Master of Science / Food Science / Kelly J. K. Getty / Dry-processing environments are particularly challenging to clean and sanitize because water introduced into systems not designed for wet cleaning can favor growth and establishment of pathogenic microorganisms such as Salmonella. The objective was to determine the efficacy of isopropyl alcohol quaternary ammonium (IPAQuat) formula and carbon dioxide (CO[subscript]2) sanitizer system for eliminating Enterococcus faecium and Salmonella on food contact surfaces. Coupons of stainless steel and conveyor belting material used in dry-processing environments were spot-inoculated in the center of 5 × 5 cm coupons with approximately 7.0 log CFU/ml of E. faecium and up to 10 log CFU/ml of a six-serotype composite of Salmonella and subjected to IPAQuat-CO[subscript]2 sanitation treatments using exposure times of 30 s, 1 or 5 min. After sanitation treatments, wet coupons were swabbed for post-treatment survivors. Preliminary experiments included coupons which were soiled with a flour and water solution prior to inoculation and subsequent sanitation treatments. For the main study, inoculated surfaces were soiled with a breadcrumb flour blend and allowed to sit on the lab bench for a minimum of 16 h before sanitation. Preliminary results showed that IPAQuat-CO[subscript]2 sanitizing system was effective in reducing approximately 3.0 logs of E. faecium and Salmonella from clean and soiled surfaces after 1 min exposure but higher initial inoculum levels were needed to demonstrate >5 log reductions. For the main study, pre-treatment Salmonella populations were approximately 7.0 log CFU/25 cm[superscript]2 and post-treatment survivors were 1.3, < 0.7 (detection limit), and < 0.7 log CFU/25 cm[superscript]2 after 30 s, 1 or 5 min sanitizer exposures, respectively, for both clean and soiled surfaces. Treatment with IPAQuat-CO[subscript]2 sanitation system using 30 s sanitizer exposures resulted in 5.7 log CFU/25 cm[superscript]2 reductions whereas, greater than 6.0 log CFU/25 cm[superscript]2 reductions were observed for sanitizer exposures of 1 and 5 min. The IPAQuat-CO[subscript]2 sanitation system reduced 6 logs CFU/25 cm[superscript]2 of Salmonella with sanitizer exposure times of at least 1 min. The IPAQuat-CO[subscript]2 system would, therefore, be an effective sanitation system to eliminate potential contamination from Salmonella on food contact surfaces and have application in facilities that process dry ingredients or low-moisture products.

Este estudo avaliou a eficiência da oleuropeína (OLE) (composto fenólico extraído das folhas de Oliveira) isolada e associada aos sanitizantes comerciais ácido peracético 2% (APA), hipoclorito de sódio 2% (HS), peróxido de hidrogênio 3% (PH), digluconato de clorexidina 2% (DC), cloreto de benzalcônio 1% (CB) e iodofor 2% (IO), para inativação de células em suspensão e biofilmes monoespécie e multiespécie formados em superfícies de aço inoxidável ou microplaca de poliestireno por Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923) e Escherichia coli (ATCC 25922), todas classificadas como fortes produtores de biofilmes. Os isolados foram semeados em caldo TSB (caldo tripticase soja), incubados (37°C/24h) e corrigidos a ~108células/mL (escala 0,5 McFarland). Para bactérias em suspensão, a resistência a sanitizantes foi determinada pela Concentração Inibitória Mínima (CIM) em tubos e pelo método de Disco Difusão em Ágar (DDA), no qual as bactérias foram plaqueadas em ágar TSA contendo discos de 6mm de papel filtro embebidos nos sanitizantes. Após a incubação, a medição dos halos de inibição foi feita com paquímetro. Para os ensaios de resistência dos biofilmes aos compostos sanitizantes, foram utilizadas microplacas de poliestireno 96 poços, as quais foram preparadas para incubação-fixação dos biofilmes e submetidas à leitura em espectrofotômetro de ELISA (600 nm). Em seguida, as placas foram lavadas com solução salina tamponada (PBS, pH 7.4) e os sanitizantes inseridos por 1 minuto. Após neutralização com tiossulfato de sódio (5 minutos), as placas foram lavadas com PBS e metanol, coradas com cristal violeta 1% e coradas com ácido acético glacial (33%) para nova leitura a 570nm. A eficácia da remoção do biofilme pelos sanitizantes foi comparada pelo índice de formação de biofilme (IFB). As imagens do aço inoxidável após tratamento