Stainless Steel Coupons Research Articles

High voltage atmospheric cold plasma technology as a food safety intervention for decontamination of cutting tools during ready-to-eat poultry meat slicing

This study aimed to build a prototype that integrates a cold plasma capability into a manual meat slicer while it is in operation to minimize cross-contamination from the circular cutting blade. A high voltage atmospheric cold plasma (HVACP) generator was attached to a meat slicer with a rotating 22.5-cm diameter stainless steel slicing blade. The meat slicer with the installed cold plasma generator was contained within a sealed, clear acrylic box. Listeria innocua (LI) was selected as the test organism, and initial testing was performed on stainless-steel coupons in room air at a relative humidity ranging from 30 to 90% for treatment times up to 240 s. Results found that 240 s and 90% RH showed a 3.0-log reduction of LI. These conditions were then replicated using “dirty” stainless-steel knife while rotating. The results showed only a 0.5-log LI reduction. The gas was changed from air to MA65 gas blend (65% O2, 30% CO2 and 5% N2) and then a 1.91 log LI reduction was achieved on the “dirty” knife. Industrial relevanceDue to the rapid processing speeds required in the food industry, it is difficult to assure the complete cleanliness of the tools and surfaces used at all times, and then, operations like the cutting /slicing step are still one of the critical points for cross-contamination during meat processing. High voltage atmospheric cold plasma (HVACP) is a practical intervention that has the potential to deliver a continuous, in-situ decontamination of the knife during operation to mitigate the risk of cross-contamination. The developed prototype, as shown in this study, can deliver beneficial results in short treatment time and with minimal inputs of air and electricity, without heating effects, minimizing the use of water, or the use of sanitizers.

The physicochemical properties of fruit powders and their residence time on stainless steel surfaces are associated with their ease of removal by brushing

Dry cleaning activities such as brushing, scraping, and vacuuming have been widely used in low-moisture food production. However, the impact that product compositional differences and time between cleaning cycles have on the ease of food particulate removal is unclear. In our study, fruit powders were deposited on stainless-steel coupons, then removed using a hygienic brush. The residence time was defined as the duration between fruit powder deposition and attempted removal. Longer residence time resulted in elevated water activity (aw), which decreased the degree of removal for most fruit powders other than pumpkin powder and apple powder (sieve size: 2.36 mm). For instance, the aw of peach powder increased from 0.59 ± 0.01 on day 1 to 0.64 ± 0.01 on day 7, and residual powder after cleaning increased from 86.12 ± 1.73% to 93.26 ± 2.12% from day 1 to day 7. Peach powder and apple/pear/plum powder were the most difficult to remove, with average residual powder of 92.79 ± 4.97% and 58.41 ± 11.41%, respectively. Fruit powders that were more difficult to remove also exhibited higher cohesion and adhesion. For example, the cohesion and adhesion of peach powder at 76% relative humidity for 3 days were 4479.53 ± 681.86 N/m2 and 1347.53 ± 439.15 N/m2, respectively. By contrast, the cohesion and adhesion of pumpkin powder at 76% relative humidity for 3 days were only 102.34 ± 93.80 N/m2 and 90.64 ± 36.52 N/m2, respectively. These results suggested that aw could significantly influence the brush cleaning outcome in a manner that was dependent on the identity of the fruit powder.

Exploring communication signals inside the microbial community of a Listeria monocytogenes-carrying biofilm contamination site

