Sentinel Coronavirus environmental monitoring can contribute to detecting asymptomatic SARS-CoV-2 virus spreaders and can verify effectiveness of workplace COVID-19 controls.

Detecting all asymptomatic or presymptomatic COVID-19 virus spreaders at a workplace requires daily testing of employees by RT-PCR, which is not practical. Over a two week period, 9 Europe and USA workplace locations were chosen to test employees for SARS-CoV-2 infection (841 tests) and high-frequency-touch point environmental surfaces (5,500 tests) for Coronavirus by RT-PCR. Of the 9 locations, 3 had one or more employees infected with SARS-CoV-2 during the two week study period. None of the employees who tested positive had symptoms at the time of testing and none developed symptoms during subsequent 14 day quarantine. Locations with significant prevalence of Coronavirus contaminated environmental surfaces were 10 times more likely to have a positive employees than locations with no or very few environmental surfaces positive for Coronavirus. Break room chairs, workbenches, and break room door handles were the most frequently contaminated environmental surfaces. Surface Coronavirus RNA was detected at very low concentrations (RT-PCR 34 to 38 Cq). These results suggest that Coronavirus environmental monitoring may have potential to predict presence of asymptotic spreaders and to validate and verify COVID-19 control strategies on a regular basis.

Long-Term Depuration of Crassostrea virginica Oysters at Different Salinities and Temperatures Changes Vibrio vulnificus Counts and Microbiological Profile.

Previous short-duration depuration studies with the eastern oyster ( Crassostrea virginica) demonstrated difficulty in achieving significant naturally incurred Vibrio vulnificus population count reductions. The present study used long-duration depuration (14 days) at controlled temperatures (10 or 22°C) and salinities (12, 16, or 20 mg/g). All depuration temperature-salinity combinations significantly reduced V. vulnificus counts, with greatest reductions seen in 12 mg/g, 10°C seawater (2.7-log CFU/g reduction) and in 20 mg/g, 22°C seawater (2.8-log reduction). Mesophilic vibrios dominated the overall microflora of freshly harvested oysters, whereas refrigerated storage selected for psychrotrophic bacteria ( Pseudomonas spp., Aeromonas spp., Shewanella spp., Psychrobacter spp.) as well as did depuration at 10°C ( Pseudoalteromonas spp., Shewanella spp., Vibrio spp.). Depuration at 22°C retained dominance of mesophilic vibrios, including pathogenic species, followed by Shewanella spp., Pseudoalteromonas spp., and Photobacterium spp. Although aerobic plate counts were lower in 22°C depurated oysters (5.0 log versus 6.0 log) compared with 10°C, depuration at 10°C offered greater V. vulnificus population reductions than depuration at 22°C. This advantage was only seen at 12 mg/g salinity, with no impact at 16 and 20 mg/g salinities. No depuration treatment reduced V. vulnificus counts to nondetectable levels. Use of prolonged depuration may be a helpful intervention to control V. vulnificus populations in oysters.

Mitigation of Salmonella on Pet Food Kibbles by Using Liquid and Powdered 3-Hydroxy-3-Methylbutyric Acid.

In recent years, several pet food recalls have been attributed to Salmonella contamination. In addition to the negative impacts on animal health, Salmonella-contaminated pet foods have been linked to infection in humans. With that in mind, the U.S. Food and Drug Administration has set forth a zero-tolerance policy for Salmonella in pet foods. Typically, pet foods are extruded or processed at high temperatures that are sufficient to reduce pathogenic bacteria. However, the possibility for postextrusion contamination still exists. One potential method to reduce the risk of postextrusion contamination of pet foods with Salmonella is through the addition of a chemical additive coating. The objective of this research was to evaluate the ability of ß-hydroxy-ß-methylbutyrate (HMB), in either free acid (HMBFA) or calcium salt (CaHMB) form, to reduce postextrusion contamination of dry extruded dog kibble with Salmonella. Three trials were conducted with HMBFA and CaHMB coated onto the kibbles at levels of 0, 0.1, 0.3, 0.5, 0.9, and 1.5% (w/w). The coated kibbles were then inoculated with Salmonella enterica subsp. enterica Enteritidis (ATCC 13076), with enumeration done on days 0, 1, 2, 7, and 14 postinoculation. Subsamples on each day were serially diluted, spread plated to xylose lysine deoxycholate agar, and incubated at 37°C for 24 h. Salmonella colonies were then counted and log CFU per gram was calculated. The 1.5% HMBFA reduced counts by 4.9 ± 0.2 log units on day 1, whereas the positive control only decreased 2.2 ± 0.1 log units (P < 0.0001). The 1.5% CaHMB level decreased counts by 7.1 ± 0.04 log units by day 7 compared with the control decrease of 2.1 ± 0.1 log units (P < 0.0001). All HMBFA and CaHMB treatments resulted in the elimination of detectable Salmonella counts by day 14 (P < 0.0001 versus controls). In conclusion, HMB coating was effective at reducing Salmonella artificially inoculated to dog kibbles in a model of postextrusion contamination.

Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catfish and Vietnamese basa fillets.

Frozen fillets of Channel catfish and Vietnamese basa fish were used to compare Salmonella spp. recovery effectiveness of selective enrichment in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulfite (BS) agar. Isolate confirmation was through fatty acid methyl ester analysis. Of 60 samples analyzed, 25 were found contaminated with Salmonella (42% incidence). Salmonella spp. recovery after enrichment in RV medium was 35% on HE agar, 30% on XLD agar, and 42% on BS agar. Similarly, after enrichment in TT broth, HE and XLD agars recovered 22% each and BS agar recovered 37%. No performance difference (p>0.05) was observed in the recovery of Salmonella using the combinations of BS, HE, and XLD agars with RV broth and BS agar with TT broth. The combination of selective enrichment in RV and selective isolation on BS gave numerically greatest isolation of Salmonella from Channel catfish and Vietnamese basa fish compared to other isolation combinations.

Mechanism of synergistic inhibition of Listeria monocytogenes growth by lactic acid, monolaurin, and nisin.

The combined lactic acid, monolaurin, and nisin effects on time-to-detection (optical density at 600 nm) extension were greater (P < 0.05) than any single or paired combination effect, which demonstrates a synergistic interaction among the antimicrobials. Monolaurin exposure caused C12:0 cell membrane incorporation. Lactic acid caused increased monolaurin C12:0 membrane incorporation, while nisin had no influence. We postulate that lactic acid-enhanced monolaurin C12:0 incorporation into the cell membrane increased membrane fluidity resulting in increased nisin activity.

Immunosensors for rapid detection of Escherichia coli O157:H7 - perspectives for use in the meat processing industry.

This review critically evaluates different types of immunosensors proposed for rapid identification of Escherichia coli O157:H7. The methods are compared with approved USDA-FSIS standard procedures for determination of this pathogen in raw or ready-to-eat meat products. Major advantages and disadvantages for each method are highlighted. Our analysis suggests that application of immunosensors in the meat-processing industry may be limited to identification of uncontaminated samples after conventional selective enrichment in broth. Use for detection appears limited at the present time.

Combined effect of packaging atmosphere and storage temperature on growth of Listeria monocytogenes on ready-to-eat shrimp.

Cooked, peeled, and deveined shrimp were inoculated with a 5 strain mixture of Listeria monocytogenes and packaged in air, vacuum, and a 100% carbon dioxide modified atmosphere. The packaged shrimp were then stored at 3, 7, and 12 degrees C for 15 days to monitor the growth of L. monocytogenes and psychrotrophic bacteria. Uninoculated shrimp were also subjected to sensory evaluation by a trained panel to measure odor and appearance over the storage period. Results demonstrated that shrimp packaged in CO(2) and stored at 3 degrees C did not permit growth of L. monocytogenes during the 15-day storage period, while all other packaging/temperature combinations allowed for multiplication of the bacterium. Carbon dioxide packaging also resulted in the slowest growth of psychrotrophic bacteria and resulted in shrimp having acceptable sensory odor and appearance scores at the end of storage. When strict temperature control is difficult, such as during processing, transportation, retail display, or home use, additional antimicrobial hurdles may be necessary to ensure safety.

Effect of trisodium phosphate adaptation on changes in membrane lipid composition, verotoxin secretion, and acid resistance of Escherichia coli O157:H7 in simulated gastric fluid.

