Deciphering the Roles of Extracellular Polymeric Substances (EPS) in Shaping Disinfection Kinetics through Permanent Removal via Genetic Disruption.

Extracellular polymeric substances (EPS) ubiquitously encapsulate microbes and play crucial roles in various environmental processes. However, understanding their complex interactions with dynamic bacterial behaviors, especially during the disinfection process, remains very limited. In this work, we investigated the impact of EPS on bacterial disinfection kinetics by developing a permanent EPS removal strategy. We genetically disrupted the synthesis of exopolysaccharides, the structural components of EPS, in Pseudomonas aeruginosa, a well-known EPS-producing opportunistic pathogen found in diverse environments, creating an EPS-deficient strain. This method ensured a lasting absence of EPS while maintaining bacterial integrity and viability, allowing for real-time in situ investigations of the roles of EPS in disinfection. Our findings indicate that removing EPS from bacteria substantially lowered their susceptibility threshold to disinfectants such as ozone, chloramine B, and free chlorine. This removal also substantially accelerated disinfection kinetics, shortened the resistance time, and increased disinfection efficiency, thereby enhancing the overall bactericidal effect. The absence of EPS was found to enhance bacterial motility and increase bacterial cell vulnerability to disinfectants, resulting in greater membrane damage and intensified reactive oxygen species (ROS) production upon exposure to disinfectants. These insights highlight the central role of EPS in bacterial defenses and offer promising implications for developing more effective disinfection strategies.

Reviewing the evidence of antimicrobial activity of glycols.

In the 1940s and 1950s, researchers seeking safe and novel ways to eliminate airborne pathogens from enclosed spaces, investigated glycol vapours as a method of disinfection. More recently, the COVID-19 pandemic highlighted the need for a non-toxic aerial disinfectant that can be used in the presence of people. This scoping review is intended to analyse the early and more recent literature on glycol disinfection, scrutinizing the methodologies used, and to determine if the use of glycols as modern-day disinfectants is justified PRISMA-ScR guidelines were used to assess the 749 articles retrieved from the Web of Science platform, with 46 articles retained after the search strategy was applied. Early studies generally demonstrated good disinfection capabilities against airborne bacteria and viruses, particularly with propylene glycol (PG) vapour. Vapour pressure, relative humidity, and glycol concentration were found to be important factors affecting the efficacy of glycol vapours. Contact times depended mainly on the glycol application method (i.e. aerosolization or liquid formulation), although information on how glycol efficacy is impacted by contact time is limited. Triethylene glycol (TEG) is deemed to have low toxicity, carcinogenicity, and mutagenicity and is registered for use in air sanitization and deodorization by the US Environmental Protection Agency. Glycols are also used in liquid formulations for their antimicrobial activity against a wide range of microorganisms, although when used as a non-active excipient in products, their contribution to antimicrobial efficacy is rarely assessed. The appropriate use of liquid glycol-containing formulations was found to positively impact the antimicrobial capabilities of disinfectants when used at temperatures <0, food preservatives, and dental medicaments. Providing modern delivery technology can accurately control environmental conditions, the use of aerosolized glycol formulations should lead to successful disinfection, aiding infection prevention, and control regimens.

Deciphering Multidrug-Resistant Plasmids in Disinfection Residual Bacteria from a Wastewater Treatment Plant.

