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Introduction: Shiga toxin-producing Escherichia coli (STEC) are prominent foodborne pathogens known for their ability to form robust biofilms on food contact surfaces, contributing to contamination risks in food processing environments. The objective of the present research investigation was to evaluate the susceptibility of seven STEC strains to different sanitizers in planktonic conditions. Further, the efficacy of sanitizers against STEC single and multispecies biofilms (with lactic acid bacteria and spoilage bacteria) under varying temperatures and surface conditions will be assessed. Methods: The first study examined their responses in planktonic form, single-species, and multispecies biofilms (T1: Carnobacterium piscicola + Lactobacillus bulgaricus, T2: Comamonas koreensis + Raoultella terrigena, T3: Pseudomonas aeruginosa + C. koreensis) cultured in 96-well microplates at 10°C and 25°C, using sanitizers such as Quats, Shypo, SHyd, HyP, and PAA. The second study assessed biocide effectiveness against mature biofilms on polyurethane (TPU) or stainless steel (SS) surfaces at 10°C and 25°C under 60-90% RH, following a standardized cleaning regimen involving detergent application, optional scrubbing, rinsing, and subsequent biocide treatment. Results: The first study found that planktonic cells and single-species biofilms in microplates were highly susceptible to sanitizers, particularly Lactobacillus and Carnobacterium. O157 strains showed resistance to sodium hypochlorite at 25°C (P < 0.05), with PAA demonstrating the highest efficacy in single-species biofilms, followed by Quats, HyP, SHyd, and Shypo (P < 0.05). Multispecies biofilms in microplates were more resistant, especially to SHyd, followed by Quats, HyP, Shypo, and BioDestroy®. In the second study, a triple interaction between mechanical removal, biofilm age, and sanitizer significantly influenced the reduction of O157 on TPU and SS surfaces regardless of the cleaning condition and temperature at 6 and

The elaboration of guidelines for the industry to establish minimum concentration to prevent cross-contamination during washing practices based on operational limits is the core of the recommended criteria for the use of sanitizers. Several studies have evidenced that sanitizers reduced the levels of foodborne pathogens. However, they might lead to the progress into a viable but non-culturable (VBNC) state of the cells. This evidence has raised concerns regarding the effectiveness of the recommended washing practices for the inactivation of microbial cells present in the process wash water (PWW). The present study evaluated if the most commonly used sanitizers, including sodium hypochlorite (chlorine), peroxyacetic acid (PAA) and chlorine dioxide (ClO) at established operational limits induced the VBNC stage of Listeria monocytogenes and Escherichia coli O157:H7. Prevention of cross-contamination was examined in four different types of PWW from washing shredded lettuce and cabbage, diced onions, and baby spinach under simulated commercial conditions of high organic matter and 1 min contact time. The results obtained for chlorine showed that recommended operational limits (20–25 mg/L free chlorine) were effective in inactivating L. monocytogenes and E. coli O157:H7 in the different PWWs. However, the operational limits established for PAA (80 mg/L) and ClO (3 mg/L) reduced the levels of culturable pathogenic bacteria but induced the VBNC state of the remaining cells. Consequently, the operational limits for chlorine are satisfactory to inactivate foodborne pathogens present in PWW and prevent cross-contamination but higher concentrations or longer contact times should be needed for PAA and ClO to reduce the likelihood of the induction of VBNC bacteria cells, as it represents a hazard.

Understanding the concept of biofilms and aspects of its structure and composition is of fundamental importance for the development of new strategies for control and eradication of these micro-organisms in a sessile state. The objective of this work was to verify the ability of adhesion of Escherichia coli ATCC 25922 and of wild lineage in biofilm formation in ethylene terephthalate surface (PET). In addition, to describe and evaluate the efficiency of sanitizers quaternary ammonium, lactic acid, chlorhexidine digluconate and sodium hypochlorite on reducing microbial cells state adhered and planktonic. To analyze the efficiency of sanitizers in planktonic cells we used the method of disk diffusion and Minimum Inhibitory Concentration (MIC) and for adhered cells, only MIC testing. The results showed biofilm formation by standard strain ATCC 25922 and isolated wild from clinical origin, from the time of 4h. The sodium hypochlorite did not show effectiveness in reducing population of planktonic and adhered cells in relation to the other sanitizers tested. The biofilm with wild isolate was sensitive to all concentrations of quaternary ammonium and inhibited the growth up to a concentration of 0,002 mg/L lactic acid, while for chlorhexidine digluconate it showed growth in all concentrations. E. coli ATCC 25922 was sensitive only to the lower concentrations of quaternary ammonium, lactic acid and chlorhexidine digluconate., A compreensão do conceito de biofilmes e de aspectos inerentes a sua estrutura e composição é de fundamental importância para o desenvolvimento de novas estratégias de controle e erradicação de micro-organismos em estado séssil. Assim, o objetivo com este trabalho foi verificar a capacidade de adesão de Escherichia coli ATCC 25922 e de linhagem selvagem na formação de biofilmes em PET. Além disso, descrever e avaliar a eficiência dos sanificantes quaternário de amônia, ácido lático, digluconato de clorexidina e hipoclorito de sódio na redução microbiana das células em estado aderido e planctônico. Para análise da eficiência dos sanificantes em células planctônicas utilizou-se o método de disco-difusão e a Concentração Inibitória Mínima (CIM) e, para as células aderidas, o teste de CIM. Os resultados obtidos indicaram formação de biofilme pela linhagem padrão ATCC 25922 e isolado selvagem de origem clínica a partir do tempo de 4h. O hipoclorito de sódio não apresentou eficiência na redução populacional de células aderidas e planctônicas em relação aos demais sanificantes testados. O biofilme com isolado selvagem foi sensível a todas as concentrações de quaternário de amônia e inibiu o crescimento até a concentração de 0,002 mg/L no ácido lático, enquanto para o digluconato de clorexidina apresentou crescimento em todas as concentrações. A E. coli ATCC 25922 foi sensível somente às menores concentrações de quaternário de amônia, ácido lático e digluconato de clorexidina.

