Multidrug-Resistance of <i>Vibrio</i> Species in Bivalve Mollusks from Southern Thailand: Isolation, Identification, Pathogenicity, and Their Sensitivity toward Chitooligosaccharide-Epigallocatechin-3-Gallate Conjugate

Abstract

Vibrio spp. is a Gram-negative bacteria known for its ability to cause foodborne infection in association with eating raw or undercooked seafood. The majority of these foodborne illnesses are caused by mollusks, especially bivalves. Thus, the prevalence of Vibrio spp. in blood clams (Tegillarca granosa), baby clams (Paphia undulata), and Asian green mussels (Perna viridis) from South Thailand was determined. A total of 649 Vibrio spp. isolates were subjected to pathogenicity analysis on blood agar plates, among which 21 isolates from blood clams (15 isolates), baby clams (2 isolates), and green mussels (4 isolates) showed positive β–hemolysis. Based on the biofilm formation index (BFI) of β–hemolysis-positive Vibrio strains, nine isolates exhibited a strong biofilm formation capacity, with a BFI in the range of 1.37 to 10.13. Among the 21 isolates, 6 isolates (BL18, BL82, BL84, BL85, BL90, and BL92) were tlh-positive, while trh and tdh genes were not detected in all strains. Out of 21 strains, 5 strains showed multidrug resistance (MDR) against amoxicillin/clavulanic acid, ampicillin/sulbactam, cefotaxime, cefuroxime, meropenem, and trimethoprim/sulfamethoxazole. A phylogenetic analysis of MDR Vibrio was performed based on 16s rDNA sequences using the neighbor-joining method. The five MDR isolates were identified to be Vibrio neocaledonicus (one isolate), Vibrio fluvialis (one isolate) and, Vibrio cidicii (three isolates). In addition, the antimicrobial activity of chitooligosaccharide–epigallocatechin gallate (COS-EGCG) conjugate against MDR Vibrio strains was determined. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of COS-EGCG conjugate were in the range of 64–128 µg/mL. The antimicrobial activity of the conjugate was advocated by the cell lysis of MDR Vibrio strains, as elucidated by scanning electron microscopic images. Vibrio spp. isolated from blood clams, baby clams, and Asian green mussels were highly pathogenic, exhibiting the ability to produce biofilm and being resistant to antibiotics. However, the COS-EGCG conjugate could be used as a potential antimicrobial agent for controlling Vibrio in mollusks.

Keywords

• <i>Vibrio</i> spp.
• bivalve mollusks
• <i>β</i>–hemolysis
• biofilm formation
• multidrug resistance
• chitooligosaccharide