Biosafety of Vibrio Parahaemolyticus Biofilm From Seafood Using Herbs and Spices

The microbial safety of foods continues to be a main concern to consumer, regulatory agencies and food industries around the world. In this study, a total of 394 presumptive vibrios from seafood samples (n=394) consists of cockles (Anadara granosa) ,clams (Mya arenaria), (shrimp) Penaeous spp. and (squid) Loligo opalescens have been isolated. Polymerase chain reaction revealed the 9.14% positive toxr gene, 63.8% of the presence of toxin gene V. parahaemolyticus tdh + , 22.2% trh + , and 5.56% of positive isolates possessing both tdh+ and trh+ while pandemics strain was 83.3% . The sixteen resistant isolates toward seven antibiotics were selected for further study in terms of minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Clove (Syzygium aromaticum), star anise (Illicium verum), lemon leaves (Melissa Officinalis) and curry leaves (Murraya koenigii) have been selected from 20 others herbs and spices because of the potential inhibitory activity against the resistant isolates. Time-kill curves were constructed to assess the concentration between bacteriostatic and bactericidal activity of the respective herbs and spices at concentrations ranging from 0 MIC to 8 × MIC. The activity of cloves against biofilm- producing resistant strains also evaluated. The growth of all resistant strains (n=16) including V. parahaemolyticus ATCC 17802 were inhibited by cloves at a concentration of 39.062 µg/ml and was able to kill all resistant strains with a MBC90 at 78.125 µg/ml compared to the others. The time-kill curves demonstrated that the bactericidal endpoint for resistant strain was reached after 30 min of incubation at a antimicrobials concentration of 8 × MIC. The growth of biofilm-producing isolates can be inhibited and eradicated by clove at concentrations of 156.25 µg/ml, 625 µg/ml respectively. Clove has stronger activity against biofilm-producing resistant strains than star anise, lemon Leaves and curry leaves extracts.