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log10 cfu/well) and that they contained 1-2 log10 cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log10 cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log10 more L. plantarum than L. monocytogenes cells (total count, 9 log10 cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log10 cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log10 cfu/well inactivation after exposure for 15 min to 100 µg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log10 cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log10 cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms. Research Highlights ¿Listeria monocytogenes and Lactobacillus plantarum form mixed species biofilms. ¿Single species biofilms are more resistant than planktonic cells. ¿Mixed species bi

Chlorine dioxide (ClO2) has been postulated as an alternative to sodium hypochlorite (NaClO) for fresh-cut produce sanitization to avoid risks associated with chlorination by-products. Experiments were performed to determine the prevention of cross-contamination of fresh-cut lettuce by Escherichia coli using chlorine dioxide (3 mg/L) or sodium hypochlorite (100 mg/L) as sanitation agents. The efficacy of these sanitation solutions was evaluated simulating as much as possible the conditions of a fresh-cut processing line. Thus, to evaluate the potential risk of cross-contamination during pre-washing, inoculated fresh-cut lettuce was pre-washed and after that non-inoculated lettuce was then pre-washed in the same water. After this pre-washing, non-inoculated lettuce was cross-contaminated, changing from 0 to 3.4 log units of E. coli cells. During washing with sanitizers, none of the tested sanitation agents significantly reduced E. coli counts in both inoculated and cross-contaminated lettuce. These results suggest that when cross-contamination occurs, even if the event is recent, subsequent sanitation steps are inefficient for inactivating E. coli cells on the vegetable tissue. However, chlorine dioxide and sodium hypochlorite solutions were able to inactivate most E. coli cells that passed from inoculated product to wash water. Therefore, they might be able to avoid cross-contamination between clean and contaminated product during the washing step. Scanning electron microscopy micrographs indicated that bacterial cells were mainly located in clusters or tissue stomata where they might be protected, which explains the low efficacy of sodium hypochlorite and chlorine dioxide solutions observed in this study.

Five commercial water sanitizer generators were tested, by the U.S. Army Natick Soldier Research, Development and Engineering Center (NSRDEC), for bactericidal efficacy of their sanitizer on stainless steel surfaces. Sanitizers generated were Acid Electrolyzed Water (AEW), hydrogen peroxide, mixed oxidants, and ozone. Manually produced chlorine dioxide was also tested. Surfaces were inoculated with three strain cocktails of S. aureus and E.coli, dried and then immersed in a solution of each sanitizer. Efficacy was determined by achieving a 5 log reduction within five minutes. AEW concentrations of 80 -100 ppm free available chlorine (FAC) and chlorine dioxide at 100 ppm were effective with both adherent and non-adherent cells. Such generators would provide the Army Mobile Kitchen Trailer (MKT), the Assault Kitchen (AK) and the Containerized Kitchen (CK) with the means to produce an effective sanitizing solution in the field without re-supply. They would also eliminate the logistical problems and hazards entailed with the purchase and shipment of chemicals throughout the military supply system. The generators will require electrical power and a potable water supply.

It is well known that fresh-cut processors usually rely on wash water sanitizers to reduce microbial counts in order to maintain quality and extend shelf-life of the end product. Water is a useful tool for reducing potential contamination but it can also transfer pathogenic microorganisms. Washing with sanitizers is important in fresh-cut produce hygiene, particularly removing soil and debris, but especially in water disinfection to avoid cross-contamination between clean and contaminated product. Most of the sanitizing solutions induce higher microbial reduction after washing when compared to water washing, but after storage, epiphytic microorganisms grow rapidly, reaching similar levels. In fact, despite the general idea that sanitizers are used to reduce the microbial population on the produce, their main effect is maintaining the microbial quality of the water. The use of potable water instead of water containing chemical disinfection agents for washing fresh-cut vegetables is being advocated in some European countries. However, the problems of using an inadequate sanitizer or even none are considered in this manuscript. The need for a standardized approach to evaluate and compare the efficiency of sanitizing agents is also presented. Most new alternative techniques accentuate the problems with chlorine suggesting that the industry should move away from this traditional disinfection agent. However, the use of chlorine based sanitizers are presented as belonging to the most effective and efficient sanitizers when adequate doses are used. In this review improvements in water disinfection and sanitation strategies, including a shower pre-washing step and a final rinse of the produce, are suggested.

