Inhibitory activity and mechanism of aqueous extract from Pithecellobium clypearia against aquatic pathogenic bacteria

Abstract:

The aquaculture industry has rapidly expanded in recent years in China,whereas it faces the challenge brought by bacterial diseases.Antibacterial agents have been the primary tools to combat these diseases.However,prolonged and haphazard usage of antibacterial agents in aquaculture has exacerbated antimicrobial resistance and led to severe antimicrobial residues.Considering these challenges,scholars worldwide have been exploring natural alternatives,such as Chinese herbal medicines.Among them,Pithecellobium clypearia stands out due to its antibacterial,antiviral,and anti-inflammatory properties,coupled with its safety and lack of antimicrobial resistance.Nonetheless,the potential of P.clypearia in the prevention and control of aquatic diseases remains underexplored.[Objective]This study evaluated the in vitro inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant strain.Additionally,we investigated changes in bacterial cell membrane permeability and observed cellular alterations by transmission electron microscopy to elucidate the mechanism of the extract.Our findings are expected to pave the way for developing P.clypearia as an environmentally friendly antibacterial agent,reducing antibacterial agent dependency,and mitigating pathogen resistance in aquaculture.[Methods] We employed the microbroth method to assess the antimicrobial resistance of 107 pathogen strains attacking aquatic animals and analyzed the inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant bacterial strain.Furthermore,we determined the extracellular K⁺ content and ultrastructural changes in Streptococcus agalactiae and Vibrio parahaemolyticus after treatment with the extract.[Results]The resistance rate of 107 pathogen strains to sulfonamides was as high as 67.29%,and 46.73% of the strains showed multidrug resistance,among which Aeromonas sp.showed the most severe resistance.The aqueous extract (12.50 mg/mL) of P.clypearia exerted inhibitory effects on all the pathogenic bacteria,especially on Aeromonas sp.with the minimum inhibitory concentration (MIC) as low as 0.39 mg/mL.The MICs of the extract were similar for the strains belonging to the same genus but with different antimicrobial resistance characteristics.The aqueous extract of P.clypearia showed stronger inhibitory effect on the artificially induced enrofloxacin-resistant strain of A.schubertii than on the original strain,with the MICs of 0.78 mg/mL on the original strain and 0.20 mg/mL on the resistant strain.In addition,the treatment with P.clypearia aqueous extract significantly increased the extracellular K⁺ concentration,leading to damage to the bacterial cell membrane structure,leakage of intracellular contents,and vacuolation of cytoplasm,which suggested that P.clypearia exerted the antibacterial effect by destroying the bacterial membrane structure.[Conclusion]P.clypearia exerts in vitro inhibitory effects on aquatic pathogenic bacteria and demonstrates great potential for further research and development in the prevention and treatment of bacterial diseases in aquatic animals.The antibacterial mechanism of P.clypearia appears to involve disrupting bacterial cell membranes.The application of P.clypearia in aquaculture promises to reduce antibiotic dependency and pathogen resistance,paving the way for a healthy and sustainable aquaculture industry.

Key words:pathogenic bacterium;antimicrobial resistance;Pithecellobium clypearia;antibacterial activityin vitro;bactericidal mechanism