com sanitizante foram feitas através de Microscopia Eletrônica de Varredura (MEV) e Microscopia Confocal, para visualizar a persistência dos biofilmes. Os valores de CIM (diluição 1:2) mostraram que OLE não teve atividade bactericida. No método DDA, L. monocytogenes, foi resistente à OLE, enquanto E. coli e S. aureus apresentaram resistência intermediária. Os sanitizantes comerciais apresentaram boa atividade bactericida nos ensaios de CIM e DDA, sendo que as associações de OLE aos sanitizantes comerciais aumentaram o efeito germicida. Nos ensaios com biofilmes em monoespécie, somente os sanitizantes comerciais, isolados ou associados com OLE, foram eficazes de reduzir o valor de BFI em microplaca de poliestireno. Em biofilmes multiespécie, OLE apresentou efeito antimicrobiano, sobretudo sobre a associação de L. monocytogenes + E. coli + S. aureus (redução: 91,49%). Nenhum dos compostos avaliados foi capaz de inativar completamente os biofilmes nas superfícies de aço inoxidável, uma vez que células viáveis foram observadas após os tratamentos com os sanitizantes, indicando persistência dos biofilmes. Os resultados indicam que a oleuropeína apresentou potencial para incrementar o efeito bactericida de sanitizantes comerciais para eliminação de biofilmes em superfícies inertes, sendo necessários estudos para compreender os mecanismos de ação dessas combinações. / This study evaluated the efficiency of oleuropein (OLE) (a phenolic compound extracted from Oliveira leaves) alone or in association with commercial sanitizers peracetic acid 2% (PPA), sodium hypochlorite 2% (SH), hydrogen peroxide 3% (HP), chlorhexidine digluconate 2% (CD), benzalkonium chloride 1% (BC) and iodophor 2% (IO), for inactivation of suspended cells and monospecies or multispecies biofilms formed on stainless steel surfaces or microplate polystyrene by Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), all classified as strong producers of biofilms. The isolates were grown in TSB (trypticase soy broth), incubated (37°C/24 h) and adjusted to ~108 cells/mL (0.5 McFarland scale). For the bacterial suspensions, resistance to sanitizers was determined by Minimum Inhibitory Concentration (MIC) in tubes and by Agar Disk Diffusion (ADD), in which the bacteria were plated on TSA agar containing 6mm disks of filter paper soaked in sanitizers. After incubation, measurement of the inhibition halos was done using a caliper rule. For the sanitizer resistance assays with biofilms, polystyrene 96-well microplates were prepared for incubation-fixing of biofilms and read in an ELISA spectrophotometer (600 nm). After, the plates were washed with phosphate buffered saline (PBS, pH 7.4) and the sanitizers were placed for 1 minute. After neutralization with sodium thiosulfate (5 minutes), the plates were washed with PBS and methanol, stained with 1% crystal violet and stained with glacial acetic acid (33%) for a new reading at 570 nm. The effectiveness of biofilm removal by each sanitizer was compared using a Biofilm Formation Index (IFB). The images from stainless steel coupons after treatments with sanitizers were obtained by Scanning Electron Microscopy (SEM) and Confocal Microscopy, to view the persistence of biofilms. MIC values (dilution 1: 2) showed that OLE had no bactericidal activity. In the ADD assays, L. monocytogenes was resistant to OLE, while E. coli and S. aureus showed intermediate resistance. Commercial sanitizers showed good bactericidal activity in both CIM and DDA assays, and the associations of OLE with commercial sanitizers increased their germicidal effect. In the monospecies biofilm assays, only commercial sanitizers, isolated or associated with OLE, were effective for reducing the BFI values in polystyrene microplates. In multispecies biofilms, OLE had an antimicrobial effect, especially on the association of L. monocytogenes + E. coli and S. aureus (reduction: 91.49%). None of the compounds evaluated was able to completely inactivate the biofilms on the stainless steel surfaces, since viable cells of bacteria were observed after treatment with sanitizers, indicating persistence of biofilms. The results indicate that oleuropein has the potential to enhance the bactericidal effect of commercial sanitizers to eliminate biofilms on inert surfaces. Further studies are needed to understand the mechanisms of action of these combinations.

