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.

Keywords: Aeromonas; Citrobacter; Shiga-like toxin; antibiotic resistance; biofilm; plastic; turtle; whole-genome sequencing.

Figure 1

WGS-based phylogenetic tree for A. hydrophila RIT668, using the TYGS platform. TYGS infers trees with FastME 2.1.4 [103] based on Genome BLAST Distance Phylogeny (GBDP) distances calculated from the genome sequences. Branch lengths are scaled in terms of GBDP distance formula d5 [107]. Numbers above branches are GBDP pseudo-bootstrap support values from 100 replications.

Figure 2

WGS-based phylogenetic tree using TYGS for C. freundii RIT669. TYGS infers trees with FastME 2.1.4 [103] based on Genome BLAST Distance Phylogeny (GBDP) distances calculated from the genome sequences, whereas branch lengths are scaled in terms of GBDP distance formula d5 [107]. Numbers above branches are GBDP pseudo-bootstrap support values from 100 replications.

Figure 3

Variation in gene expression during planktonic and adherent growth of A. hydrophila using tryptic soy broth on polyethylene (PE) surface as shown in (A) and polypropylene (PP) surface as shown in (B), respectively. The 16S rRNA gene expression was used as an internal control.

Figure 4

Variation in gene expression during planktonic and adherent growth of C. freundii in tryptic soy broth on polyethylene (PE) surface as shown in (A) and polypropylene (PP) surface as shown in (B), respectively. The 16S rRNA gene expression was used as an internal control.

Figure 5

Scanning electron micrographs (A) of un-colonized PE (×4980); (B–D) of A. hydrophila in the planktonic phase (×6470), adherent phase on PE (×15,900) and biofilm phase induced on PE pressed on blood agar (×19,700), respectively.

Figure 6

Scanning electron micrographs (A) of un-colonized PP (×10,000); (B–D) of A. hydrophila in the planktonic phase (B; ×6540), adherent phase on PP (C; ×7530) and biofilm phase induced on PP pressed on blood agar (D; ×2540).

Figure 7

Scanning electron micrographs (A) of un-colonized PE (×4980); (B–D) of C. freundii in the planktonic phase (×16,800), adherent phase on PE (×10,000) and biofilm phase induced on PE pressed on MacConkey agar (×8300), respectively.

Figure 8

Scanning electron micrographs (A) of un-colonized PP (×10,000); (B–D) of C. freundii in the planktonic phase (×18,700), adherent phase on PP (×11,200) and biofilm phase induced on PP pressed on MacConkey agar (×18,600), respectively.