Assessment of Antimicrobial Activity of Synthetic Peptides on Escherichia coli Isolated from Boar Semen

Abstract

Recently, antimicrobial peptides (AMPs) are known as new potential agent which can inhibit bacteria growth in liquid-preserved boar semen and used to replace the conventional antibiotics. Previous study, our researchers found that nine peptides with derived from the seminal plasma showed the highest antimicrobial activity with Escherichia coli ATCC 25922 ranged from 70% to 100% inhibition (not published yet). It is very interesting for further investigation on their activities against Escherichia coli isolated from boar semen. The objectives of this study were to i) identify the bacterial contaminants in boar semen ii) discriminate Escherichia coli strains and iii) investigate the antimicrobial susceptibility testing and antimicrobial activity of synthetic peptides on Escherichia coli isolated from boar semen. The synthetic peptides were prepared by manufacturer as powder and only eight of nine peptides could be synthesized. Eight pooled semen kept in transport media and ten fresh boar semen samples were collected from pig farms located in the central and southern region of Thailand, respectively. The gram staining and biochemical test were conducted to identify the species of both gram negative and gram-positive bacteria. The remaining unknown species of bacteria were identified by MALDI-TOF MS technique. Antimicrobial susceptibility of Escherichia coli isolates was tested by disk-diffusion (penicillin G, ampicillin, gentamicin, amikacin and ceftazidime) and broth microdilution methods (colistin). BOX A1R PCR (primer: 5’-CTACGGCAAGGCGACGCTGACG-3’) was used to discriminate Escherichia coli strains isolated from boar semen. To determine the minimum inhibitory concentration (MIC), one of isolated Escherichia strains, randomly selected, and Escherichia coli ATCC 25922 were preliminary investigated with 2 peptides (Sam1 and Sam5) by using a broth microdilution. The MIC value of Sam1 and Sam5 with Escherichia coli ATCC 25922 and B05N44 showed higher than 400 µg/ml. So that, 100 µg/ml of all the peptides were preliminary investigated the antimicrobial activity with 4 strains of Escherichia coli selected from each group of clonal relatedness (one replication). The results revealed that Sam1, Sam4, and Sam9 showed higher activity with Escherichia coli ATCC 25922 while Sam1, Sam2, and Sam9 showed higher activity with A01N04, A06N16, B05N44, and B07N62. Then, the peptides which showed the highest activity were conducted the time-kill assay by OD measurement and colony count at 0, 4, 8, 16, and 24 h of incubation (triplicate). The results showed that all the samples were contaminated with gram-negative and gram positive bacteria (n=18, 100%). The dominant species were Escherichia coli (100 %, n=18) and Staphylococcus spp. (100%, n=18) followed by Serratia marcescens (33.33%, n=6), Klebsiella pneumoniae (22.22%, n=4), Enterobacter cloacea, Citrobacter koseri and Enterobacter aerogenes and Streptococcus spp. (equally 11.11%, n=2), Aeromonas hydrophila, Edwardsiella tarda, Providencia stuartii, Providencia rettgeri, Klebseilla oxytoca, Klebseilla aerogenes and Pseudomonas aeruginosa (equally 5.55%, n=1). Moreover, the colony forming unit of gram-positive and gram-negative bacteria isolated from fresh boar semen varied from 4.00×102 to 8.50×103 and 1.33×102 to 4.17×103 CFU/ mL, respectively. Escherichia coli isolates were resistant to penicillin G (100%), ampicillin (97.96%), gentamicin (12.24%) and colistin (8.16%). All of Escherichia coli were sensitive to amikacin (0%) and ceftazidime (0%). According to 75% similarity levels of clonal relatedness, 49 strains of Escherichia coli were distinguished into 8 clusters. Sam1, Sam4, and Sam9 showed high antimicrobial activity, in term of inhibition percentage, with Escherichia coli ATCC 25922 (99.99%, 98.14%, and 97.57%, respectively) whereas Sam1, Sam2, and Sam9 showed high activity with A01N04 (98.75%, 85%, and 87.5%, respectively), A06N16 (84.29%, 90.86%, and 95.71%, respectively), B05N44 (99.88%, 95.21%, and 98.70%, respectively), and B07N62 (99.79%, 99.97%, and 80%, respectively). In conclusion, Sam1 had the highest antibacterial activity against Escherichia coli ATCC 25922 and Escherichia coli strains isolated from boar semen.