Escherichia coli O157:H7 (HEC), E. coli O157:H7 rpoS mutant (HEC-RM), and nonpathogenic E. coli (NPEC) were step-wise adapted to trisodium phosphate (TSP) by incubation in broths of increasing concentration, from 0% to 0.6%, at 37 degrees C for 24 h. After incubation at each concentration, each population was examined for acid resistance (D value) in simulated gastric fluid of pH 1.5, cell envelope membrane lipid composition, and intracellular and extracellular verotoxin concentrations. The ratio of cis-vaccenic acid (18:1omega7c) to palmitic acid (16:0) increased, indicating increased membrane fluidity with increasing TSP concentration up to 0.4%, but decreased at 0.6%. HEC and HEC-RM adapted at 0.4% TSP had the highest verotoxin concentrations of 1805 and 1879 ng/ml, respectively. In addition, with HEC the ratio of extracellular to intracellular verotoxin concentration decreased at higher TSP concentrations. In contrast, the ratio for HEC-RM increased at 0.4% TSP. HEC adapted to 0.4% TSP had the greatest survival in gastric fluid (58 min D value) among all treatments. For HEC, the increase in membrane fluidity was associated with increased acid resistance and extracellular verotoxin concentration for cells adapted to 0.4% TSP. In contrast, the increase in membrane fluidity was associated with decreased acid resistance of TSP adapted HEC-RM although the extracellular verotoxin concentration increased. Therefore, the deletion of the rpoS gene appeared to affect the changes in verotoxin concentration and acid resistance of TSP adapted E. coli O157:H7.

Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria.

The occurrence of antibiotic-resistant bacteria in foods of animal origin is a potential health threat because resistance can be transferred among bacteria, and antibiotic-resistant pathogens may not respond to antibiotic treatments. Thirteen brands of ready-to-eat shrimp representing four countries of origin were obtained from local grocery stores. Total heterotrophic plate counts were determined, and antibiotic-resistant bacteria were isolated. Total heterotrophic colony counts ranged from 3.3 to 5.6 log CFU/g, which was within approved quality limits. A total of 1,564 isolates representing 162 bacterial species were recovered during screening of resistance to 10 antibiotics: ampicillin, ceftriaxone, chloramphenicol, clindamycin, erythromycin, nalidixic acid, streptomycin, tetracycline, trimethoprim, and vancomycin. Six hundred fifty-seven (42%) of the isolates and 131 (81%) of the species had acquired resistance to antibiotics. Numerous resistant human pathogens were isolated, including Escherichia coli, Enterococcus spp., Salmonella, Shigella flexneri, Staphylococcus spp., and Vibrio spp. Nonresistant Yersinia spp. also were isolated. Ready-to-eat shrimp is sold with instructions to thaw the product before serving, which may result in consumer exposure to antibiotic-resistant bacteria. Widespread trade of this product provides an avenue for international dissemination of antibiotic-resistant pathogens.

Influence of acetic, citric, and lactic acids on Escherichia coli O157:H7 membrane lipid composition, verotoxin secretion, and acid resistance in simulated gastric fluid.

The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid-adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid-adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid-adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid-adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid-adapted cells, but the ratio increased for acetic acid-adapted cells at pH 5.4. Organic acid-adapted cells produced less total verotoxin than did nonadapted cells at approximately 10(8) CFU/ml. Extracellular verotoxin concentration proportionally decreased with decreasing pH for both HEC and RM. Changes in membrane lipid composition, verotoxin concentration, and acid resistance in HEC and RM were dependent on both pH and organic acid. Deletion of the rpoS gene did not affect these changes but did decrease acid resistance in citric acid-adapted cells. Results indicate that decreased membrane fluidity may have caused increased acid resistance and decreased verotoxin secretion.

Adaptation of Escherichia coli O157:H7 to pH alters membrane lipid composition, verotoxin secretion, and resistance to simulated gastric fluid acid.

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.

Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production.

The influence of heat adaptation (growth at 42 and 45 degrees C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57 degrees C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1omega7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.

Rapid determination of sanitizer concentration using impedance-based methods.