In nature, bacterial pathogens like L. monocytogenes, live in nature associated with other microbial species in spatially-structured communities called biofilms. In the food industry, biofilms contribute to the survival and persistence of L. monocytogenes within processing facilities, thereby enhancing its risk of cross-contaminating food products. The challenge of combating biofilms has triggered the search for new antibiofilm strategies including devising ways to interfere with cell communication mechanisms (quorum-sensing) that are known to be involved with biofilm development regulation. The aim of this study was to explore cell communication signals in a L. monocytogenes-carrying microbial community isolated from a meat processing plant (location No. 96) in order to elucidate the ecological interactions that could serve as a starting point for the development of new antibiofilm strategies. Quorum quenching (QQ) and quorum sensing (QS) activities were screened among 31 bacterial strains isolated from location No. 96. Whereas no QQ activity was detected against short-chain lactone N-hexanoyl-DL-homoserine lactone (C6-HSL), it was detected against N-dodecanoyl-DL-homoserine lactone (C12-HSL) in 7 isolates (23%), particularly in Pseudomonas monteilli, Rhodococcus sp. and Rhodococcus erythropolis. QS activity assays detected HC4, C4, C6, OC6, HC10 and C16 in all the extracts, being C4, C6 and OC6 with predominantly produced by Pseudomonas monteilli, Pseudomonas gesardii, Psychrobacter maritimus and Paracoccus sp. High production levels of C16-HSL by Paracoccus sp. and the role of this long-chain lactone as a self-inhibitor of cell aggregation led us to carry out further studies focused on the effects of a Paracoccus lactone extract (PLE) against the biofilm formation by L. monocytogenes. A quantitative microscopic analysis demonstrated a significant decrease (p < 0.05) in the area occupied by biofilms formed on stainless steel (SS) coupons by different strains of L. monocytogenes in the presence of PLEs. Conversely, no significant differences were observed in the total number of viable adhered cells on SS coupons with or without PLE. The observed effect was partially reproduced by the addition of pure C16-HSL to 24 h-biofilms of L. monocytogenes L1.96. These results demonstrate that the observed effects can be attributed, at least partially, to the HSLs contained in the PLE. Overall, the present results highlight how interspecies communication within a biofilm can open up new insights for the development of new ways to combat biofilm.

Improved assay for detecting SARS-CoV-2 from nonporous hospital surfaces using surrogate human coronavirus OC43

Background: Understanding SARS-CoV-2 persistence on surfaces can help inform transmission risk from surfaces in healthcare and community settings. A sensitive viral infectivity assay is crucial for the detection of infective virus in environmental investigations. The conventional cell culture-based infectivity assay is limited by the time dependence, subjectivity, and insensitivity of cytopathic effect (CPE) scoring. We validated an integrated cell-culture and reverse-transcription quantitative RT-PCR method (cc-RT-qPCR) to improve SARS-CoV-2 detection and reduce detection time. We compared cc-RT-qPCR with CPE-scored cell culture to evaluate assay sensitivity of recovered virus from stainless-steel coupons simulating nonporous healthcare surfaces. Method: Human β-coronavirus OC43, a model strain for SARS-CoV-2, was propagated on HRT-18G cells in growth medium at 33°C in a 5% CO2 incubator. The OC43 infectivity was determined by cell culture with a 10-fold dilution series of viral samples in 96-well plates, and incubation for 7 days at 33°C to confirm CPE. Plates were CPE-scored and TCID50 was calculated using the Reed-Muench method. For the cc-RT-qPCR assay, CPE-negative wells were interrogated for viral intracellular replication using RT-PCR; infectivity was based on a titer increase of ≥ 2 logs 7 days after inoculation using RT-qPCR. CPE-positive or replicative virus-harboring cells were enumerated to determine TCID50. The sensitivity of both CPE-scored cell culture and cc-RT-qPCR assays were evaluated by inoculating 105 TCID50/mL OC43 in infection media and artificial saliva matrices onto coupons and dried in an environmental chamber at 26°C and 57% relative humidity for 6 hours. Viral eluates from coupons served as test samples. Results: Low-titer infectious OC43 (0.75 log10) was detected by both methods 7 days after incubation; however, infectivity confirmation required 4 and 6 days after incubation, respectively, for cc-RT-qPCR and CPE-scored cell culture methods. When cells were inoculated with OC43 at titer range 1.75–4.75 log10, CPE presented at 4–5 days after incubation, while viral replication was already detected at 3 days after incubation via RT-PCR. Upon virus titration, cc-RT-qPCR demonstrated greater sensitivity, detecting up to 1 log10 higher of infectious OC43 than cell culture alone at 0 and 6 hours (P ≤ .05) dried in infection medium and 0 hours (P ≤ .05) in saliva. Conclusions: Our data demonstrated greater sensitivity and shorter times to detect viral replication by cc-RT-qPCR, minimizing potential for false-negative results with cell culture alone. This sensitive assay may provide investigators with quicker results for informing infection control practices to reduce risk of transmission from deposited bodily fluids on surfaces, eg, coughing and sneezing.Funding: NoneDisclosures: None