Este estudo avaliou a eficiência da oleuropeína (OLE) (composto fenólico extraído das folhas de Oliveira) isolada e associada aos sanitizantes comerciais ácido peracético 2% (APA), hipoclorito de sódio 2% (HS), peróxido de hidrogênio 3% (PH), digluconato de clorexidina 2% (DC), cloreto de benzalcônio 1% (CB) e iodofor 2% (IO), para inativação de células em suspensão e biofilmes monoespécie e multiespécie formados em superfícies de aço inoxidável ou microplaca de poliestireno por Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923) e Escherichia coli (ATCC 25922), todas classificadas como fortes produtores de biofilmes. Os isolados foram semeados em caldo TSB (caldo tripticase soja), incubados (37°C/24h) e corrigidos a ~108células/mL (escala 0,5 McFarland). Para bactérias em suspensão, a resistência a sanitizantes foi determinada pela Concentração Inibitória Mínima (CIM) em tubos e pelo método de Disco Difusão em Ágar (DDA), no qual as bactérias foram plaqueadas em ágar TSA contendo discos de 6mm de papel filtro embebidos nos sanitizantes. Após a incubação, a medição dos halos de inibição foi feita com paquímetro. Para os ensaios de resistência dos biofilmes aos compostos sanitizantes, foram utilizadas microplacas de poliestireno 96 poços, as quais foram preparadas para incubação-fixação dos biofilmes e submetidas à leitura em espectrofotômetro de ELISA (600 nm). Em seguida, as placas foram lavadas com solução salina tamponada (PBS, pH 7.4) e os sanitizantes inseridos por 1 minuto. Após neutralização com tiossulfato de sódio (5 minutos), as placas foram lavadas com PBS e metanol, coradas com cristal violeta 1% e coradas com ácido acético glacial (33%) para nova leitura a 570nm. A eficácia da remoção do biofilme pelos sanitizantes foi comparada pelo índice de formação de biofilme (IFB). As imagens do aço inoxidável após tratamento com sanitizante foram feitas através de Microscopia Eletrônica de Varredura (MEV) e Microscopia Confocal, para visualizar a persistência dos biofilmes. Os valores de CIM (diluição 1:2) mostraram que OLE não teve atividade bactericida. No método DDA, L. monocytogenes, foi resistente à OLE, enquanto E. coli e S. aureus apresentaram resistência intermediária. Os sanitizantes comerciais apresentaram boa atividade bactericida nos ensaios de CIM e DDA, sendo que as associações de OLE aos sanitizantes comerciais aumentaram o efeito germicida. Nos ensaios com biofilmes em monoespécie, somente os sanitizantes comerciais, isolados ou associados com OLE, foram eficazes de reduzir o valor de BFI em microplaca de poliestireno. Em biofilmes multiespécie, OLE apresentou efeito antimicrobiano, sobretudo sobre a associação de L. monocytogenes + E. coli + S. aureus (redução: 91,49%). Nenhum dos compostos avaliados foi capaz de inativar completamente os biofilmes nas superfícies de aço inoxidável, uma vez que células viáveis foram observadas após os tratamentos com os sanitizantes, indicando persistência dos biofilmes. Os resultados indicam que a oleuropeína apresentou potencial para incrementar o efeito bactericida de sanitizantes comerciais para eliminação de biofilmes em superfícies inertes, sendo necessários estudos para compreender os mecanismos de ação dessas combinações. / This study evaluated the efficiency of oleuropein (OLE) (a phenolic compound extracted from Oliveira leaves) alone or in association with commercial sanitizers peracetic acid 2% (PPA), sodium hypochlorite 2% (SH), hydrogen peroxide 3% (HP), chlorhexidine digluconate 2% (CD), benzalkonium chloride 1% (BC) and iodophor 2% (IO), for inactivation of suspended cells and monospecies or multispecies biofilms formed on stainless steel surfaces or microplate polystyrene by Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), all classified as strong producers of biofilms. The isolates were grown in TSB (trypticase soy broth), incubated (37°C/24 h) and adjusted to ~108 cells/mL (0.5 McFarland scale). For the bacterial suspensions, resistance to sanitizers was determined by Minimum Inhibitory Concentration (MIC) in tubes and by Agar Disk Diffusion (ADD), in which the bacteria were plated on TSA agar containing 6mm disks of filter paper soaked in sanitizers. After incubation, measurement of the inhibition halos was done using a caliper rule. For the sanitizer resistance assays with biofilms, polystyrene 96-well microplates were prepared for incubation-fixing of biofilms and read in an ELISA spectrophotometer (600 nm). After, the plates were washed with phosphate buffered saline (PBS, pH 7.4) and the sanitizers were placed for 1 minute. After neutralization with sodium thiosulfate (5 minutes), the plates were washed with PBS and methanol, stained with 1% crystal violet and stained with glacial acetic acid (33%) for a new reading at 570 nm. The effectiveness of biofilm removal by each sanitizer was compared using a Biofilm Formation Index (IFB). The images from stainless steel coupons after treatments with sanitizers were obtained by Scanning Electron Microscopy (SEM) and Confocal Microscopy, to view the persistence of biofilms. MIC values (dilution 1: 2) showed that OLE had no bactericidal activity. In the ADD assays, L. monocytogenes was resistant to OLE, while E. coli and S. aureus showed intermediate resistance. Commercial sanitizers showed good bactericidal activity in both CIM and DDA assays, and the associations of OLE with commercial sanitizers increased their germicidal effect. In the monospecies biofilm assays, only commercial sanitizers, isolated or associated with OLE, were effective for reducing the BFI values in polystyrene microplates. In multispecies biofilms, OLE had an antimicrobial effect, especially on the association of L. monocytogenes + E. coli and S. aureus (reduction: 91.49%). None of the compounds evaluated was able to completely inactivate the biofilms on the stainless steel surfaces, since viable cells of bacteria were observed after treatment with sanitizers, indicating persistence of biofilms. The results indicate that oleuropein has the potential to enhance the bactericidal effect of commercial sanitizers to eliminate biofilms on inert surfaces. Further studies are needed to understand the mechanisms of action of these combinations.