The industry of fresh-cut fruits and vegetables is constantly growing due to consumers demand. New techniques for maintaining quality and inhibiting undesired microbial growth are demanded in all the steps of the production and distribution chain. In this review, we summarize some of the new processing and preservation techniques that are available in the fresh-cut industry. The combination of sanitizers with other intervention methods is discussed. The use of ultraviolet-C, modified-atmospheres, heat shocks and ozone treatments, alone or in different combinations have proved useful in controlling microbial growth and maintaining quality during storage of fresh-cut produce. In addition, combinations of physical and chemical treatments are also reviewed. The use of acidic or alkaline electrolyzed water (AcEW), chlorine dioxide, power ultrasound and bacteriocins and the potential applications to the fresh-cut products industry to control human microbial pathogens are presented.

The use of ozonated water as a sanitizer to extend the shelf life of fresh-cut lettuce and the effect on the antioxidant constituents (polyphenols and vitamin C) were investigated. Fresh-cut iceberg lettuce (Lactuca sativa L.) was washed at 4 °C using three different ozonated water dips [10, 20, and 10 activated by ultraviolet C (UV-C) light mg L-1 min total ozone dose], and the dips were compared with water and chlorine rinses. Treated lettuce was packaged in air or active modified atmosphere packaging (MAP) (4 kPa of O2 + 12 kPa of CO2 balanced with N2) and stored for 13 days at 4 °C. Despite its strong oxidizing activity, ozonated water did not stimulate the respiratory activity of fresh-cut lettuce. Moreover, ozonated water maintained the initial visual appearance of fresh-cut lettuce and controlled browning during storage in air. Initially, ozonated water and chlorine reduced the total mesophilic population by 1.6 and 2.1 log, respectively, when compared with water. Active MAP was effective in controlling total microbial growth, achieving 2.0 log reduction in relation to samples stored in air at the end of storage. On the other hand, active MAP caused a 2.0−3.5 reduction of coliforms on sanitized samples compared with water-washed samples. The most efficient treatments were ozone 20 and ozone 10 activated by UV-C, which were as effective as chlorine. Changes in individual phenolic compounds were independent of the washing treatments. In air, chlorogenic and isochlorogenic acid contents increased noticeably after 13 days while monocaffeoyltartaric and dicaffeoyltartaric acids remained unchanged. MAP effectively suppressed accumulation of caffeoylquinic derivatives, whereas caffeoyltartaric derivatives decreased during MAP storage to reach similar levels. The content of vitamin C (ascorbic acid and dehydroascorbic acid) decreased during storage, particularly under MAP. Ozonated water could be an alternative sanitizer to chlorine for fresh-cut lettuce due to good

Three studies regarding Escherichia coli O157:H7 in apple cider were conducted. The objectives were: to evaluate the effectiveness of wash and sanitizers for removing E. coli O157:H7 from apples; to survey cider producer practices; and to determine the efficacy of ultraviolet light for reducing E. coli O157:H7 in cider. Apples with a five-strain acid resistant mixture of E. coli O157:H7 were treated with 200 ppm hypochlorite, a phosphoric acid-based fruit wash, 5% acetic acid, 5% acetic acid followed by 3% hydrogen peroxide, a peroxyacetic acid-based solution, and distilled water. The water wash caused insignificant reductions. All other treatments caused significant reductions. Acetic acid and peroxyacetic acid were the most effective with reductions of 3.1 and 2.6 logs, respectively. The survey determined that most producers are small, seasonal operations. Most use sound orchard management practices, clean and sanitize daily, sort and wash apples, use refrigeration, and try to prevent contamination. However, some use drop and damaged apples. Few use chemical sanitizers on apples, preservatives, pasteurize cider, or have HACCP programs. Cider inoculated with the same mixture of E. coli O157:H7 was processed using a thin- film ultraviolet disinfection unit operating at 254 nm. Dosages ranged from 9,402 to 61,005 æW- sec/cm2. Treatment significantly reduced E. coli O157:H7 (pó 0.0001) with a mean reduction of 3.81 log CFU/ml. Reduction was also affected by the level of background microflora in cider. Results indicate that ultraviolet light can reduce this pathogen in cider. However, additional reduction measures are necessary to achieve the required 5 log reduction.

This NebGuide provides cleaning, sanitizing, mechanical, environmental and other procedures to follow to assure a low bacterial count in milk. Bacteria are tiny, rapidly reproducing microorganisms that are too small to be seen without a high power microscope. Their very tiny size makes it very difficult to eliminate them from dairy equipment. Because bacterial growth in milk may cause spoilage and possibly human illnesses, a low bacterial count is one of the best indicators of top quality milk. Milk will almost always be of top quality if properly harvested from healthy, clean cows with clean equipment, cooled quickly and kept cold.