Current disinfection processes pose an emerging environmental risk due to the ineffective removal of antibiotic-resistant bacteria, especially disinfection residual bacteria (DRB) carrying multidrug-resistant plasmids (MRPs). However, the characteristics of DRB-carried MRPs are poorly understood. In this study, qPCR analysis reveals that the total absolute abundance of four plasmids in postdisinfection effluent decreases by 1.15 log units, while their relative abundance increases by 0.11 copies/cell compared to investigated wastewater treatment plant (WWTP) influent. We obtain three distinctive DRB-carried MRPs (pWWTP-01-03) from postdisinfection effluent, each carrying 9-11 antibiotic-resistant genes (ARGs). pWWTP-01 contains all 11 ARGs within an ∼25 Kbp chimeric genomic island showing strong patterns of recombination with MRPs from foodborne outbreaks and hospitals. Antibiotic-, disinfectant-, and heavy-metal-resistant genes on the same plasmid underscore the potential roles of disinfectants and heavy metals in the coselection of ARGs. Additionally, pWWTP-02 harbors an adhesin-type virulence operon, implying risks of both antibiotic resistance and pathogenicity upon entering environments. Furthermore, some MRPs from DRB are capable of transferring and could confer selective advantages to recipients under environmentally relevant antibiotic pressure. Overall, this study advances our understanding of DRB-carried MRPs and highlights the imminent need to monitor and control wastewater MRPs for environmental security.

Virucidal efficacy of hypochlorous acid water for aqueous phase and atomization against SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is an infectious viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged at the end of 2019. SARS-CoV-2 can be transmitted through droplets, aerosols, and fomites. Disinfectants such as alcohol, quaternary ammonium salts, and chlorine-releasing agents, including hypochlorous acid, are used to prevent the spread of SARS-CoV-2 infection. In the present study, we investigated the efficacy of ionless hypochlorous acid water (HOCl) in suspension and by spraying to inactivate SARS-CoV-2. The virucidal efficacy of HOCl solution in tests against SARS-CoV-2 was evaluated as 50% tissue culture infectious dose. Although the presence of organic compounds influenced the virucidal efficacy, HOCl treatment for 20 s was significantly effective to inactivate Wuhan and Delta strains in the suspension test. HOCl atomization for several hours significantly reduced the SARS-CoV-2 attached to plastic plates. These results indicate that HOCl solution with elimination containing NaCl and other ions may have high virucidal efficacy against SARS-CoV-2. This study provides important information about the virucidal efficacy and use of HOCl solution.

Efficacy evaluation of hydrogen peroxide disinfectant based zinc oxide nanoparticles against diarrhea causing Escherichia coli in ruminant animals and broiler chickens.

Different strains of Escherichia coli that exhibit genetic characteristics linked to diarrhea pose a major threat to both human and animal health. The purpose of this study was to determine the prevalence of pathogenic Escherichia coli (E. coli), the genetic linkages and routes of transmission between E. coli isolates from different animal species. The efficiency of disinfectants such as hydrogen peroxide (H2O2), Virkon®S, TH4+, nano zinc oxide (ZnO NPs), and H2O2-based zinc oxide nanoparticles (H2O2/ZnO NPs) against isolated strains of E. coli was evaluated. Using 100 fecal samples from different diarrheal species (cow n = 30, sheep n = 40, and broiler chicken n = 30) for E. coli isolation and identification using the entero-bacterial repetitive intergenic consensus (ERIC-PCR) fingerprinting technique. The E. coli properties isolated from several diarrheal species were examined for their pathogenicity in vitro. Scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), Fourier-transform infrared spectrum (FT-IR), X-ray diffraction (XRD), zeta potential, and particle size distribution were used for the synthesis and characterization of ZnO NPs and H2O2/ZnO NPs. The broth macro-dilution method was used to assess the effectiveness of disinfectants and disinfectant-based nanoparticles against E. coli strains. Regarding the results, the hemolytic activity and Congo red binding assays of pathogenic E. coli isolates were 55.3 and 44.7%, respectively. Eleven virulent E. coli isolates were typed into five ERIC-types (A1, A2, B1, B2, and B3) using the ERIC-PCR method. These types clustered into two main clusters (A and B) with 75% similarity. In conclusion, there was 90% similarity between the sheep samples' ERIC types A1 and A2. On the other hand, 89% of the ERIC types B1, B2, and B3 of cows and poultry samples were comparable. The H2O2/ZnO NPs composite exhibits potential antibacterial action against E. coli isolates at 0.04 mg/ml after 120 min of exposure.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Controlling Legionella pneumophila growth in hot water systems by reducing dissolved oxygen levels.

