Dry surface biofilm of Salmonella and Cronobacter sakazakii: a real concern for the low moisture food industry.

In this study, Salmonella Typhimurium dry surface biofilm (DSB) formation was investigated in comparison with wet surface biofilm (WSB) development. Confocal laser scanning microscopic analysis revealed a prominent green cell signal during WSB formation, whereas a red signal predominated during DSB formation. Electron microscopy was also used to compare the features of DSB and WSB. Overall, WSB was unevenly scattered over the surface, whereas DSB was evenly dispersed. In contrast to WSB cells, which have a distinct plasma membrane and outer membrane layer, DSB cells are contained in large capsules and compressed. Next, microbiome single-cell transcriptomics was used to investigate the functional heterogeneity of the Salmonella DSB microbiome, with nine clusters successfully identified. Although over 60% of the dried cells were metabolically inactive, the rest of the Salmonella cells still demonstrated specific antioxidative and virulence capabilities, suggesting a possible concern for low-moisture food (LMF) safety. Finally, because sanitization in LMF industries must be conducted without water, a list of 39 flavonoids was tested for their combined effect with 70% isopropyl alcohol (IPA) against DSB, and morin induced the greatest reduction in the green:red ratio from 3.67 to 0.43. Significantly higher reductions of Salmonella viability in DSB were achieved by 10-, 100-, 1,000-, and 10,000-µg/mL morin (1.69 ± 0.25, 3.21 ± 0.23, 4.32 ± 0.24, and 5.18 ± 0.16 log CFU/sample reductions) than 70% IPA alone (1.55 ± 0.20 log CFU/sample reduction) (P < 0.05), indicating the potential to be formulated as a dry sanitizer for the LMF industry.IMPORTANCEDSB growth of foodborne pathogens in LMF processing environments is associated with food safety, financial loss, and compromised consumer trust. This work is the first comprehensive examination of the characteristics of Salmonella DSB while exploring its underlying survival mechanisms. Furthermore, morin dissolved in 70% IPA was proposed as an efficient dry sanitizer against DSB to provide insights into biofilm control during LMF processing.

Development of Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Pseudomonas aeruginosa multi-species in vitro dry surface biofilm models: Insights into resilience and persistence in low-moisture environments

Bacterial biofilm formation in low moisture food processing (LMF) plants is related to matters of food safety, production efficiency, economic loss, and reduced consumer trust. Dry surfaces may appear dry to the naked eye, however, it is common to find a coverage of thin liquid films and microdroplets, known as microscopic surface wetness (MSW). The MSW may favor dry surface biofilm (DSB) formation. DSB formation is similar in other industries, it occurs through the processes of adhesion, production of extracellular polymeric substances, development of microcolonies and maturation, it is mediated by a quorum sensing (QS) system and is followed by dispersal, leading to disaggregation. Species that survive on dry surfaces develop tolerance to different stresses. DSB are recalcitrant and contribute to higher resistance to sanitation, becoming potential sources of contamination, related to the spoilage of processed products and foodborne disease outbreaks. In LMF industries, sanitization is performed using physical methods without the presence of water. Although alternative dry sanitizing methods can be efficiently used, additional studies are still required to develop and assess the effect of emerging technologies, and to propose possible combinations with traditional methods to enhance their effects on the sanitization process. Overall, more information about the different technologies can help to find the most appropriate method/s, contributing to the development of new sanitization protocols. Thus, this review aimed to identify the main characteristics and challenges of biofilm management in low moisture food industries, and summarizes the mechanisms of action of different dry sanitizing methods (alcohol, hot air, UV-C light, pulsed light, gaseous ozone, and cold plasma) and their effects on microbial metabolism.

Biofilms on dry hospital surfaces can enhance the persistence of microorganisms on dry harsh clinical surfaces and can potentially act as reservoirs of infectious agents on contaminated surfaces. This study was conducted to quantify the transfer of viable Staphylococcus aureus cells from dry biofilms through touching and to investigate the impact of nutrient and moisture deprivation on virulence levels in S. aureus. Dry biofilms of S. aureus ATCC 25923 and a defective biofilm-forming ability mutant, S. aureus 1132 were formed in 24 well plates under optimised conditions mimicking dry biofilm formation on clinical surfaces. Microbial cell transfer was induced through the touching of the dry biofilms, which were quantified on nutrient agar. To investigate the impact of nutrient and moisture deprivation on virulence levels, dry and standard biofilms as well as planktonic cells of S. aureus ATCC 25923 were inoculated into Galleria mellonella and their kill rates compared. Results of this study showed that viable cells from dry biofilms of S. aureus ATCC 25923 were significantly more virulent and readily transferrable from dry biofilms through a touch test, therefore representing a greater risk of infection. The biofilm-forming capability of S. aureus strains had no significant impact on their transferability with more cells transferring when biofilm surfaces were wet. These findings indicate that dry biofilms on hospital surfaces may serve as a reservoir for the dissemination of pathogenic microorganisms in hospitals, thus highlighting the importance of regular cleaning and adequate disinfection of hospital surfaces.

Enterococcus spp. ability to form a dry surface biofilm: a route to persistence on environmental surfaces.

