O gênero Aeromonas está amplamente distribuído em ambientes aquáticos, e estudos recentes incluem o gênero no grupo de patógenos emergentes, devido à sua freqüente associação com infecções locais e sistêmicas em humanos. Este trabalho foi realizado com o objetivo de pesquisar por meio da PCR e confirmar por meio de seqüenciamento, a ocorrência dos genes de virulência act, alt e ast, e resistência cphA, blaIMP, blaVIM, blaSPM-1, blaCTXM, blaTEM e blaSHV, verificando também o perfil de resistência a partir de antibiogramas, e a ocorrência de plasmídios nas cepas estudadas. A partir dos resultados observou-se que das 100 cepas selecionadas inicialmente, 87 pertenciam às espécies A. jandaei (46) e A. hydrophila (41). Dentre as quais pôde-se observar a ocorrência de act, alt e ast, respectivamente em 70,7 por cento (29), 97,6 por cento (40) e 26,8 por cento (11) das cepas de A. hydrophila, e em 4,4 por cento (2), 0 por cento (0) e 32,6 por cento (15) nas cepas de A. jandaei. Os genes blaIMP, blaVIM, blaSPM-1, blaCTX-M, e blaSHV não foram encontrados em nenhuma cepa. O gene cphA foi encontrado em 97,6 por cento (40) e 100 por cento (46) das cepas de A. hydrophila e A. jandaei, respectivamente e o gene blaTEM foi encontrado em 97,6 por cento (40) das cepas de A. hydrophila e em 85 por cento (39) das cepas de A. jandaei. Foi verificada a presença de plasmídio em 10/41 (24,4 por cento) das cepas de A. hydrophila e em 16/46 (34,9 por cento) das cepas de A. jandaei / The genus Aeromonas is widely distributed in aquatic environments, and recent studies include the genus in the emergent pathogens group, due to its frequent association with local and systemic human infections. This work was carried out aiming the investigation and sequencing virulence (act, alt and ast) and resistance (cphA, blaIMP, blaVIM, blaSPM-1, blaCTX-M, blaTEM e blaSHV) genes, also verifying the resistance profile of the strains using antibiograms and the occurrence of plasmids. From the 100 strains analyzed in this study, 87 belonged to A. jandaei (46) and A. hydrophila (41) species. Out of which it was observed the occurrence of act, alt and ast, respectively in 70.7 per cent (29), 97.6 per cent (40) and 26.8 per cent (11) of A. hydrophila strains, and in 4.4 per cent (2), 0 per cent (0) e 32.6 per cent (15) of A. jandaei strains. The genes blaIMP, blaVIM, blaSPM-1, blaCTX-M e blaSHV were not found. CphA gene was found in 97.6 per cent (40) and 100 per cent (46) of A. hydrophila and A. jandaei strains, respectively and blaTEM gene was found in 97.6 per cent (40) of A. hydrophila strains and in 85 per cent (39) of A. jandaei strains. Presence of plasmid was found in 10/41 (24.4 per cent) of A. hydrophila strains and in 16/46 (34,9 per cent) of A. jandaei strains

O gênero Aeromonas está amplamente distribuído em ambientes aquáticos, e estudos recentes incluem o gênero no grupo de patógenos emergentes, devido à sua freqüente associação com infecções locais e sistêmicas em humanos. Este trabalho foi realizado com o objetivo de pesquisar por meio da PCR e confirmar por meio de seqüenciamento, a ocorrência dos genes de virulência act, alt e ast, e resistência cphA, blaIMP, blaVIM, blaSPM-1, blaCTXM, blaTEM e blaSHV, verificando também o perfil de resistência a partir de antibiogramas, e a ocorrência de plasmídios nas cepas estudadas. A partir dos resultados observou-se que das 100 cepas selecionadas inicialmente, 87 pertenciam às espécies