Occurrence of ingression of Salmonella spp. in Betel leaf (Piper betle L.)

Background: Salmonella spp. is one of the most common pathogens associated with fresh produce related foodborne illness. This study aimed to determine Salmonella spp. contamination level in betel leaf, internalization potential and possible decontamination process. Results: A total of 77% betel leaf sample collected from local market was found to be contaminated with Salmonella spp. Of all the Salmonella spp. isolated and identified, 28.5% belong to Salmonella enterica subsp. enterica serovar Enteritidis, 19.5% belong to Salmonella Typhimurium, 15.6% to Salmonella Paratyphi, 10.4% to Salmonella Schottmuelleri, 9.1% to Salmonella Gallinarum, 10.4% to Salmonella Choleraesuis and 6.5% belong to Salmonella Bongori. Internalized Salmonella spp. showed moderate resistance to commonly used antibiotics. Treatment with common surface food disinfectants could not remove Salmonella spp. completely from betel leaf indicating the possibility that the bacteria may be in internal tissue of the leaf. Assessment of internalization potential showed that Salmonella spp. isolated from inner part of betel leaf pose better internalization potential (6.7–7.4 logCFU/gm) comparing with ATCC (American Type Culture Collection) strains (0.86–0.6 logCFU/gm). The isolates also showed better survivability in internalized condition, biofilm formation ability and motility than ATCC strains. Prevalence and expression of invasion (sefA and invA) and type 3 secretion system (TTSS) associated genes (hila, avrA and sopE) were high in internalized Salmonella isolates. Commercial disinfectants as well as H2O2 were found to have poor efficacy (log reduction around 2 CFU/gm) against internalized Salmonella. Ozonated water showed better decontamination efficiency (log reduction around 3 CFU/gm) whereas ethanolic extract of Terminalia arjuna stem bark showed higher decontamination (log reduction around 4.5 CFU/gm) of internalized Salmonella. Conclusion: Salmonella spp. can ingress into betel leaf and better decontamination treatment is needed to be established.