Legionella pneumophila, the leading cause of Legionnaires' disease in the United States, is found in lakes, ponds, and streams but poses a health risk when it grows in building water systems. The growth of L. pneumophila in hot water systems of healthcare facilities poses a significant risk to patients, staff, and visitors. Hospitals and long-term care facilities account for 76% of reported Legionnaires' disease cases with mortality rates of 25%. Controlling L. pneumophila growth in hot water systems serving healthcare and hospitality buildings is currently achieved primarily by adding oxidizing chemical disinfectants. Chemical oxidants generate disinfection byproducts and can accelerate corrosion of premise plumbing materials and equipment. Alternative control methods that do not generate hazardous disinfection byproducts or accelerate corrosion are needed. L. pneumophila is an obligate aerobe that cannot sustain cellular respiration, amplify, or remain culturable when dissolved oxygen (DO) concentrations are too low (< 0.3 mg/L). An alternative method of controlling L. pneumophila growth by reducing DO levels in a hot water model system using a gas transfer membrane contactor was evaluated. A hot water model system was constructed and inoculated with L. pneumophila at DO concentrations above 0.5 mg/L. Once the model system was colonized, DO levels were incrementally reduced. Water samples were collected each week to evaluate the effect of reducing dissolved oxygen levels when all other conditions favored Legionella amplification. At DO concentrations below 0.3 mg/L, L. pneumophila concentrations were reduced by 1-log over 7 days. Under conditions in the hot water model system, at favorable temperatures and with no residual chlorine disinfectant, L. pneumophila concentrations were reduced by 1-log, indicating growth inhibition by reducing DO levels as the sole control measure. In sections of the model system where DO levels were not lowered L. pneumophila continued to grow. Reducing dissolved oxygen levels in hot water systems of healthcare and other large buildings to control L. pneumophila could also lower the risk of supplemental chemical treatment methods currently in use.

Sunlight-triggered prebiotic nanomotors for inhibition and elimination of pathogen and biofilm in aquatic environment.

Pathogen contamination in drinking water sources causes waterborne infectious diseases, seriously threatening human health. Nowadays, stimuli-responsive self-propelled nanomotors are appealing therapeutic agents for antibacterial therapy in vivo. However, achieving water disinfection using these nanobots is still a great challenge. Herein, we report on prebiotic galactooligosaccharide-based nanomotors for sunlight-regulated water disinfection. The nanomotors can utilize galactooligosaccharide-based N-nitrosamines as sunlight-responsive fuels for the spontaneous production of antibacterial nitric oxide. Such a solar-to-chemical energy conversion would power the nanomotors for self-diffusiophoresis, which could promote the diffusion of the nanomotors in water and their penetration in the biofilm, significantly enhancing the inhibition and elimination of the pathogens and their biofilms in aquatic environments. After water treatments, the prebiotic-based residual disinfectants can be selectively utilized by beneficial bacteria to effectively relieve safety risks to the environment and human health. The low-energy-cost, green and potent antibacterial nanobots show promising potential in water disinfection.

Effective elimination of bacteria on hard surfaces by the combined use of bacteriophages and chemical disinfectants.

