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.
Highlights
Antibiotic resistance is an emerging crisis in bacterial diseases, as the frequency of resistance in clinical as well as environmental settings increases, and the development of new antibiotics slows down [1,2,3]
An earlier work on the growth at 35 ◦C of A. hydrophila strains in Brain Heart Infusion broth buffered with 3-[N-morpholino] propane sulfonic acid (MOPS) and adjusted to pH 7.0, reported generation times of 19.8–23.4 min [93]
C. portucalensis RIT669 activated our broad range acyl-homoserine lactone (AHL)-dependent whole cell biosensor but further work is need to identify the potential two AHLs identified as well as the biosynthetic pathway used since a luxI homolog was not readily located in our whole genome sequence of this strain
Summary
Antibiotic resistance is an emerging crisis in bacterial diseases, as the frequency of resistance in clinical as well as environmental settings increases, and the development of new antibiotics slows down [1,2,3]. Aeromonas and Citrobacter are two widely distributed Gram-negative genera containing several pathogenic species of clinical concern. Aeromonas hydrophila and Citrobacter freundii strains are considered emerging pathogens [4,5], which can both form biofilms [6,7,8,9]. A. hydrophila and C. freundii infect both freshwater and marine turtles, and antibiotic resistance among the turtle strains has been reported [10,11,12]. We reported the biofilm formation and Shiga-toxin expression by A. hydrophila and C. freundii strains isolated from infected spotted turtles (Clemmys guttata) [17]. Bacteria associated with reptiles could infect warm-blooded mammals, since reptile and clinical strains of Aeromonas spp. were identical in some cases [18]
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