Rugose Morphotype in Salmonella Typhimurium and Salmonella Heidelberg Induced by Sequential Exposure to Subinhibitory Sodium Hypochlorite Aids in Biofilm Tolerance to Lethal Sodium Hypochlorite on Polystyrene and Stainless Steel Surfaces

Abstract

Salmonella biofilms act as a continuous source for cross-contamination in the food processing environments. In this study, a stable rugose morphotype of Salmonella was first induced by sequential exposure to subinhibitory concentrations (SICs) of sodium hypochlorite (NaOCl) (ranging from 50 to 300 ppm over 18-day period) in tryptic soy broth. Then, rugose and smooth morphotypes of Salmonella Typhimurium ATCC 14028 and Salmonella Heidelberg ATCC 8326 were characterized for biofilm forming abilities on polystyrene and stainless steel surfaces. Rugose morphotype of both ATCC 14028 and ATCC 8326 exhibited higher Exopolysaccharide (EPS) formation than smooth morphotype (p ≤ 0.05). Also, the SICs of NaOCl (200 or 300 ppm in broth model) increased the biofilm formation ability of rugose morphotype of ATCC 8326 (p ≤ 0.05) but decreased that of ATCC 14028. The 2-day-old Salmonella biofilms were treated with biocidal concentrations of 50, 100, or 200 ppm NaOCl (pH 6.15) in water for 5, 10, or 20 min at room temperature. The biofilm reduction in CFU/cm2 for the rugose was lower than the smooth morphotype on both surfaces (p ≤ 0.05) by lethal NaOCl in water. Scanning electron micrographs on both polystyrene and stainless steel surfaces demonstrated that the rugose morphotype produced a denser biofilm than the smooth morphotype. Transmission electron micrographs revealed the cell wall roughness in rugose morphotype, which may help in tolerance to NaOCl. The gene expression data indicate that the expression of biofilm regulator (csgD), curli (csgA, csgB, and csgC), and cellulose (bcsE) was significantly increased in rugose morphotype when induced by sequential exposure of NaOCl SICs. These findings reveal that the rugose morphotype of S. Typhimurium and S. Heidelberg produced significantly denser biofilm on food contact surfaces, which also increased with sequential exposure to SICs of NaOCl in the case of S. Heidelberg, and these biofilms were more tolerant to biocidal NaOCl concentrations commonly used in the food processing plants.

Surviving Reactive Chlorine Stress: Responses of Gram-Negative Bacteria to Hypochlorous Acid

“…Moreover, a higher amount of EPS and an increased expression of the genes involved in environmental stress response ( envZ and csrA ), curli synthesis ( csgA , csgB , and csgC ), cellulose formation ( bscE ), biofilm regulators ( csgD and hns ), and stress resistance and biofilm formation ( rpos ), were detected in rugose S. Heidelberg biofilms in comparison to smooth cells. In this case, the adaptative response of S. Heidelberg biofilms to NaClO could be due to the increased production of EPS, which protects the cells against the disinfectant, and the expression of cellulose and curli, which is important for biofilm attachment [ 160 ]. The EPS matrix provides not only a physical but also chemical protection against environmental stressors, which is crucial for adaptation in hostile conditions.…”

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro

“…The effect of SIC of sodium butyrate on the expression of SE virulence genes was determined using real-time quantitative PCR (RT-qPCR) as described previously (Upadhyaya et al, 2015;Sun and Jia, 2018;Bansal et al, 2019). SE was cultured to mid-log phase with or without SIC of sodium butyrate in TSB at 37 • C for 10 h. The total RNA was extracted using TRIzol reagents (Thermo Fisher Scientific) as per manufacturer's protocol.…”

Effect of atmospheric cold plasma (ACP) on chlorine-adapted Salmonella enterica on spring onion

“…So, stops in bacterial growth were observed at 3125 ppm. So, bacterial colonies obtained from 3100 ppm (25 ppm lower than the concentration at the bacterial growth stops) were used as adapted S. enterica cells in this study (Bansal et al 2019).…”

“…In particular, lately increasing concerns have been reported on Salmonella enterica serovar occurrence in leafy green vegetables through inappropriate agricultural and environmental practices (Azimirad et al 2021). Among the approved disinfectants in food industry, chlorine and chlorine-based materials are the most available disinfectants due to their lower cost and broad inactivation range on bacteria (Bansal et al 2019). Sodium hypochlorite as chlorine-based disinfectant is the ideal disinfectant with high efficacy against most pathogens (Gere et al 2022).…”