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Characterization of Aeromonas species isolated from an estuarine environment

Thirty water samples were collected, at two week intervals, from the estuary of the River Cocó. The aim was to characterize the presence, distribution and types of Aeromonas spp, in the estuary of the River Cocó, Ceara, Brazil (03°46’28.83’’S e 38°26’36.52’’S). Aeromonas were identified in 19 (63%) samples analyzed by plating and CFU counts. Presence/absence tests were positive for 11 (37%) of the samples resulting in the detection of Aeromonas in a total of 23 (77%) of samples. CFU counts varied from < 10 to 1.4 x 104 CFU mL-1. From the isolated strains seven species of Aeromonas were identified: A. caviae (29/69), A. veronii bv. sobria (13/69), A. veronii bv. veronii (8/69), A. trota (6/69), A. media (5/69), A. sobria (4/69) and A. hydrophila and Aeromonas sp. (2/69). Of the 38 strains tested, 23 (60%) showed resistance to at least one of the eight antimicrobials. Multiple resistance to antibiotics was observed in A. caviae, A. media , A. sóbria and A. veronii bv. sobria. Aeromonas caviae showed the highest multiple resistance, being resistant to four antibiotics. The presence of those microorganisms may contribute to the occurrence of gastroenteritis, mainly in children, since they are considered opportunists.

In Brazil, several studies appoint the occurrence of these pathogens isolated from aquatic ecosystem, food origin (fish, oysters, mussels and crabs) and coetaneous lesions in humans caused by fisheries (8,31). These studies are important to Public Health due to the association of some of these microorganisms on foodborne disease and extraintestinal infections (14,30). Previous studies have demonstrated that the genus Aeromonas is the second cause of gastroenteritis in children and the fifth in adult patients (15).

Sample collection
Thirty water samples were collected on a weekly basis from april to october from one point in the Cocó river estuary Samples were transported in refrigerated isothermal boxes to the microbiology laboratory at the Instituto de Ciências do Mar/LABOMAR/UFC and analyzed immediately. Physicochemical parameters such as pH, salinity and water temperature were determined using standard methods (2).

Bacterial Isolation
The strains were isolated by the direct plating (DP) and the presence/absence methods (P/A) on Pseudomonas Aeromonas selective agar (GSP, Merck), with 20 g mL -1 of ampicillin (GSPA) added.
For Aeromonas enumeration the DP method was used by Aeromonas isolates were counted using a Phoenix mod.
EC 550A counter from plates with between 25-250 colonies.
Aeromonas spp were initially identified based on being amylase positive, yellow in colour and showing a clear halo around each colony.

Bacterial identification
From each plate typical colonies (2 to 5) were transferred to trypticase soy agar (Difco) for biochemical identification tests according to Palumbo et al. (28). The positive control was Aeromonas ATCC 7966.

RESULTS AND DISCUSSION
Of the 30 water samples, 23 (77%) contained culturable While there is no specific legislation which stipulates limits concerning the numbers of these microorganisms, we must not forget that their presence in this area of the estuary is of social importance. These waters are used for leisure activities and they are also used for oyster fishing, which is normally eaten raw. Generally, Aeromonas spp are the most commonly found contaminants in fish and marine products, which to an extent is explained by their ubiquitous nature in aquatic environments Hãnninen et al. (10). In rivers, they form part of the normal microbiota being able to multiply under normal environmental conditions.
The microbiological properties of waterways are altered depending on source, type and quantity of pollutant entering them. That noted, the presence of Aeromonas spp can not always be directly related to fecal pollution Dumonter et al. (6). In this study, it is important to highlight that upstream of the water sample site the river flows through several shantytowns without any basic sanitation system, thus increasing environmental contamination.

Species P/A (%) CFU mL -1 (%) Total identified strains (%)
A  Nalidixic acid is an antimicrobial which can be applied with great success in the treatment of "traveller's diarrhoea" caused by these microorganisms (7). In this study, only 13% of Characterization of Aeromonas species the strains showed resistance to this antimicrobial. The resistance to quinolones and nalidixic acid is considered to be chromosomally mediated, as a result of drug resistant isolates selective pressure (11), or even multiplication of resistant clones (18). The relation between the increasing number of aquatic bacteria resistant to antibiotics and their hability to uptake and transfer resistance genes, is a well known fact (33).