Effect of chlorine sanitizer on metabolic responses of Escherichia coli biofilms “big six” during cross-contamination from abiotic surface to sponge cake

The effect of chlorine on Escherichia coli biofilm O157:H7 are well established; however, the effect on biofilm adhesion to food as well as the six emerging E. coli serotypes (“big six”) have not been fully understood. Chlorine sanitization with 1-min 100 mg/L was applied against seven pathogenic E. coli (O111, O121:H19, O45:H2, O26:H11, O103:H11, O145, and O157:H7) biofilms on high-density polyethylene (HDPE) and stainless steel (SS) coupons, respectively. Using sponge cake as a food model, the adhesion behavior was evaluated by comparison of bacteria transfer rate before and after treatment. Besides, the metabolic profiles of biofilms were analyzed by nuclear magnetic resonance (NMR) spectrometer. A significant decrease in transfer rate (79% decline on SS and 33% decline on HDPE) was recorded as well as the distinctive pattern between SS and HDPE coupons was also noticed, with a low population (6–7 log CFU/coupon) attached and low survivals (0–3 log CFU/coupon) upon chlorine on SS, while high population (7–8 log CFU/coupon) attached and high survivals (5–7 log CFU/coupon) on HDPE. Moreover, O121:H19 and O26:H11 demonstrated the highest resistance to chlorine with the least metabolic status and pathways affected. O103:H11, O145, and O111 followed similar metabolic patterns on both surfaces. Distinct metabolic patterns were found in O45:H2 and O157:H7, where the former had more affected metabolic status and pathways on SS but less on HDPE, whereas the latter showed an opposite trend. Overall, a potential contamination source of STEC infection in flour products was demonstrated and metabolic changes induced by chlorine were revealed by NMR-based metabolomics, which provides insights to avoid “big six” biofilms contamination in food.

Direct microbial electron uptake as a mechanism for stainless steel corrosion in aerobic environments

Shewanella oneidensis MR-1 is an attractive model microbe for elucidating the biofilm-metal interactions that contribute to the billions of dollars in corrosion damage to industrial applications each year. Multiple mechanisms for S. oneidensis-enhanced corrosion have been proposed, but none of these mechanisms have previously been rigorously investigated with methods that rule out alternative routes for electron transfer. We found that S. oneidensis grown under aerobic conditions formed thick biofilms (∼50 µm) on stainless steel coupons, accelerating corrosion over sterile controls. H2 and flavins were ruled out as intermediary electron carriers because stainless steel did not reduce riboflavin and previous studies have demonstrated stainless does not generate H2. Strain ∆mtrCBA, in which the genes for the most abundant porin-cytochrome conduit in S. oneidensis were deleted, corroded stainless steel substantially less than wild-type in aerobic cultures. Wild-type biofilms readily reduced nitrate with stainless steel as the sole electron donor under anaerobic conditions, but strain ∆mtrCBA did not. These results demonstrate that S. oneidensis can directly consume electrons from iron-containing metals and illustrate how direct metal-to-microbe electron transfer can be an important route for corrosion, even in aerobic environments.

The reproducibility of corrosion testing in supercritical water—Results of a second international interlaboratory comparison exercise

Supercritical water-cooled reactor candidate materials are typically corrosion-resistant alloys whose mass changes in supercritical water fluctuate around zero. A previous interlaboratory corrosion experiment exercise revealed large scatter in mass change results between laboratories. Here, we reduced systemic differences between laboratories by unilateral preparation of coupons and unilateral chemical cleaning. Type 310S stainless steel and Alloy 800HT test coupons were exposed for 1000 h to 550 °C, 25 MPa, deaerated water. Average mass loss for 310S and 800HT was 38 ± 26 and 51 ± 31 mg/dm2, respectively. Differences in mass transfer, galvanic and local corrosion, and average coupon temperature may explain the poor reproducibility.

Desiccation-Tolerance of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium abscessus and Mycobacterium chelonae.

