SCREENING OF SYNTHETIC PEPTIDES FOR ANTIMICROBIAL ACTIVITY AGAINST FISH PATHOGENIC BACTERIA

There is an immediate requirement for the novel antimicrobials in aquaculture, mainly because of the high rate of evolving antibiotic-resistant bacteria. Screening compounds for antimicrobial activity against fish pathogenic bacteria is a cost-effective and sustainable solution to address this challenge. In the present work,we investigated the in vitro antibacterial activity of 7 synthetic antimicrobial peptides (AMPs) against 12 clinical isolates of pathogenic bacteria, and two reference strains, Aeromonas hydrophila (ATCC 7966), and Escherichia coli ATCC 25922. The clinical isolates were previously recovered from the internal organs of 4 species of freshwater fish (Colossoma macropomum, Oreochromis niloticus, Pseudoplatystoma fasciatum, and Pterophyllum scalare) positively diagnosed with a bacterial infection, sampled during disease outbreaks at six fish farms or facilities located in South, and Southeast Brazil. The bacteria identities were confirmed by 16S rRNA gene sequencing, being 1 Aeromonas caviae, 1 A. hydrophila, 3 A. jandaei, 1 Citrobacter freundii, 1 Edwardsiella tarda, 2 Streptococcus agalactiae, 2 Lactococcus garvieae, and 1 Vibrio fluvialis. Antimicrobial activity (minimum inhibitory and bactericidal concentrations (MIC and MBC)) was determined using the broth microdilution protocol recommended by the Clinical and Laboratory Standards Institute (CLSI). All isolates were recovered and cultured prior to being analyzed using a specific medium to each strain. AMPs (Table 1) were synthesized, dissolved and diluted in phosphate-buffered saline to prepare 5,000 µg/mL antimicrobial stock solutions. The ranges of antimicrobials concentrations in the tests were 250 - 0.4883 µg/mL. Bacteria at a final concentration of 5x10⁵ CFU/mL were added to each well and incubated at 28 ºC for 24 h. MICs were determined in triplicate, with two wells in each MIC test plate used as a control for growth and sterility. Florfenicol and E. coli ATCC 25922 was used for quality control (QC).

QC MICs were within the concentration range established in CLSI guidelines. The MICs and MBCs of 7 AMPs for 12 bacterial isolates are summarized in Table 2. AMPs demonstrated potent antibacterial activity against A. jandaei (Kr12a5), C. freundii (Kr12a5; [Trp^3,5,10] des-Cys¹¹, Lys¹², Lys¹³-(pBthTX-I)₂K; des-Cys¹¹, Lys¹², Lys¹³-(pBthTX-I)₂K; LL 37; and Hylin a1), S. agalactiae (Hylin a1; [Trp^3,5,10] des-Cys¹¹, Lys¹², Lys¹³-(pBthTX-I)₂K; and LL 37), L. garviae (Hylin a1; and [Trp^3,5,10] des-Cys¹¹, Lys¹², Lys¹³-(pBthTX-I)₂K), and V. fluvialis (Kr12a5).

These findings suggest the potential of some AMPs as a treatment option for bacterial diseases in aquaculture.