Biofilms on instruments and environmental surfaces: Do they interfere with instrument reprocessing and surface disinfection? Review of the literature

BackgroundBacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS).MethodsS. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB.ResultsOverall, a minimum of six mean log10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS.ConclusionA rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Dry surface biofilms (DSB) harbouring pathogens are widespread in healthcare settings, are difficult to detect and are resistant to cleaning and disinfection interventions. Here, we describe a practical test protocol to palliate the lack of standard efficacy test methods for DSB. Staphylococcus aureus DSB were produced over a 12-day period, grown with or without the presence of organic matter, and their composition and viability were evaluated. Disinfectant treatment was conducted with a modified ASTM2967-15 test and reduction in viability, transferability and biofilm regrowth post-treatment weremeasured. Dry surface biofilms produced over a 12-day period had a similar carbohydrates, proteins and DNA content, regardless of the presence or absence of organic matter. The combination of sodium hypochlorite (1000ppm) and a microfiber cloth was only effective against DSB in the absence of organic load. With the increasing concerns of the uncontrolled presence of DSB in healthcare settings, the development of effective intervention model in the presence of organic load is appropriate for the testing of biocidal products, while the use of three parameters, log10 reduction, transferability and regrowth, provides an accurate and practical measurement of product efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The widespread presence of biofilms on dry surfaces in healthcare settings has been recently documented. These dry surface biofilms (DSB) present an unprecedented challenge to cleaning and disinfection processes. Here, we describe a practical efficacy protocol based on an invitro Staphylococcus aureus DSB model. The protocol measures reduction in viability, transferability and biofilm regrowth post-treatment to provide altogether a practical assessment of product efficacy against dry surface biofilms.

Impact of Wiping Materials on the Elimination from Surfaces of Dry Surface Biofilm of Bacteria of Food Safety Concern.

Opposing regulation of Gig2 by RecA and GlrK controls redox-linked desiccation tolerance in Cronobacter sakazakii.

In food preparation and manufacturing environments, surfaces contaminated with Salmonella can lead to outbreaks of Salmonellosis. We hypothesise that Salmonella resides on dry surfaces in a biofilm form leading to potential environmental persistence and transfer following contact. This is the first study reporting that Salmonella Typhimurium can form dry surface biofilm (DSB). Six disinfectants commonly used in the food industry were evaluated for their efficacy against the DSB. The two most efficacious formulations reduced bacterial viability in DSB by &gt;99.99% when combined with mechanical removal (5 sec wiping; 300 g weight). Five out of six formulations significantly reduced bacterial transfer when combined with wiping. Complete eradication of Salmonella Typhimurium DSB was challenging, and mechanical removal was essential to produce a &gt;99.99% reduction in bacterial viability within DSB. This study highlights a potential mode of survival of Salmonella Typhimurium on food‐contact surfaces and DSB challenges for disinfection.

Candida auris is an enigmatic fungal pathogen, recently elevated as a critical priority group pathogen by the World Health Organisation, linked with its ability to cause outbreaks within nosocomial care units, facilitated through environmental persistence. We investigated the susceptibility of phenotypically distinct C. auris isolates to sodium hypochlorite (NaOCl), and evaluated the role of biofilms in surviving disinfection using a dry-surface biofilm (DSB) model and transcriptomic profiling. Planktonic cells were tested for susceptibility to NaOCl, with biofilm formation using the 12-day DSB model, assessed using viable counts, biomass assays and microscopy. Disinfection efficacy was assessed using clinical protocols of 500-1,000 ppm for 1-5 min. RNA sequencing was performed on untreated DSBs in comparison to planktonic cells. Isolates were found to be susceptible planktonically, but grew NaOCl-tolerant biofilms, with only 2-4 log10 reductions in viable cells observed at highest concentrations. Transcriptomics identified DSB upregulation of ABC transporters and iron acquisition pathways relative to planktonic cells. Our findings optimized a DSB protocol in which C. auris can mediate tolerance to NaOCl disinfection, suggesting a lifestyle through which this problematic yeast can environmentally persist. Mechanistically, it has been shown for the first time that upregulation of small-molecule and iron transport pathways are potential facilitators of environmental survival.

Dry surface biofilms (DSB) have been found to persist on hospital beds and pillows. This study aimed to investigate whether cotton bedsheets prevent transmission of DSB-related pathogens to patients. Staphylococcus aureus DSB was cultured on polycarbonate coupons. DSB transmission was simulated via 150 or 250 threads/in2 cotton bedsheets directly onto surfaces, or through cotton sheets onto hands and then onto surfaces, both before and after neutral detergent treatment by CFU counts. qPCR and SEM were used to evaluate bacterial biofilm contamination on hospital mattresses and pillow covers. Lab simulations: Bacterial transfer counts (CFU) differed significantly across all experimental conditions. Specifically, bacterial transmission was significantly higher for cotton sheets with a thread count of 150 threads/in2 compared to those with 250 threads/in2 (p < 0.001). Additionally, transmission was significantly greater for wet sheets (post-detergent treatment) than for dry sheets (p < 0.001), and significantly higher for direct touch than for contact through the sheets (p < 0.001). CLINICAL SAMPLES: qPCR detected 1.1×105±6.8×104 bacteria/cm2 on mattress covers and 7.5±3.7×104 bacteria/cm2 on pillow covers; SEM directly visualized DSB embedded in surface pits of mattress covers, confirming real-world clinical contamination. Cotton bedsheets show high transmissibility for DSB, especially when wet. DSB can persist on mattresses and could be transmitted through bedsheets, highlighting the need for rigorous cleaning, frequent sheet changes, and higher-thread-count sheets to reduce pathogen transmission and hospital-acquired infections.

Transfer of dry surface biofilm in the healthcare environment: the role of healthcare workers' hands as vehicles