Desiccation-tolerance of cells of four strains of Mycobacterium chimaera and individual strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium chelonae were measured by two methods. The survival of water-acclimated cells both in filter paper and on the surface of stainless-steel coupons were measured. In filter paper at 40% relative humidity at 25 °C, survival of patient isolates of M. avium and M. chimaera cells was 28% and 34% after 21 days of incubation, whereas it was 100% for the Sorin 3T isolate of M. chimaera. On stainless-steel biofilms after 42 days of incubation at 40% relative humidity at 25 °C, survival of water-acclimated cells of M. intracellulare was above 100%, while M. chelonae cells did not survive beyond 21 days, and survival of water-acclimated cells of M. avium and M. abscessus was 18% and 14%, respectively. On stainless-steel coupons, survival of patient and Sorin 3T isolates of M. chimaera was quite similar, specifically between 14% and 28% survival, after 42 days of incubation at 40% relative humidity at 25 °C. The experiments would support the hypothesis that some nontuberculous mycobacterial species are relatively desiccation-tolerant, whereas others are not. Further, long-term survival of the two M. chimaera strains is consistent with the presence of that species in Sorin 3T heater-coolers shipped throughout the world.

53 Evaluating the Efficacy of Boot Baths with Wet and Dry Disinfectants for Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus

Abstract Maintaining biosecurity between barns is challenging. Boot baths, either wet or dry, can be implemented to limit pathogen spread. The objective was to evaluate the efficacy of boot baths using wet or dry disinfectants for porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). Treatments included 1) control, 2) wet disinfectant (Synergize, Neogen, Lexington, KY), and 3) dry disinfectant (Traffic C.O.P., PSP, LLC, Rainsville, AL). Prior to disinfection, 0.5 mL of both PRRSV (~1×105 TCID50/mL) and PEDV (~1×105 TCID50/mL) was placed onto a new boot with a layer of autoclaved corn dust and allowed to dry for 15 minutes. After the mixture dried, the boot was put on and stepped into its respective boot bath. After 3 seconds, the boot was lifted out of the bath and stepped onto a stainless-steel coupon to simulate walking through a facility. Both boot and coupon were allowed to dry for 1 minute before swabs were taken from both surfaces. Samples were analyzed in a duplex PCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a treatment×surface×virus interaction (P &amp;gt; 0.10). The interaction between treatment×surface impacted (P &amp;lt; 0.05) the quantity of detectable RNA. The control had greater concentration of virus on the coupon than the boot. The reverse was true for boots treated with wet disinfectant, where the boot had a greater concentration of virus than the coupon. Treatment×virus also impacted detectable RNA (P &amp;lt; 0.05), where wet and control boots had greater quantities of PEDV RNA than PRRSV. There was no detectable virus when dry disinfectant was used. For this trial, dry disinfectant was the most efficacious in reducing the viral RNA on both boots and subsequent surfaces; however, further research in commercial settings is warranted.

Controlling Listeria monocytogenes Growth and Biofilm Formation Using Flavonoids

The aim of this study was to investigate the ability of natural plant-derivate products (flavonoid compounds) to inhibit the growth and biofilm-forming ability of Listeria monocytogenes. A collection of 500 synthetic and natural flavonoids were tested individually on strains of L. monocytogenes for their antimicrobial and antibiofilm activity. The flavonoids were tested against a L. monocytogenes cocktail of five strains at a concentration of 100 μM to determine their effect on planktonic growth. The optical density was measured every hour for 24 h at 37°C, and every hour for 48 h at 22°C. A total of 17 flavonoids were chosen for further study because of their ability to significantly reduce the growth of L. monocytogenes up to 97%. An additional two flavonoids that increased planktonic growth were chosen as well to investigate whether they had the same effect on biofilm growth. A lower concentration of flavonoid compounds (50 μM) was selected to investigate the individual effects on L. monocytogenes biofilm formation using (i) stainless steel coupons to quantify biomass using crystal violet staining and (ii) glass slides using confocal laser scanning microscopic (CLSM) imaging to observe the biofilm architecture. The 19 flavonoids showed various levels of L. monocytogenes biofilm growth inhibition, ranging from 2 to 100% after 48 h of incubation at 22 or 10°C. This includes 18 of the 19 flavonoids significantly (P ≤ 0.05) inhibiting L. monocytogenes biofilm formation on stainless steel coupons under at least one of the testing conditions. However, only one flavonoid compound demonstrated significant biofilm inhibition (P ≤ 0.05) under all conditions tested. Furthermore, 8 of the selected 19 flavonoid compounds showed visible reductions through CLSM in L. monocytogenes biofilm formation. Overall, we identified five flavonoid compounds to be promising antibiofilm and antimicrobial agents against L. monocytogenes.