Hospital-acquired infections (HAIs) represent one of the significant causes of morbidity and mortality worldwide, and controlling pathogens in the hospital environment is of great importance. Currently, the standard disinfection method in the hospital environment is chemical disinfection. However, disinfectants are usually not used strictly according to the label, making them less effective in disinfection. Therefore, there is an emergent need to find a better approach that can be used in hospitals to control pathogenic bacteria in the clinical environment. Bacteriophages (phages) are effective in killing bacteria and have been applied in the treatment of bacterial infections but have not received enough attention regarding the control of contamination in the clinical environment. In this study, we found that various phages remain active in the presence of chemical disinfectants. Moreover, the combined use of specific phages and chemical disinfectants is more effective in removing bacterial biofilms and eliminating bacteria on hard surfaces. Thus, this proof-of-concept study indicates that adding phages directly to chemical disinfectants might be an effective and economical approach to enhance clinical environment disinfection. IMPORTANCE: In this study, we investigated whether the combination of bacteriophages and chemical disinfectants can enhance the efficacy of reducing bacterial contamination on hard surfaces in the clinical setting. We found that specific phages are active in chemical disinfectants and that the combined use of phages and chemical disinfectants was highly effective in reducing bacterial presence on hard surfaces. As a proof-of-concept, we demonstrated that adding specific phages directly to chemical disinfectants is an effective and cost-efficient strategy for clinical environment disinfection.

A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Nonthermal plasma air disinfection for the inactivation of airborne microorganisms in an experimental chamber and indoor air.

AIMS: Airborne transmission of diseases presents a serious threat to human health, so effective air disinfection technology to eliminate microorganisms in indoor air is very important. This study evaluated the effectiveness of a non-thermal plasma (NTP) air disinfector in both laboratory experiments and real environments. METHODS AND RESULTS: An experimental chamber was artificially polluted with a bioaerosol containing bacteria or viruses. Additionally, classroom environments with and without people present were used in field tests. Airborne microbial and particle concentrations were quantified. A 3.0 log10 reduction in the initial load was achieved when a virus-containing aerosol was disinfected for 60 min and a bacteria-containing aerosol was disinfected for 90 min. In the field test, when no people were present in the room, NTP disinfection decreased the airborne microbial and particle concentrations (P < 0.05). When people were present in the room, their constant activity continuously contaminated the indoor air, but all airborne indicators decreased (P < 0.05) except for planktonic bacteria (P = 0.094). CONCLUSIONS: NTP effectively inactivated microorganisms and particles in indoor air.

Persistence and free chlorine disinfection of human coronaviruses and their surrogates in water.

The coronavirus disease 2019 pandemic illustrates the importance of understanding the behavior and control of human pathogenic viruses in the environment. Exposure via water (drinking, bathing, and recreation) is a known route of transmission of viruses to humans, but the literature is relatively void of studies on the persistence of many viruses, especially coronaviruses, in water and their susceptibility to chlorine disinfection. To fill that knowledge gap, we evaluated the persistence and free chlorine disinfection of human coronavirus OC43 (HCoV-OC43) and its surrogates, murine hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV), in drinking water and laboratory buffer using cell culture methods. The decay rate constants of human coronavirus and its surrogates in water varied, depending on virus and water matrix. In drinking water without disinfectant addition, MHV showed the largest decay rate constant (estimate ± standard error, 2.25 ± 0.09 day-1) followed by HCoV-OC43 (0.99 ± 0.12 day-1) and TGEV (0.65 ± 0.06 day-1), while in phosphate buffer without disinfectant addition, HCoV-OC43 (0.51 ± 0.10 day-1) had a larger decay rate constant than MHV (0.28 ± 0.03 day-1) and TGEV (0.24 ± 0.02 day-1). Upon free chlorine disinfection, the inactivation rates of coronaviruses were independent of free chlorine concentration and were not affected by water matrix, though they still varied between viruses. TGEV showed the highest susceptibility to free chlorine disinfection with the inactivation rate constant of 113.50 ± 7.50 mg-1 min-1 L, followed by MHV (81.33 ± 4.90 mg-1 min-1 L) and HCoV-OC43 (59.42 ± 4.41 mg-1 min-1 L). IMPORTANCE: This study addresses an important knowledge gap on enveloped virus persistence and disinfection in water. Results have immediate practical applications for shaping evidence-based water policies, particularly in the development of disinfection strategies for pathogenic virus control.

