Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata)

Abstract

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes—biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)—and two virulence factors—Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)—was investigated by RT-PCR. A. hydrophila displayed a >2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a >2-fold, and on polypropylene a >6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669—Potential Zoonotic Pathogens Isolated from Spotted Turtles

“…Shiga-like toxin production causing bloody diarrhea and hemolytic uremic syndrome is reported from Aeromonas spp. [ 101 ], and there is evidence that surface colonization upregulates the expression of the Shiga-like toxin genes [ 17 ].…”

“…The overexpression of genes encoding for ATP-binding cassette (ABC) transporter membrane proteins or multidrug efflux pumps, which enable the export of drugs out of the cell is a prevalent cause of MDR [ 107 , 108 , 109 ]. Analysis of the genomes using the RGI server suggest that efflux pumps may be present in both isolates [ 17 ]. Future studies should address the mechanisms of MDR in the isolated A. hydrophila and C. portucalensis strains.…”

“…Turtles have been regarded as “sentinel species” for assessing ecosystem health [ 13 , 14 ] and the illegal trade in reptiles at “wet markets” could pose a significant risk of zoonotic transmission [ 15 , 16 ]. In an earlier study, we reported the biofilm formation and Shiga-toxin expression by A. hydrophila and C. freundii strains isolated from infected spotted turtles ( Clemmys guttata ) [ 17 ]. In this study, we have reclassified our C. freundii strain whose classification was based on the 16S-rDNA gene phylogeny, as a C. portucalensis strain on the basis of whole genome comparison.…”

“…Both A. hydrophila and C. freundii have been shown to be susceptible to neomycin and cotrimoxazole in the planktonic phase [ 60 , 61 , 62 , 63 , 64 ]. We have previously predicted using whole genome sequencing that A. hydrophila RIT668 contains resistance genes for six classes of antibiotics, while C. freundii RIT669 contains resistance genes for 19 classes of antibiotics [ 17 ]. In the same report, we also showed the growth of biofilms of A. hydrophila RIT668 and C. freundii (now C. portucalensis ) RIT669, and the upregulated expression of a Shiga-like toxin during adhesion on the polymers, polyethylene (PE) and polypropylene (PP) [ 17 ].…”

“…We have previously predicted using whole genome sequencing that A. hydrophila RIT668 contains resistance genes for six classes of antibiotics, while C. freundii RIT669 contains resistance genes for 19 classes of antibiotics [ 17 ]. In the same report, we also showed the growth of biofilms of A. hydrophila RIT668 and C. freundii (now C. portucalensis ) RIT669, and the upregulated expression of a Shiga-like toxin during adhesion on the polymers, polyethylene (PE) and polypropylene (PP) [ 17 ]. However, experimental verification of the antibiotic resistance in the planktonic and biofilm forms was missing.…”