Residual antimicrobial coating efficacy against SARS-CoV-2.

This study evaluated the residual efficacy of commercially available antimicrobial coatings or films against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on non-porous surfaces. Products were applied to stainless steel or ABS plastic coupons and dried overnight. Coupons were inoculated with SARS-CoV-2 in the presence of 5% soil load. Recovered infectious SARS-CoV-2 was quantified by TCID50 assay. Tested product efficacies ranged from <1.0 to >3.0log10 reduction at a 2-h contact time. The log10 reduction in recovered infectious SARS-CoV-2 ranged from 0.44 to 3log10 reduction on stainless steel and 0.25 to >1.67log10 on ABS plastic. The most effective products tested contained varying concentrations (0.5%-1.3%) of the same active ingredient: 3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride. Products formulated with other quaternary ammonium compounds were less effective against SARS-CoV-2 in this test. The residual antimicrobial products tested showed varied effectiveness against SARS-CoV-2 as a function of product tested. Several products were identified as efficacious against SARS-CoV-2 on both stainless steel and ABS plastic surfaces under the conditions evaluated. Differences in observed efficacy may be due to variation in active ingredient formulation; efficacy is, therefore, difficult to predict based upon listed active ingredient and its concentration. This study highlights the formulation-specific efficacy of several products against SARS-CoV-2 and may inform future development of residual antiviral products for use on non-porous surfaces. The identification of antimicrobial coatings or films showing promise to inactivate SARS-CoV-2 suggests that these products may be worth future testing and consideration.

Inhibition of corrosion causing Pseudomonas aeruginosa using plasma-activated water.

The cost of Microbiologically Influenced Corrosion (MIC) significantly affects a wide range of sectors. This study aims to assess the efficiency of a novel technology based on the use of plasma-activated water (PAW) in inhibiting corrosion caused by bacteria. This study evaluated the effectiveness of PAW, produced by a plasma bubble reactor, in reducing corrosion causing Pseudomonas aeruginosa planktonic cells in tap water and biofilms were grown onto stainless steel (SS) coupons. Planktonic cells and biofilms were treated with PAW at different discharge frequencies (500-1500Hz) and exposure times (0-20min). P. aeruginosa cells in tap water were significantly reduced after treatment, with higher exposure times and discharge frequencies achieving higher reductions. Also, PAW treatment led to a gradual reduction for young and mature biofilms, achieving >4-Log reductions after 20min. Results were also used to develop two predictive inactivation models. This work presents evidence that PAW can be used to inactivate both planktonic cells and biofilms of P. aeruginosa. Experimental and theoretical results also demonstrate that reduction is dependent on discharge frequency and exposure time. This work demonstrates the potential of using PAW as means to control MIC.

Evaluation of the Pathogenic-Mixed Biofilm Formation of Pseudomonas aeruginosa/Staphylococcus aureus and Treatment with Limonene on Three Different Materials by a Dynamic Model.

Background: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. Methods: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). Results: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. Conclusions: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Virucidal efficacy of antimicrobial surface coatings against the enveloped bacteriophage Φ6.

Antimicrobial coatings, for use in combination with routine cleaning and disinfection, were evaluated for their effectiveness in reducing virus concentration on stainless steel surfaces. Twenty antimicrobial coating products, predominantly composed of organosilane quaternary ammonium compounds, were applied to stainless steel coupons, dried overnight and evaluated for efficacy against Φ6, an enveloped bacteriophage. Additionally, two peel and stick polymer-based films, a copper-based film and three copper alloys were evaluated. Efficacy was determined by comparison of recoveries from uncoated (positive control) and coated (test) surfaces. The results indicated that some of the coating products initially demonstrated >3-log reduction of Φ6; no direct correlation of efficacy was observed with an active ingredient or its concentration. The peel and stick films and copper alloys each demonstrated efficacy in initial testing. However, none of the spray-based products retained efficacy after subjecting the coating to abrasion with either a hypochlorite or quaternary ammonium-based solution applied in accordance with EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings. Of the products tested for this durability, only one peel and stick polymeric film retained efficacy; the copper alloys were not tested for their durability in this study. These results suggest that while some organosilane quaternary ammonium compound-based products demonstrate antiviral efficacy, more research and development is needed to understand effective formulations with sufficient durability to perform as supplements to routine cleaning and disinfection.