The synergistic effect of periodate/ferrate (VI) system on disinfection of antibiotic resistant bacteria and removal of antibiotic resistant genes: The dominance of Fe (IV)/Fe (V).

The proliferation of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) caused by antibiotic abuse has raised concerns about the global infectious-disease crisis. This study employed periodate (PI)/ferrate (VI) (Fe (VI)) system to disinfect Gram-negative ARB (Escherichia coli DH5α) and Gram-positive bacteria (Bacillus subtilis ATCC6633). The PI/Fe (VI) system could inactivate 1 × 108 CFU/mL of Gram-negative ARB and Gram-positive bacteria by 4.0 and 2.8 log in 30 min. Neutral and acidic pH, increase of PI dosage and Fe (VI) dosage had positive impacts on the inactivation efficiency of ARB, while alkaline solution and the coexistence of 10 mM Cl-, NO3-, SO42- and 20 mg/L humic acid had slightly negative impacts. The reactive species generated by PI/Fe (VI) system could disrupt the integrity of cell membrane and wall, leading to oxidative stress and lipid peroxidation. Intracellular hereditary substance, including DNA and ARGs (tetA), would leak into the external environment through damaged cells and be degraded. The electron spin resonance analysis and quenching experiments indicated that Fe (IV)/Fe (V) played a leading role in disinfection. Meanwhile, PI/Fe (VI) system also had an efficient removal effect on sulfadiazine, which was expected to inhibit the ARGs transmission from the source.

New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

The Role of Natural and Synthetic Flavonoids in the Prevention of Marine Biofouling.

Marine biofouling is a major concern for the maritime industry, environment, and human health. Biocides which are currently used in marine coatings to prevent this phenomenon are toxic to the marine environment, and therefore a search for antifoulants with environmentally safe properties is needed. A large number of scientific papers have been published showing natural and synthetic compounds with potential to prevent the attachment of macro- and microfouling marine organisms on submerged surfaces. Flavonoids are a class of compounds which are highly present in nature, including in marine organisms, and have been found in a wide range of biological activities. Some natural and synthetic flavonoids have been evaluated over the last few years for their potential to prevent the settlement and/or the growth of marine organisms on submerged structures, thereby preventing marine biofouling. This review compiles, for the first-time, natural flavonoids as well as their synthetic analogues with attributed antifouling activity against macrofouling and microfouling marine organisms.

Comparison of antimicrobial efficacy of different disinfectants on the biofilm formation in dental unit water systems using dip slide and conventional methods: A pilot study.

OBJECTIVE: Biofilm formation in dental waterlines brings opportunistic infections, especially for immunosuppressive patients. This study aimed to determine biofilm-forming microorganisms by various methods and investigate disinfectants' effects on biofilm. MATERIALS & METHODS: In the study, samples were obtained from the waterlines of 10-15 aged six dental units, before (0 min.) and after chlorine dioxide (ClO2) and hypochlorous acid (HOCl) treatment (1, 5, 10, 20, and 30 min.), and total colony counts were performed using conventional surface smear method (SSM) and dip slide method (DSM). The Congo red agar and Christensen methods were used to examine the biofilm-forming properties of the isolates. Monitoring of biofilm presence was also visualized by SEM scanning. RESULTS: When DSM and SSM are compared in all units where ClO2 and HOCl are applied, DSM can detect bacterial growth even during periods of greater exposure to disinfectant application. Although DSM can achieve a value approaching 3% even at the 10th minute in units treated with HOCl; SSM does not show reproduction at the same disinfectant exposure and duration; It was observed that in the units where ClO2 was applied, the growth was no longer observed at the 10th minute with DSM, and SSM, 50% growth in the first minute of the units treated with ClO2 could not be detected in the 5th minute. CONCLUSIONS: It is concluded that it can be advisable to routinely disinfect the dental unit water systems with non-toxic doses of ClO2 application before patient treatments in clinics and also to perform contamination controls at regular intervals with DSM, which is a sensitive and very practical method. RESEARCH HIGHLIGHTS: It has been observed that the dip slide method can count bacteria more sensitively than conventional methods in dental water systems without the need for experienced personnel and equipment. The difference between biofilm formation in water systems before and after disinfectant exposure in SEM examinations is remarkable. The effects of ClO2 and HOCl on biofilm were investigated and bacterial growth was inhibited in dental units between 5 and 10 minutes with both disinfectants.

