The Use of Vortex and Ultrasound Techniques for the in vitro Removal of Salmonella spp. Biofilms

Background : The presence of biofilm is common to all types of surfaces, such as stainless steel. Once formed, biofilms act as a point of constant contamination releasing fragments or planktonic cells of microorganisms, such as Salmonella spp., and may impair the microbiological quality of products. The laboratory methods are being used and tested in vitro for removal and the subsequent quantification of biofilms, including the use of vortex and ultrasound. The aim of the study was to evaluate the effectiveness of these two laboratory methods for removing Salmonella spp. biofilms, in vitro cultured on stainless steel surface from the food industry. Materials, Methods & Results : Three strains were analyzed for biofilm formation by Salmonella spp., and they are S. Typhimurium ATCC 14028, S. Enteritidis ATCC 13076 and S. Enteritidis isolated from drag swab on poultry farm and genetically confirmed by Microarray, denominated P106. Coupons of stainless steel AISI 316, with an area of 1 cm² were used. These materials used for the coupons were obtained from the equipments at the cutting room in the poultry slaughterhouse. Biofilms were formed using TSB broth without glucose and incubated at 36°C for 24 h. Six replicates were performed for each microorganism in each removal method. After the biofilm formation, two methods were used in the removal stage, the vortexing, performed for 2 min, and the sonication method, with coupons maintained for 10 min in an ultrasound bath, at a frequency of 40 kHz and potency 81 W. Serial dilutions were made and transferred to PCA agar for quantification in log 10 CFU.mL-1. The microtopography was performed by scanning electron microscopy (SEM) of surfaces before the removal step. Statistical analysis of the results showed no significant difference between the two removal methods and between the three strains studied ( P > 0.05). Discussion : Biofilms can be attached to any surface, biotic or abiotic. This adhesion of bacterias, especially pathogenic, can cause serious hygiene problems and economic losses due to impairment the microbiological quality of the products. Food safety is a concern for consumers and for the food industry, as well as the agencies responsible for public health. Research involving studies with the formation of microbial biofilms has become increasingly present. Laboratory methods are used for removing biofilms in vitro and subsequent quantification, including the use of the vortexing and ultrasound. The vortex­ing acts through tourbillon movements, removing or reducing the adhesion of microorganisms on a surface. Ultrasound destabilizes the structure of the biofilm, dislodging bacterial aggregates through physical, chemical and mechanical results of the cavitation process. These processes are responsible by the cleaning of surfaces with presence of adhered particles. In our study there was no statistical difference between the two removal methods, vortexing and ultrasound. There was also no significant difference between S. Typhimurium, S. Enteritidis and SE P106, which confirms the similarity between the two methods and among the studied strains. Although there was no statistical difference between the tested methods, the use of ultrasound in moderate frequency (40 kHz) and for 10 min, should be used as standard for detachment of biofilms formed in vitro , due to the ease of use and to their hydrodynamic properties that destabilize the structure of the biofilm.