Performance Augmentation of Epoxy Adhesives with TiN Nanoparticles.

In the current study, nanoparticles (NPs) of titanium nitride (50–70 nm) in varying amounts (0–4 wt %) were uniformly suspended in an epoxy solution and then used to cast the films of nanocomposites. The same formulations were used to prepare the lap shear strength joints using stainless-steel coupons with the help of standard molds and then employing the compression molding technique. The nanocomposites films were characterized for their physical properties, thermal stability, friction performance, and scratch hardness, while the lap shear strength of joints prepared using nanocomposites as nanoadhesives was evaluated. The failed surfaces of joints were investigated using scanning electron microscopy (SEM) to understand the failure modes, that is, micro-failure mechanisms, while the cross-sectional surfaces of fractured nanocomposites were investigated using SEM to identify the distribution of NPs. The increase in the contents of NPs in the epoxy led to an almost linear increase in the selected performance properties. The highest (70%) improvement in the lap shear strength was observed with 4 wt % NPs, which was correlated with an increase in the hardness of composites.

N-acyl-homoserine lactone produced by Rahnella inusitata isolated from the gut of Galleria mellonella influences Salmonella phenotypes.

The most studied mechanism of quorum sensing in Gram-negative bacteria is mediated by autoinducer 1 (AI-1), namely, acyl-homoserine lactone (AHL). This system allows communication among different bacterial species and regulates the expression of virulence genes in many pathogens. Although AHL-producing bacteria have been detected in the intestines of humans and other animals, no report was found about AHL-producing bacteria in the insect gut and the possible effects of these autoinducers on enteropathogenic bacteria. Therefore, this study aimed to identify AHL-producing bacteria in the gut of larvae of Galleria mellonella and to evaluate the influence of this quorum sensing signal on the regulation of adhesion and motility phenotypes in the intestinal pathogen Salmonella. Sequencing of the 16S rRNA gene, 16S rRNA gene-based phylogenetic analyses, and phenotypic characterization of gut isolates was performed. The profile of AHLs produced by the isolates was determined using thin-layer chromatography (TLC) and revealed with the biosensor strain Chromobacterium violaceum CV026. Sequencing, phylogenetic analyses and phenotypic characterization of gut isolates showed that the three AHL-producing strains belong to the species Rahnella inusitata, named GM34, GM56, and GM60. The TLC showed that R. inusitata produces a six-carbon AHL. In the presence of cell-free extract of R. inusitata containing AHL and under anaerobic conditions, Salmonella enterica increased the adhesion to stainless steel coupons and presented swarming motility. Extracts from the culture medium of R. inusitata isolates containing AHL increased the adhesion on stainless steel coupons and swarming motility of Salmonella enterica serovar Enteritidis PT4 under anaerobic conditions. The results suggest the possibility of communication between members of the G. mellonella intestinal microbiota with pathogens such as Salmonella.

Combined Effect of Temperature and Relative Humidity on the Survival of Salmonella Isolates on Stainless Steel Coupons.

The survival on stainless steel of ten Salmonella isolates from food factory, clinical and veterinary sources was investigated. Stainless steel coupons inoculated with Salmonella were dried and stored at a range of temperatures and relative humidity (RH) levels representing factory conditions. Viability was determined from 1 to 22 days. Survival curves obtained for most isolates and storage conditions displayed exponential inactivation described by a log-linear model. Survival was affected by environmental temperatures and RH with decimal reduction times (DRTs) ranging from <1 day to 18 days. At 25 °C/15% RH, all isolates survived at levels of 103 to 105 cfu for >22 days. Furthermore, temperatures and RH independently influenced survival on stainless steel; increasing temperatures between 10 °C and 37 °C and increasing RH levels from 30–70% both decreased the DRT values. Survival curves displaying a shoulder followed by exponential death were obtained for three isolates at 10 °C/70% RH. Inactivation kinetics for these were described by modified Weibull models, suggesting that cumulative injury occurs before cellular inactivation. This study highlights the need to control temperature and RH to limit microbial persistence in the food manufacturing environment, particularly during the factory shut-down period for cleaning when higher temperature/humidity levels could be introduced.

