Determination of Microbial Diversity of Aeromonas Strains on the Basis of Multilocus Sequence Typing, Phenotype, and Presence of Putative Virulence Genes

Abstract

The genus Aeromonas has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir. Aeromonas spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the Aeromonas genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of Aeromonas spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (ast, ahh1, act, asa1, eno, ascV, and aexT) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus Aeromonas, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (http: //pubmlst.org/aeromonas) specific for the Aeromonas genus was developed and implemented with all the results.

Detection and diversity of aeromonads from treated wastewater and fish inhabiting effluent and downstream waters

“…For example, over 6% of retrieved aeromonads in this work were identified as A. bestiarum by MALDI TOF MS, but were attributed to A. hydrophila by API 20E. This identification disparity can be attributed to close relatedness of the two species, belonging to the same phenogroup, described as the A. hydrophila complex (Martino et al, 2011). MALDI-TOF MS can however provide their good separation at the genospecies level comparable with the phylogenetic tree obtained by gyrB gene sequencing; it categorizes in clusters well differentiated A. bestiarum and A. hydrophila (Benagli et al, 2012).…”

Multilocus sequence typing revealed a clonal lineage of Aeromonas hydrophila caused motile Aeromonas septicemia outbreaks in pond-cultured cyprinid fish in an epidemic area in central China

“…The taxonomy of the genus Aeromonas is complex and accompanied with confusion and controversy (Janda and Abbott, 2010;Martinez-Murcia et al, 2011;Martino et al, 2011). At present, 24 validly published species in the genus Aeromonas have been reported Fig.…”

“…MLST can achieve precise strain genotyping, and is a powerful tool for outbreak traceability (Martino et al, 2011). Six housekeeping genes of all strains, namely, gyrB, groL, gltA, metG, ppsA, and recA, were sequenced with the previously described primers and procedures (Martino et al, 2011). The sequences of distinct alleles were deposited in the Aeromonas MLST database (http://pubmlst.org/aeromonas) (Jolley and Maiden, 2010) and GenBank under accession nos.…”

“…Each gene fragment is translated into a distinct allele, and each isolate is classified as a sequence type (ST) by the combination of alleles of the housekeeping loci (Urwin and Maiden, 2003). MLST can achieve precise strain genotyping, and is a powerful tool for outbreak traceability (Martino et al, 2011). Six housekeeping genes of all strains, namely, gyrB, groL, gltA, metG, ppsA, and recA, were sequenced with the previously described primers and procedures (Martino et al, 2011).…”

Molecular Detection and Characterization of Furunculosis and Other Aeromonas Fish Infections

“…However, the importance of the species A. hydrophila is overestimated because biochemical identification systems erroneously identify up to 70-80% of the strains of Aeromonas as belonging to this species, when in fact they are many different species when identified by molecular methods (Beaz-Hidalgo et al, 2010;Figueras, 2005;2011b;Soler et al, 2003a). Prevalent motile species associated to diseased fish following molecular identification include A. veronii, associated with catfish, A. sobria with tilapia and trout, A. hydrophila with trout, A. bestiarum with carp and the recently described A. piscicola recovered from diseased salmonids and turbots (Beaz-Hidalgo et al, 2010;Kozińska, 2007;Li & Cai, 2011;Martino et al, 2011;Nawaz et al, 2006;Soriano-Vargas et al, 2010). Other motile species, such as A.…”