Resistant bacteria from aquatic environments, a retrospective study from loggerhead sea turtle Caretta caretta

In sea turtle rescue medicine, the most frequent occurrences are traumatic injuries (fishing net entrapment, ingestion of hooks and lines, shell fractures, stranding, abnormal buoyancy) or infectious opportunistic pathologies in immunosuppressed animals (e.g., juveniles in cold stunning) (1), and the use of antimicrobial drugs is mandatory. The paucity of data on therapeutic protocols supported by scientific evidence (pharmacokinetics and efficacy trials), together with the repeated reports of antimicrobial resistance in strains isolated from free-ranging sea turtles (2), suggest isolation/sensitivity testing before a therapeutic plan is established as the most correct approach (3). In the present study the results of a four-year investigation about isolation, characterization, and sensitivity testing of isolates from Caretta caretta are reported, to explore the diffusion of antimicrobial resistance (AMR) in bacteria form wild species with no history of previous therapies. The animals were presented at the Sea Turtle Rescue Center “Luigi Cantoro” of Torre Guaceto (BR, Italy, Adriatic Sea) for noninfectious occurrences, with lesions at different location (lesions of limbs or carapace with exposition of bone plates, shell fractures, gastrointestinal lesions from hooks/lines). Amies transport swabs were collected from superficial lesions with deep blade scrub, while bioptic samples were collected during surgery for hook/lines removal. Samples were cultured on Columbia Blood agar and McConkey agar. The isolates were identified by Gram staining, oxidase test, API 20E and 20NE systems, then screened for antimicrobial susceptibility by the disk diffusion method (4) with amoxicillin-clavulanic acid (AMC), ampicillin-sulbactam (AMS), cefuroxime (CXM), ceftazidime (CAZ), ciprofloxacin (CIP), norfloxacin (NOR), gentamycin (GEN), and doxycycline (DOX). Samples were cultured also for Mycoplasma on Hayflick agar and tested with microbroth dilution for tetracycline, doxycycline, erythromycin, clarithromycin, tylosin, azithromycin, enrofloxacin and marbofloxacin. The prevalent isolates were Gram negative, and 30 of them, belonging to genera Aeromonas, Morganella, Pseudomonas, Serratia, and Vibrio, are potentially pathogenic. Many isolates resulted resistant to the association of semisynthetic penicillin and beta-lactamase inhibitors (18/32 to AMS and 27/32 to AMC), and to a second-generation cephalosporin (7/32 to CXM), while only two strains of Vibrio alginolyticus and Serratia marcescens were resistant to the third-generation cephalosporin CAZ. Noteworthy, the same V. alginolyticus strain was also resistant to all the tested antibiotics, including GEN and the fluoroquinolone CIP. Four more strains of Aeromonas, Pseudomonas, and Serratia genera were multidrug resistant (MDR) (semisynthetic penicillins+beta-lactamase inhibitors, second-generation cephalosporin, and doxycycline or fluoroquinolones). A single Mycoplasma spp. strain was isolated, proving resistant to tetracyclines and macrolides, with the only susceptibility to fluoroquinolones. The present data confirm the progressive spreading of AMR and MDR bacterial strains in wild animals without previous contacts with antimicrobial compounds, together with the concern about the potential for AMR diffusion linked to mobile genetic elements, frequently reported in many strains of the present isolates (5, 6). At the same time the sea turtles’ rehab suffers from the progressive reduction of therapeutic tools and adds zoonotic concerns for operators.