Inhibition of bacterial adherence on stainless steel coupons by surface conditioning with selected polar lipids

AbstractPolar lipids were recently found to have the surfactant property that could interfere with the bacterial adherence process during biofilm formation, a promising effect against the bacterial contamination on food processing surfaces. This study aimed to investigate the effect of surface conditioning of polar lipids such as phosphatidylcholine and sphingomyelin on stainless steel surfaces and subsequently on bacterial adherence. A confocal laser scanning microscope was used to examine formed conditioning layers and bacterial adherence. The thinnest conditioning layer was created by dilute skim milk and by sphingomyelin, while thicker layers were created by phosphatidylcholine, dilute buttermilk, and butter serum. Among surface conditioning treatments of stainless steel coupons, those performed with diluted buttermilk, butter serum, and polar lipids such as phosphatidylcholine, and sphingomyelin were able to scale down the adherence of Lactococcus lactis subsp. lactis NBRC 100676 and Leuconostoc mesenteroides subsp. cremoris NBRC 102479 compared to the surface conditioning with dilute skim milk. For both strains, the higher the phosphatidylcholine concentration (over 0.01%) and the longer the adhesion time (over 12.0 h), the more the adherence‐reducing ability of conditioned surfaces. The higher the sphingomyelin concentration used in surface conditioning, the higher the adherence‐reducing activity on stainless‐steel surfaces was at all adhesion times (0.5, 12.0, and 24.0 h). Our results suggested that, for short bacterial exposure period (0.5, 12.0, and 24.0 h), dilute buttermilk, butter serum, and individual polar lipids may have a bacterial adherence‐reducing effect, and can potentially be used for reducing biofilm formation.

Quaternized chitosan as a biopolymer sanitizer for leafy vegetables: synthesis, characteristics, and traditional vs. dry nano-aerosol applications

A series of quaternary dimethyl-(alkyl)-ammonium chitosan derivatives (QACs) was synthesized and studied for physicochemical properties and bioactivity. The QACs tended to spontaneously self-assembly into nanoaggregates. Antimicrobial activity was examined in vitro on Gram-negative Escherichia coli (E. coli) and Gram-positive Listeria innocua (L. innocua) bacteria as well as phytopathogenic fungus Botrytis cinerea. The hexyl chain-substituted QAC-6 demonstrated the highest potency causing 3.0- and 4.5-log CFU mL-1 reduction of E. coli and L. innocua, respectively. QAC-6 was tested for antimicrobial activity on stainless steel coupons and fresh spinach leaves. A traditional ‘wet’ application (spray) and dry Engineered Water Nanostructure (EWNS) approach were used for spinach decontamination. With both approaches, significant reduction of microbial load on the treated produce was achieved. The wet application showed a greater reduction of microbial load, while the advantages of EWNS were reaching the antimicrobial effect with miniscule dose of active agent leaving treated surface visibly dry.

Effect of Mauritia flexuosa L. leaf extract on Staphylococcus aureus and Staphylococcus haemolyticus biofilms adhered to stainless steel surface.

Staphylococcus spp. represents the main mastitis agents in ruminants and contaminants of milk due to their expressive capacity to make biofilms. The aims in this study was evaluate evaluated the antimicrobial activity of Mauritia flexuosa L. extracts against Staphylococcus spp. adhered to a stainless steel surface. Two isolates from cows with clinical mastitis were evaluated; one was identified as Staphylococcus aureus, and the other Staphylococcus haemolyticus. Additionally the ATCC 25923 strain, S. aureus from human was evaluated. The chemical profile obtained from gas chromatography revealed the presence of carbohydrates, organic acids, and flavonoids. The minimum bactericidal concentrations of the ethanolic extract (EE) and aqueous extract (AE) were 4.4 and 5.82 mg/mL, respectively. After EE treatment at 4.4 mg/mL for 2.5 min, total removal of mature biofilms grown on stainless steel coupons was observed (reduction by 3.85-4.81 log units). This extract from M. flexuosa shows potential as an effective sanitizer and may represent a natural alternative against Staphylococcus spp.