A. jandaei (46) e A. hydrophila (41). Dentre as quais pôde-se observar a ocorrência de act, alt e ast, respectivamente em 70,7 por cento (29), 97,6 por cento (40) e 26,8 por cento (11) das cepas de A. hydrophila, e em 4,4 por cento (2), 0 por cento (0) e 32,6 por cento (15) nas cepas de A. jandaei. Os genes blaIMP, blaVIM, blaSPM-1, blaCTX-M, e blaSHV não foram encontrados em nenhuma cepa. O gene cphA foi encontrado em 97,6 por cento (40) e 100 por cento (46) das cepas de A. hydrophila e A. jandaei, respectivamente e o gene blaTEM foi encontrado em 97,6 por cento (40) das cepas de A. hydrophila e em 85 por cento (39) das cepas de A. jandaei. Foi verificada a presença de plasmídio em 10/41 (24,4 por cento) das cepas de A. hydrophila e em 16/46 (34,9 por cento) das cepas de A. jandaei / The genus Aeromonas is widely distributed in aquatic environments, and recent studies include the genus in the emergent pathogens group, due to its frequent association with local and systemic human infections. This work was carried out aiming the investigation and sequencing virulence (act, alt and ast) and resistance (cphA, blaIMP, blaVIM, blaSPM-1, blaCTX-M, blaTEM e blaSHV) genes, also verifying the resistance profile of the strains using antibiograms and the occurrence of plasmids. From the 100 strains analyzed in this study, 87 belonged to A. jandaei (46) and A. hydrophila (41) species. Out of which it was observed the occurrence of act, alt and ast, respectively in 70.7 per cent (29), 97.6 per cent (40) and 26.8 per cent (11) of A. hydrophila strains, and in 4.4 per cent (2), 0 per cent (0) e 32.6 per cent (15) of A. jandaei strains. The genes blaIMP, blaVIM, blaSPM-1, blaCTX-M e blaSHV were not found. CphA gene was found in 97.6 per cent (40) and 100 per cent (46) of A. hydrophila and A. jandaei strains, respectively and blaTEM gene was found in 97.6 per cent (40) of A. hydrophila strains and in 85 per cent (39) of A. jandaei strains. Presence of plasmid was found in 10/41 (24.4 per cent) of A. hydrophila strains and in 16/46 (34,9 per cent) of A. jandaei strains

Τρεις ευρέως χρησιμοποιούμενες μέθοδοι τυποποίησης, μια βιοχημική (API20NE), μια φαινοτυπική (SDS-PAGE) και μια μοριακή (RAPD) χρησιμοποιήθηκαν για την ταυτοποίηση και ταξινόμηση 160 περιβαλλοντικών ψευδομονάδων που απομονώθηκαν από το υδάτινο περιβάλλον της Νοτιοδυτικής Ελλάδας και συγκεκριμένα από εμφιαλωμένα νερά (46%), νερά δικτύου ύδρευσης (16%), κολυμβητικών δεξαμενών (9%) και θαλασσών (29%). Οι ψευδομονάδες ταυτοποιήθηκαν με βάση το βιοχημικό τους αποτύπωμα δια μέσου του συστήματος ΑΡΙ20ΝΕ, και στη συνέχεια υποβλήθηκαν σε ηλεκτροφόρηση των ολικών πρωτεϊνών τους (SDS-PAGE) και σε ανάλυση του γενετικού τους υλικού με τη μέθοδο RAPD (Random Amplified Polymorphic DNAs) με χρήση δύο διαφορετικών δεκαμερών εκκινητών (primers). Το σύστημα API20NE ταυτοποίησε το 88% των στελεχών διακρίνοντας 14 ομάδες-είδη, ενώ η SDS-PAGE ταξινόμησε το 98.1% σε 20 ομάδες και η RAPD το 94% των στελεχών σε 22 και 34 ομάδες, με εκκινητή τον OPA-13 και τον OPD-13 αντίστοιχα. Η ταξινόμηση προέκυψε με εφαρμογή της ανάλυσης κατά συστάδες (cluster analysis) των αποτυπωμάτων (πρωτεϊνικών και γενετικών) που παρήγαγαν τα στελέχη. Τα 20 στελέχη που δεν ταυτοποιήθηκαν σε επίπεδο είδους με το API20NE, ταξινομήθηκαν με την SDS-PAGE σε ποσοστό 100%, ενώ με την RAPD σε ποσοστό 90%. Οι τρεις μέθοδοι συγκρίθηκαν ως προς την επαναληψιμότητα (reproducibility), την ικανότητα τυποποίησης (typeability) και τη διακριτική ικανότητα (discriminatory power). Την μεγαλύτερη επαναληψιμότητα έδωσαν το API20NE και η RAPD με τον εκκινητή OPA-13, την μεγαλύτερη ικανότητα τυποποίησης η SDS-PAGE, ενώ τη μεγαλύτερη διακριτική ικανότητα έδωσε η RAPD με τον εκκινητή OPD-13. Η πλέον σωστή ταξινόμηση, όπως προέκυψε από τη διακριτή ανάλυση, επιτεύχθη με τη μέθοδο SDS-PAGE. Η παρούσα εργασία αποδεικνύει ότι τα βιοχημικά συστήματα ταυτοποίησης (όπως το API20NE) μπορούν να χρησιμοποιηθούν με αξιοπιστία μόνο για αδρή αναγνώριση των περιβαλλοντικών ψευδομονάδων. Πληροφορίες σε βάθος για την ταυτότητα και τη φύση τους μπορούν να εξαχθούν με τη περαιτέρω εφαρμογή ηλεκτροφορητικών και μοριακών μεθόδων. Δεδομένης της ευρείας διασποράς, της ετερογένειας και της, έστω και δυνητικής, παθογόνου δράσης των ψευδομονάδων, είναι σημαντικό, από πλευράς δημόσιας υγείας, ο προσδιορισμός της ταυτότητάς τους να γίνεται με συνδυασμένη εφαρμογή βιοχημικών, ηλεκτροφορητικών και μοριακών μεθόδων ώστε να καθίσταται δυνατή η αναγνώριση στελεχών που μπορούν να αποτελέσουν αιτιολογικούς παράγοντες ασθενειών, ιδιαίτερα σε ομάδες υψηλού κινδύνου. / Three broadly used typing techniques, one biochemical (API20NE), one phenotypic (SDS-PAGE) and one molecular (RAPD), were employed for the identification and taxonomy of 160 environmental pseudomonas isolated from the aquatic environment in Southwestern Greece. In particular, the isolates were obtained from bottled waters (46%), potable waters (16%), waters from swimming pools (9%) and seawaters (29%). The isolates were identified by the system API20NE and then subjected to whole-cell protein electrophoresis (SDS-PAGE) and Random Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. The API20NE system identified 88% of the whole bacterial population and classified them in 14 species, while SDS-PAGE classified 98.1% of the isolates in 20 groups and RAPD classified 94% of the strains in 22 groups using the primer OPA-13 and 34 groups using the primer OPD-13. The classification was achieved by applying cluster analysis in the protein or RAPD fingerprints of the isolates. Twenty isolates that could not be identified by the API20NE system, at least to the species level, were classified by the SDS-PAGE and the RAPD in a percentage of 100% and 90%, respectively. The reproducibility, typeability and discriminatory power of the three methods were compared to evaluate their application. The API20NE and the RAPD assay with primer OPA-13 showed better reproducibility in comparison with the other methods; the higher typeability was achieved by the SDS-PAGE assay while the higher discriminatory power was that obtained by the RAPD method with the primer OPD-13. The SDS-PAGE gave the higher percentage of “correctly classified” isolates, as it was assessed by discriminant analysis. This study shows that the rapid identification systems, such as the API20NE, may be reliable only for a rough characterization of environmental Pseudomonas. In order to acquire further information about their identities, other phenotypic and molecular techniques have to be applied. Given the ubiquity, heterogeneity and pathogenicity, either established or potential, of the environmental pseudomonas it is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using the combination of specific methods, so as to be possible for strains, which can serve as causative agents of diseases, especially in high risk population, to be recognizable.