Chlorine, iodophor, and quaternary ammonium solutions of various concentrations were assayed with rapid test kits and with three Bactometer impedimetric test codes (the impedance, conductance, and capacitance test codes). An initial study was conducted to determine which test code was most suitable for each sanitizer. Impedance was shown to be the best for sodium hypochlorite solutions, conductance for iodophor solutions, and capacitance for quaternary ammonium solutions. When Bactometer results were compared with test kit results, linear regression revealed strong correlations for all three sanitizer solutions. For sodium hypochlorite concentrations of 0 to 100 ppm and 100 to 1,000 ppm, R2 values of 0.87 and 0.99, respectively, were obtained. For iodophor concentrations between 25 to 150 ppm, an R2 value of 0.95 was obtained. For quaternary ammonium compound concentrations of 100 to 1,000 ppm, an R2 value of 0.94 was obtained. The impedimetric methods proved to be simple and rapid (6 min) alternatives for measuring concentrations of the sanitizer solutions with a high level of certainty (P < 0.0002). The Bactometer will save time when multiple samples are tested.

Influence of catfish skin mucus on trisodium phosphate inactivation of attached Salmonella Typhimurium, Edwardsiella tarda, and Listeria monocytogenes.

This study examined the antimicrobial effectiveness of trisodium phosphate (TSP) on Edwardsiella tarda, Listeria monocytogenes, and Salmonella Typhimurium attached to catfish skin with and without mucus. Salmonella Typhimurium and E. tarda attached more readily to catfish skin than did L monocytogenes. At high inoculum levels (10(7) CFU/ml), TSP treatments (at 2 to 6%) for 10 min reduced bacterial counts of E. tarda by >2.5 to >3.3 log10 CFU per skin sample for firmly attached cells and by 3.5 to 3.6 log10 CFU per skin sample for loosely attached cells. Counts of L. monocytogenes declined by 0.6 to >1.8 log10 CFU per skin sample for firmly attached cells and by 1.2 to 2.2 log10 CFU per skin sample for loosely attached cells. Counts of Salmonella Typhimurium were reduced by 3.6 to >3.8 log10 CFU per skin sample for firmly attached cells and by 3.5 to >3.8 log10 CFU per skin sample for loosely attached cells. Overall, counts of firmly attached bacteria on TSP-treated skins with mucus were higher than counts on skin without mucus. Firmly attached L. monocytogenes was more resistant to TSP than was firmly attached Salmonella Typhimurium or E. tarda. The presence of mucus on skins slightly decreased the antimicrobial effect of TSP Significant (P < 0.05) reduction in the numbers of all three bacteria can be achieved by treatment with 6% TSP for 10 min.

Microbiological Quality of Catfish Frames Treated with Selected Phosphates.

This study examined the effects of trisodium phosphate (TSP), sodium tripolyphosphate (STPP), and sodium metaphosphate (SMP) dipping solutions on the microbiological quality of catfish frames (the carcasses remaining after skinless boneless fillets are removed). Frames were dipped for 5 min in 10% phosphate solutions at 5°C, drained for 2 min, and analyzed for aerobic plate counts and total coliform counts. TSP reduced aerobic plate and total coliform counts by 1.0 and 2.5 log CFU/ml of rinse buffer, respectively. STPP reduced aerobic plate and total coliform counts by 0.3 and 1.0 logs, respectively. SMP did not reduce aerobic plate counts, but did decrease total coliform counts by 0.7 logs. The microbiological shelf life (time to reach 107 CFU/ml) of the frames treated with TSP was 3 days longer than controls. Rinsing frames in water after phosphate treatment reduced the effectiveness of the dips. The results demonstrate that TSP was more effective than either STPP or SMP in reducing microbial numbers on the surface of the frames and provided a subsequent shelf life extension.

Use of Double-Gradient Plates To Study Combined Effects of Salt, pH, Monolaurin, and Temperature on Listeria monocytogenes †.

This study evaluated the combined effects of pH, NaCl, incubation temperature, and sublethal concentrations of monolaurin on the survival of Listeria monocytogenes using the double-gradient diffusion technique. L. monocytogenes tolerance to NaCl was greatest (>78 g/liter) at neutral pH (6.8 to 7.2) and increased in the pH range 7.0 to 5.4 as the incubation temperature was lowered. Monolaurin at 2 µg/ml lowered the salt tolerance of L. monocytogenes to 60 g/liter independently of pH. At 4 µg of monolaurin per ml, salt tolerance was reduced to approximately 40 g/liter with no growth occurring at pH 6.0 to 5.4 and 25 g of NaCl per liter. At 8 µg of monolaurin per ml, only a subpopulation of the initial inoculum tolerated 25 g of NaCl per liter at neutral pH (6.5 to 7.5). Monolaurin reduced the tolerance of L. monocytogenes to NaCl and low pH.

