Aeromonas hydrophila and Aeromonas veronii predominate among potentially pathogenic ciprofloxacin- and tetracycline-resistant aeromonas isolates from Lake Erie

Abstract

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.

FIG 1

Prevalence of Aeromonas in Presque Isle Bay, Lake Erie. Abundances (CFU/100 ml water) of cultivatable bacteria (light bars) and presumptive Aeromonas organisms (dark bars) were determined by filter plating water samples onto tryptic soy agar and ADA-VI, respectively. The value above each pair of bars is the relative abundance (%) of presumptive Aeromonas. The mean relative abundance of Aeromonas at the site was 16.0% (SD = 10.2%) for the study period.

FIG 2

Prevalences of Aeromonas species among tetracycline- and ciprofloxacin-resistant populations. Isolates (n = 119 total) were randomly picked from filter plates containing ADA-VI (white bars; n = 51), ADA-VI supplemented with 4 μg/ml tetracycline (gray bars; n = 50), and ADA-VI supplemented with 0.16 μg/ml ciprofloxacin (black bars; n = 18). Species identity was determined using RFLP analysis of the 16S rRNA gene and was confirmed by gyrB sequencing analysis. A. allosacch., A. allosaccharophila.

FIG 3

Cytotoxic effects of Aeromonas cell-free supernatants on human epithelial cells. HeLa cells were incubated with cell-free bacterial supernatants for 4 h and fixed to identify cytotoxic phenotypes. (A) Control HeLa cells. The inset shows an intact membrane. (B) HeLa cells following 4 h of incubation with Aeromonas supernatant, showing evident blebbing of the plasma membrane. The inset shows that the bleb is contiguous with the cell membrane. (C) HeLa cells following 4 h of incubation with another Aeromonas isolate supernatant, showing an irregular shape. The inset demonstrates pores in the plasma membrane (marked by arrowheads). Bars, 7.5 μm (main panels) and 1.5 μm (insets).

FIG 4

Aerolysin and serine protease association with cytotoxic phenotypes. Multiplex PCR was performed on all Aeromonas isolates, and the isolates were grouped into virulence gene profiles based on the presence or absence of aerolysin and serine protease. Cytotoxic activity is represented by dark bars, and noncytotoxicity is represented by white bars. The percent cytotoxicity against HeLa cells for each genotypic pattern was determined by dividing the number of cytotoxic isolates by the total number of isolates. The total numbers of isolates exhibiting the described genotypes are given above the bars. A, aerolysin; S, serine protease.