Insights into microbial contamination and antibiotic resistome traits in pork wholesale market: An evaluation of the disinfection effect of sodium hypochlorite.

Chlorine and its derivatives, such as sodium hypochlorite (NaClO) and chlorine dioxide, are frequently employed as disinfectants throughout the pork supply chain in China. Nevertheless, the extensive use of NaClO has the potential to cause the creation of 'chlorine-tolerant bacteria' and accelerate the evolution of antibiotic resistance. This study evaluated the efficacy of NaClO disinfection by examining alterations in the microbiome and resistome of a pork wholesale market (PWM), and bacteria isolation and analysis were performed to validate the findings. As expected, the taxonomic compositions of bacteria was significantly different before and after disinfection. Notably, Salmonella enterica (S. enterica), Salmonella bongori (S. bongori), Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) were observed on all surfaces, indicating that the application of NaClO disinfection treatment in PWM environments for pathogenic bacteria is limited. Correlations were identified between antibiotic resistance genes (ARGs) associated with aminoglycosides (aph(3'')-I, aph(6')-I), quinolone (qnrB, abaQ), polymyxin (arnA, mcr-4) and disinfectant resistance genes (emrA/BD, mdtA/B/C/E/F). Furthermore, correlations were found between risk Rank I ARGs associated with aminoglycoside (aph(3')-I), tetracycline (tetH), beta_lactam (TEM-171), and disinfectant resistance genes (mdtB/C/E/F, emrA, acrB, qacG). Importantly, we found that Acinetobacter and Salmonella were the main hosts of disinfectant resistance genes. The resistance mechanisms of the ARGs identified in PWM were dominated by antibiotic deactivation (38.7%), antibiotic efflux (27.2%), and antibiotic target protection (14.4%). The proportion of genes encoding efflux pumps in the PWM resistome increased after disinfection. Microbial cultures demonstrated that the traits of microbial contamination and antibiotic resistane were consistent with those observed by metagenomic sequencing. This study highlights the possibility of cross-resistance between NaClO disinfectants and antibiotics, which should not be ignored.

Biomimetic leaves with immobilized catalase for machine learning-enabled validating fresh produce sanitation processes.

Washing and sanitation are vital steps during the postharvest processing of fresh produce to reduce the microbial load on the produce surface. Although current process control and validation tools effectively predict sanitizer concentrations in wash water, they have significant limitations in assessing sanitizer effectiveness for reducing microbial counts on produce surfaces. These challenges highlight the urgent need to improve the validation of sanitation processes, especially considering the presence of dynamic organic contaminants and complex surface topographies. This study aims to provide the fresh produce industry with a novel, reliable, and highly accurate method for validating the sanitation efficacy on the produce surface. Our results demonstrate the feasibility of using a food-grade, catalase (CAT)-immobilized biomimetic leaf in combination with vibrational spectroscopy and machine learning to predict microbial inactivation on microgreen surfaces. This was tested using two sanitizers: sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2). The developed CAT-immobilized leaf-replicated PDMS (CAT@L-PDMS) effectively mimics the microscale topographies and bacterial distribution on the leaf surface. Alterations in the FTIR spectra of CAT@L-PDMS, following simulated sanitation processes, indicate chemical changes due to CAT oxidation induced by NaClO or H2O2 treatments, facilitating the subsequent machine learning modeling. Among the five algorithms tested, the competitive adaptive reweighted sampling partial least squares discriminant analysis (CARS-PLSDA) algorithm was the most effective for classifying the inactivation efficacy of E. coli on microgreen leaf surfaces. It predicted bacterial reduction on microgreen surfaces with 100% accuracy in both training and prediction sets for NaClO, and 95% in the training set and 86% in the prediction set for H2O2. This approach can improve the validation of fresh produce sanitation processes and pave the way for future research.

