ReviewA DNA probe specific for Aeromonas colonies
Introduction
The genus Aeromonas comprises oxidase-positive, facultative anaerobic, Gram-negative bacilli. Some species have been described as causative agents of various gastrointestinal and, to a lesser extent, extraintestinal diseases Janda and Abbott 1998, Altwegg 1999. The commercial biochemical systems currently employed in clinical laboratories for the identification of Gram-negative enterobacteria (API 20E, Vitek, MicroScan etc.) are unable to correctly identify Aeromonas spp. and confusion with Vibrio spp. (V. fluvialis, V. damsela or V. cholerae) is common Overman et al 1985, Overman and Overley 1986, Abbott et al 1998, Vivas J et al 2000. The confusion with V. cholerae is of special relevance due to its public health significance (Abbott et al., 1998).
Given the increasing importance of Aeromonas in clinical infections (Janda & Abbot, 1998) and the limitations of the existing methods, there is a need to design accurate systems that can unequivocally identify these microorganisms.
Molecular methods tend to be more accurate, the vast majority of them having been focused on species identification by using either the 16S rRNA gene Ash et al 1993, Dorsch et al 1994, Borrell et al 1997, Demarta et al 1999, Figueras et al 2000 or virulence associated genes such aerolysin Husslein et al 1992, Khan et al 1999 and lipases (Cascon et al., 1996). These methods are useful and suitable in well-equipped research laboratories, but clinical laboratories need cheaper and faster methods for unequivocal detection of Aeromonas, especially at the genus level.
Glycerophospholipid-cholesterol acyltransferase (GCAT) is an unusual extracellular lipase released by members of the genus Vibrio and Aeromonas (MacIntyre et al., 1979). The enzyme is considered one of the potential virulence factors that can contribute to the pathogenesis of Aeromonas in fish (Lee and Ellis, 1990). The gene has been cloned and sequenced in strains of A. salmonicida and A. hydrophila Thorton et al 1988, Nerlan 1996. In a previous study, where the gene presence was investigated by PCR, we found it in practically all Aeromonas strains tested, including representatives of all species (Chacón et al., 2001, submitted for publication).
The aim of the present study was to evaluate if the 237 bp fragment of the GCAT gene could be used to distinguish Aeromonas sp. from other common enteropathogens in colony hybridization assays.
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Bacteria strains and media
A total of 36 bacteria strains belonging to 8 different genera were tested, although from 5 of them only one strain was assayed. The strains used in this study are listed in Table 1. The strains of the different species (up to 5 colonies) were inoculated in groups of 9 onto petri dishes (9 cm diameter). The media used were: tryptic soy agar (TSA) (Difco, Barcelona, Spain), 5% human agar blood, MacConkey agar (Difco), Salmonella-Shigella agar (SS) (Difco), xylose lysine desoxycholate agar (XLD)
Results and Discussion
In a previous study we demonstrated that the GCAT gene was present in 98% of the 234 strains of Aeromonas of environmental and clinical origin representing all species (Chacon et al., 2001, submitted for publication). This demonstrates that this gene is widely distributed. Therefore, it was considered a good candidate to be tested as a genus probe. The primers designed, for the GCAT gene, amplified a fragment of the expected size (237 bp) and no other amplicons were generated. Lower annealing
Acknowledgements
This work has been supported by the grants: FIS 99/0944 from the Spanish Ministry of Health, from Fundació Ciència i Salut and by a fellowship PSR/99–01 from the Universitat Rovira i Virgili. We would like to thank the Colección Española de Cultivos Tipo for kindly providing isolates.
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