Competitive Interactions Inside Mixed-culture Biofilms of Salmonella Typhimurium and Cultivable Indigenous Microorganisms on Lettuce Enhance Microbial Resistance of Their Sessile Cells to Ultraviolet C (UV-C) Irradiation

Introduction: Salmonella Typhimurium (ST) is one of the leading causes of foodborne diseases in fresh produce, such as lettuce. Despite this, the role of the possible interactions between lettuce indigenous microorganisms and ST on their ability to form biofilm on lettuce and subsequently on the sensitivity of their sessile cells to ultraviolet C (UV-C) irradiation, remains relatively unexplored.

Purpose: Here, the interaction of a mixed-culture of ST and cultivable indigenous microorganisms (CIMs) was examined, as well as the efficacy of UV-C.

Methods: Initially, the CIMs were isolated and cultured with ST at 15°C either planktonically or left to form biofilms on stainless steel (SS) and lettuce leaves. Microbial growth, biofilms formation, and survival following UV-C treatment were monitored using traditional plate count methods while biofilm formation, production of extracellular polymeric substance (EPS), and stomatal colonization were also observed by field emission scanning electron microscopy (FESEM). Internalization strength, color, and texture were analyzed by standard methods.

Results: It was revealed that the mixed-culture of ST and CIMs presented significantly (P <0.05) decreased biofilms formation on lettuce leaves compared to mono-cultures (i.e., ST or CIMs alone), which indicated competitive interaction between them, while no interactions were observed for biofilms on SS and for the planktonic cultures.  It was also demonstrated that a mixed-culture biofilm on lettuce presented significantly higher resistance (P < 0.05) to UV-C treatment compared to mono-cultures biofilms, but such an effect was not observed for biofilms formed on SS and for the planktonic cultures. The Weibull model fitted well to microbial inactivation curves with R² values that ranged from 0.90 to 0.97. Regarding the mixed-culture conditions, a UV-C fluency of 35 mJ/cm² was required to achieve a 5.0 log CFU/ml or cm² reduction in planktonic and biofilms on the SS for the mixed-culture, while 360 mJ/cm² was required to reduce biofilm cell number by approximately 2.0 log CFU/cm² on lettuce. Furthermore, FESEM analysis indicated higher EPS production, and greater stomatal colonization on lettuce mixed-cultures compared to mono-cultures. Finally, internalization strength was significantly higher (P < 0.05) for the mixed-culture on lettuce, thus supporting the notion that internalization in lettuce is a factor contributing to microbial UV-C resistance. The UV-C doses (360 mJ/cm²) did not change the color and texture of lettuce leaves.

Significance: Therefore, UV-C could be an alternative to chlorine disinfectants for lettuce leaves, even high efficacy is required as lettuce possess indigenous microorganisms.