Human intestinal enteroids and predictive models validate the operational limits of sanitizers used for viral disinfection of vegetable process wash water.

Vegetables are globally associated with a considerable number of foodborne outbreaks caused by viral infections, specifically human norovirus. In fresh produce industry, washing represents a critical step for food safety as process wash water (PWW) needs to be maintained at appropriate microbial quality to prevent water-mediated cross-contamination. This study aimed to explore the disinfection efficacy of chlorine (free chlorine, FC), chlorine dioxide (ClO2) and peracetic acid (PAA) in PWW against infectious human norovirus and Tulane virus (TV). First, we tested the extent of TV inactivation in baby leaf, bell pepper, and vegetables mix PWW and monitored the viral decay by cell culture. Then, inactivation kinetics were defined for infectious human norovirus exposed to FC, ClO2 and PAA in baby leaves PWW using the human intestinal enteroids (HIE) system. Finally, kinetic inactivation models were fitted to TV reduction and decay of sanitizers to aid the implementation of disinfection strategies. Results showed that >8 log10 human norovirus and 3.9 log10 TV were inactivated by 20 ppm FC within 1 min; and by 3 ppm ClO2 in 1 min (TV) or 5 min (norovirus). PAA treatment at 80 ppm reduced ca. 2 log10 TV but not completely inactivated the virus even after 20 min exposure, while 5 min treatment prevented norovirus replication in HIE. TV inactivation in PWWs was described using an exponential decay model. Taking these data together, we demonstrated the value of applying the HIE model to validate current operational limits for the most commonly used sanitizers. The inactivation kinetics for human norovirus and TV, along with the predictive model described in this study expand the current knowledge to implement post-harvest produce safety procedures in industry settings.

Efficacy of disinfectant and bacteriophage mixture against planktonic and biofilm state of Listeria monocytogenes to control in the food industry.

Fresh produce and animal-based products contaminated with Listeria monocytogenes have been the main cause of listeriosis outbreaks for many years. The present investigation explored the potential of combination treatment of disinfectants with a bacteriophage cocktail to control L. monocytogenes contamination in the food industry. A mixture of 1 minimal inhibitory concentration (MIC) of disinfectants (sodium hypochlorite [NaOCl], hydrogen peroxide [H2O2], and lactic acid [LA]) and multiplicity of infection (MOI) 100 of phage cocktail was applied to both planktonic cells in vitro and already-formed biofilm cells on food contact materials (FCMs; polyethylene, polypropylene, and stainless steel) and foods (celery and chicken meat). All the combinations significantly lowered the population, biofilm-forming ability, and the expression of flaA, motB, hlyA, prfA, actA, and sigB genes of L. monocytogenes. Additionally, in the antibiofilm test, approximately 4 log CFU/cm2 was eradicated by 6 h treatment on FCMs, and 3 log CFU/g was eradicated within 3 days on celery. However, <2 log CFU/g was eradicated in chicken meat, and regrowth of L. monocytogenes was observed on foods after 5 days. The biofilm eradication efficacy of the combination treatment was proven through visualization using scanning electron microscopy (SEM) and confocal microscopy. In the SEM images, the unusual behavior of L. monocytogenes invading from the surface to the inside was observed after treating celery with NaOCl+P or H2O2 + P. These results suggested that combination of disinfectants (NaOCl, H2O2, and LA) with Listeria-specific phage cocktail can be employed in the food industry as a novel antimicrobial and antibiofilm approach, and further research of L. monocytogenes behavior after disinfection is needed.