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

Abstract

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500-1000, and the minimum biofilm eradication concentration (MBEC) > 1000 μg/mL, using cotrimoxazole, and MIC = 32.3-62.5, and MBEC > 1000 μg/mL, using neomycin. For C. freundii MIC = 7.8-15.6, and, MBEC > 1000 μg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 μg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.

Keywords: Aeromonas hydrophila; Citrobacter portucalensis; antibiotics; antibiotics resistance; quorum sensing.

Figure 1

Schematic representation of the high throughput in vitro antimicrobial testing model to evaluate efficacy of cotrimoxazole (sulfmethoxazole and trimethoprim) and neomycin, estimated via the MIC and MBEC values against A. hydrophila and C. portucalensis. OSB refers to organism specific broth; in this work trypticase soy broth (TSB) was used.

Figure 2

Fisher individual 95% confidence intervals for the MIC values from the challenge plate of A. hydrophila containing (A) cotrimoxazole; the MIC is determined to be between 1000 and 500 μg/mL (rows A and B) due to lack of growth and a significant difference in the absorbance between rows A and B, but not rows B and C, (B) neomycin; the MIC is determined to be between 62.5 and 32.3 μg/mL (rows E and F) due to lack of growth and a significant difference in the absorbance between rows E and F.

Figure 3

Scanning electron micrographs of A. hydrophila biofilm inoculation pegs at (A) the lowest concentration of cotrimaxazole (7.8 μg/mL; magnified 7070×), (B) the highest concentration of cotrimaxazole (1000 μg/mL; magnified 7120×), (C) the lowest concentration of neomycin (7.8 μg/mL; magnified 6540×), and (D) the highest concentrations of neomycin (1000 μg/mL; magnified 6470×).

Figure 4

Quorum sensing signals of the acyl-homoserine lactone (AHL) class are produced by A. hydrophila RIT668 and C. portucalensis RIT669. Three formats using two complementary AHL-dependent whole cell biosensor strains were used, Chromobacterium violaceum CV026 (panel A) and Agrobacterium tumefaciens NTL4 (pZLR4) (panels (B,C)) [70]. T-streak bioassay using biosensor CV026 (A) C+ strain, DH5a (pT140A) and C− strain, CV026. Disc-diffusion bioassay using increasing volumes of ethyl acetate extract from early stationary stage cultures of A. hydrophila (668) and C. portucalensis (669), C+ N-(3-Oxooctanoyl)-L-homoserine lactone (3OC8), 4 µL 10 nm; C− 30 µL ethyl acetate (B); and Thin layer chromatography separation and detection of AHL signals from ethyl acetate extracts of cultures of A. hydrophila (668) and C. portucalensis (669) (C). AHL standards include: lane 1, N-(3-Oxohexanoyl)-L-homoserine lactone, (3OC6) (4 µL, 100 nM); lane 2, N-(3-Hydroxyhexanoyl)-L-homoserine lactone (3OHC6) (10 µL, 1 mM); lane 3, N-Hexanoyl-L-homoserine lactone (C6) (2 µL, 1 mM); lane 4, N-Butyryl-L-homoserine lactone (C4) (6 µL, 1 mM); lane 5, RIT668 ethyl acetate extract (10 µL of 200× concentrate); lane 6, RIT669 ethyl acetate extract (10 µL, 200× concentrate), lane 7, N-(3-Oxooctanoyl)-L-homoserine lactone (3OC8) (5 µL, 10 nM); lane 8, N-Octanoyl-L-homoserine lactone (C8) (10 µL, 100 mM); lane 9, N-(3-Hydroxyoctanoyl)-L-homoserine lactone (3OHC8) (4 µL, 1 mM). C+ = positive control; C− = negative control; 668 = A. hydrophila RIT668, and 669 = C. portucalensis RIT669.

Figure 5

Fisher individual 95% confidence intervals for the MIC values from the challenge plate of C. portucalensis containing (A) cotrimoxazole; the MIC is determined to be between 15.6 and 7.8 μg/mL (rows G and H) due to effect of antibiotic causing a lack of growth and a significant difference in the absorbance between rows G and H; (B) neomycin; the MIC is determined to be between 31.3 and 7.8 μg/mL (rows F and H) due to a lack of growth. More data is required to determine if the MIC is between 31.3 and 15.6 μg/mL (rows F and G) due to near significant differences in the absorbance between rows F and G.

Figure 6

Scanning electron micrographs of C. portucalensis biofilm inoculation pegs at (A) the lowest (7.8 μg/mL) and (B) the highest (1000 μg/mL) concentrations of cotrimoxazole. The images were magnified 3380× and 4430×, respectively. (C) (18,700×), and (D) (21,300×), show further magnification of cells in (A,B), which received the lowest and highest cotrimoxazole concentrations, respectively.

Figure 7

Scanning electron micrographs of C. portucalensis biofilm inoculation pegs at (A) the lowest (7.8 μg/mL), and (B) the highest (1000 μg/mL) concentrations of neomycin. The images are magnified 3600× and 4880×, respectively.