Destruction of Listeria monocytogenes Biofilms on Stainless Steel Using Monolaurin and Heat 1.

Individual and combined antimicrobial effects of monolaurin and heat on planktonic, 1-day adherent, or 7-day adherent cells of Listeria monocytogenes were determined to evaluate biofilm removal from stainless steel. Planktonic cells were more sensitive to heat and monolaurin than were cells attached to stainless steel. Young (1-day) biofilm cells were more sensitive to each treatment than were old (7-day) biofilm cells. Adherent cells were destroyed by 50 µg/ml monolaurin combined with heating at 65°C for 5 min. Cells in a rich nutrient environment were more resistant to treatment than were cells in a depleted nutrient environment. Results demonstrate the usefulness of combining chemical and physical treatments to control L. monocytogenes biofilm problems in the food industry.

Radiosensitivity of Listeria monocytogenes at Various Temperatures and Cell Concentrations.

Among food-borne pathogens, Listeria monocytogenes is more radiation resistant than gram-negative bacteria of the genera Salmonella and Vibrio . This study was designed to determine if initial cell concentration and/or temperature at the time of irradiation influences the radiosensitivity of L. monocytogenes . Concentrations of 103, 106, and 109 CFU (colony-forming units)/ml of L. monocytogenes Scott A were suspended in tryptic soy broth and exposed to 0 to 5 kGy of gamma radiation (1.25 MeV) at 20, 4, and -80°C. Survivors were enumerated and irradiation D-values were calculated using regression analysis and total-dose methods. A 103 CFU/ml population was destroyed with a <2 kGy dose. The irradiation D-value of 0.43 kGy when calculated by regression analysis for frozen (-80°C) cultures of 106 CFU/ml was significantly lower (P < 0.05) than those (0.58 and 0.62 kGy) at 20° and 4°C, respectively. However, the -80°C D-value was not significantly different (0.61 kGy) when calculated by the total dose required to eliminate all recovery. At 109 CFU/ml, a D-value (calculated by both methods) of 0.42 kGy was obtained at both 4° and -80°C, which was significantly lower (P < 0.05) than 0.50 kGy for 20°C suspensions. The temperature of irradiation only influenced the radiosensitivity of L. monocytogenes at 109 CFU/ml.

Extending Shelf Life of Refrigerated Catfish Fillets Using Sodium Acetate and Monopotassium Phosphate.

The effects of sodium acetate (SA) and monopotassium phosphate (MKP) on total aerobic plate counts (APC), pH, odor, and appearance of catfish fillets during storage at 4°C were determined. Use of 0.75% and 1.0% SA lowered (P < 0.05) initial APC by 0.6 to 0.7 log units compared to the control. Microbial counts of SA-treated fillets remained lower than the control during storage, resulting in a 6-day shelf-life increase. MKP alone had no effect on APC values, but it did influence the activity of SA. The results indicate that a combination of SA and MKP could prolong the microbiological shelf life of catfish to 12 days at 4°C. Fillets treated with 1% SA alone or SA-MKP combinations had pH values and odor scores that were similar to fresh controls for up to 9 days; however, appearance scores were lower after 3 days, probably due to a brownish and watery appearance. MKP alone is not recommended for shelf-life extension of catfish fillets. Conversely, SA alone or combined with MKP is recommended to extend the microbiological shelf life of refrigerated catfish fillets.

Growth of Selected Cross-Contaminating Bacterial Pathogens on Beef and Fish at 15 and 35°C.

Isolates of Escherichia coli and Clostridium perfringens from beef and Aeromonas hydrophila from fish were examined for their ability to survive and grow as cross-contaminates on nonnative tissues at simulated ambient (35°C) and aging/conditioning (15°C) temperatures of handling and retailing found in the tropics. Growth of all isolates over a 10-h period was greater (P < 0.05) on their native tissues at both temperatures. The aging/conditioning temperature effectively limited growth of E. coli and A. hydrophila to less than l-logl0 CFU/g and prevented growth of C. perfringens on beef and fish samples. All three isolates demonstrated characteristic mesophilic growth response on both tissues at 35°C during the 10-h retail period. The study suggests that two muscle food products could be jointly handled to efficiently use available storage/haulage capacity in tropical countries. Potential savings in space, labor and energy would be made if cross-contamination between the two products is minimized by available packaging and sanitizing technologies.