials in both relative efficacy, rate of effect and for some antimicrobials, marked differences in sensitivity toward growing and non-growing cells.

Prevalence, persistence, and phenotypic variation of Aspergillus fumigatus in the outdoor environment in Manchester, UK, over a 2-year period.

Aspergillus fumigatus, an opportunistic fungal pathogen that causes invasive aspergillosis in immunosuppressed patients, is considered to be the world's most dangerous mould. It is widely distributed in the environment, and airborne asexual conidia serve as the main mode of transport for pulmonary lung infection. It is important to monitor seasonal airborne conidia levels when assessing the risk of acquiring this infection. In this study, air was sampled for total viable fungal spores and viable A. fumigatus conidia monthly over a 2-year period (2009 and 2010) close to Manchester, UK, city center. Total viable airborne fungal counts varied seasonally, peaking in the summer and autumn for both years and reaching levels of approximately 1100-1400 colony-forming units (CFU)/m(3); counts were strongly positively correlated to mean temperature (R(2) = 0.697). By contrast, A. fumigatus viable airborne counts were not seasonally associated; persistent low levels were between 3 and 20 CFU/m(3) and were not correlated with mean temperature (R(2) = 0.018). A total of 220 isolates of A. fumigatus were recovered on potato dextrose agar (PDA) at 45°C, and internal transcribed spacer sequencing and restriction digestion of a partial polymerase chain reaction amplicon of the ß-tubulin gene (benA) of 34 randomly selected isolates were used to confirm the isolates as A. fumigatus. When the colony radial growth rates (Kr) were determined, the highest rates were observed on PDA, followed by Vogel's medium supplemented with phosphatidylcholine and Vogel's medium alone. Clinical isolates had a significantly higher mean colony Kr on PDA compared with environmental isolates.

Genetic and virulence variation in an environmental population of the opportunistic pathogen Aspergillus fumigatus.

Environmental populations of the opportunistic pathogen Aspergillus fumigatus have been shown to be genotypically diverse and to contain a range of isolates with varying pathogenic potential. In this study, we combined two RAPD primers to investigate the genetic diversity of environmental isolates from Manchester collected monthly over 1 year alongside Dublin environmental isolates and clinical isolates from patients. RAPD analysis revealed a diverse genotype, but with three major clinical isolate clusters. When the pathogenicity of clinical and Dublin isolates was compared with a random selection of Manchester isolates in a Galleria mellonella larvae model, as a group, clinical isolates were significantly more pathogenic than environmental isolates. Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were the most pathogenic, Dublin isolates were the least pathogenic and Manchester isolates showed a range in pathogenicity. Overall, this suggests that the environmental population is genetically diverse, displaying a range in pathogenicity, and that the most pathogenic strains from the environment are selected during patient infection.