Mechanisms of action of microbicides commonly used in infection prevention and control.

SUMMARYUnderstanding how commonly used chemical microbicides affect pathogenic microorganisms is important for formulation of microbicides. This review focuses on the mechanism(s) of action of chemical microbicides commonly used in infection prevention and control. Contrary to the typical site-specific mode of action of antibiotics, microbicides often act via multiple targets, causing rapid and irreversible damage to microbes. In the case of viruses, the envelope or protein capsid is usually the primary structural target, resulting in loss of envelope integrity or denaturation of proteins in the capsid, causing loss of the receptor-binding domain for host cell receptors, and/or breakdown of other viral proteins or nucleic acids. However, for certain virucidal microbicides, the nucleic acid may be a significant site of action. The region of primary damage to the protein or nucleic acid is site-specific and may vary with the virus type. Due to their greater complexity and metabolism, bacteria and fungi offer more targets. The rapid and irreversible damage to microbes may result from solubilization of lipid components and denaturation of enzymes involved in the transport of nutrients. Formulation of microbicidal actives that attack multiple sites on microbes, or control of the pH, addition of preservatives or potentiators, and so on, can increase the spectrum of action against pathogens and reduce both the concentrations and times needed to achieve microbicidal activity against the target pathogens.

Environmental dissemination of respiratory viruses: dynamic interdependencies of respiratory droplets, aerosols, aerial particulates, environmental surfaces, and contribution of viral re-aerosolization.

During the recent pandemic of COVID-19 (SARS-CoV-2), influential public health agencies such as the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) have favored the view that SARS CoV-2 spreads predominantly via droplets. Many experts in aerobiology have openly opposed that stance, forcing a vigorous debate on the topic. In this review, we discuss the various proposed modes of viral transmission, stressing the interdependencies between droplet, aerosol, and fomite spread. Relative humidity and temperature prevailing determine the rates at which respiratory aerosols and droplets emitted from an expiratory event (sneezing, coughing, etc.) evaporate to form smaller droplets or aerosols, or experience hygroscopic growth. Gravitational settling of droplets may result in contamination of environmental surfaces (fomites). Depending upon human, animal and mechanical activities in the occupied space indoors, viruses deposited on environmental surfaces may be re-aerosolized (re-suspended) to contribute to aerosols, and can be conveyed on aerial particulate matter such as dust and allergens. The transmission of respiratory viruses may then best be viewed as resulting from dynamic virus spread from infected individuals to susceptible individuals by various physical states of active respiratory emissions, instead of the current paradigm that emphasizes separate dissemination by respiratory droplets, aerosols or by contaminated fomites. To achieve the optimum outcome in terms of risk mitigation and infection prevention and control (IPAC) during seasonal infection peaks, outbreaks, and pandemics, this holistic view emphasizes the importance of dealing with all interdependent transmission modalities, rather than focusing on one modality.

Decontamination of high-touch environmental surfaces (HITES) by wiping: quantitative assessment of a carrier platform simulating pathogen removal, inactivation and transfer in the field.

We report here a carrier platform (Teflon; 30.0 × 60.0 × 0.9 cm) and a carrier retrieval device to assess pathogen decontamination of high-touch environmental surfaces (HITES) by wiping. Each one of the nine metallic disks (1 cm diameter and 0.7 mm thick) received 10 µL of the microbial suspension in a soil load, the inocula dried and the platform then wiped with a piece of fabric presoaked in a control or disinfectant fluid; the used wipe was immediately applied on a second platform with sterile disks to assess microbial transfer. Each test and control disk from a given platform was separately and simultaneously retrieved into 10 mL of an eluent/neutralizer for assays at the end of the contact time (total of 5 min, starting from the beginning of the wiping). Staphylococcus aureus and Acinetobacter baumannii were used as representative HITES-borne pathogens. The wipes tested separately contained 0.26% of a quaternary ammonium compound (Product A), and 250 ppm sodium hypochlorite at neutral pH (Product B). The control fabric (Product C) was dampened with a buffer containing a detergent. Product A achieved a >4 log10 (>99.99%) reduction in the viability of the bacteria on wiping with a barely detectable level of transfer of CFUs to clean disks. Product B achieved a >2 log10 (>99.00%) reduction in the viability of the test microbes while transferring a higher level of CFUs as compared to Product A. With Product C, there was a <1 log10 (<86.2%) reduction in the viability of the test microbes while transferring >1% of the contamination.

Quantifying pathogen infection risks from household laundry practices.

AIMS: Contaminated laundry can spread infections. However, current directives for safe laundering are limited to healthcare settings and not reflective of domestic conditions. We aimed to use quantitative microbial risk assessment to evaluate household laundering practices (e.g., detergent selection, washing and drying temperatures, and sanitizer use) relative to log10 reductions in pathogens and infection risks during the clothes sorting, washer/dryer loading, folding and storing steps. METHODS AND RESULTS: Using published data, we characterized laundry infection risks for respiratory and enteric pathogens relative to a single user contact scenario and a 1.0 × 10-6 acceptable risk threshold. For respiratory pathogens, risks following cold water wash temperatures (e.g. median 14.4℃) and standard detergents ranged from 2.2 × 10-5 to 2.2 × 10-7 . Use of advanced, enzymatic detergents reduced risks to 8.6 × 10-8 and 2.2 × 10-11 respectively. For enteric pathogens, however, hot water, advanced detergents, sanitizing agents and drying are needed to reach risk targets. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions provide guidance for household laundry practices to achieve targeted risk reductions, given a single user contact scenario. A key finding was that hand hygiene implemented at critical control points in the laundering process was the most significant driver of infection prevention, additionally reducing infection risks by up to 6 log10 .

Direct and quantitative capture of viable bacteriophages from experimentally contaminated indoor air: A model for the study of airborne vertebrate viruses including SARS-CoV-2.

AIM: The air indoors has profound health implications as it can expose us to pathogens, allergens and particulates either directly or via contaminated surfaces. There is, therefore, an upsurge in marketing of air decontamination technologies, but with no proper validation of their claims. We addressed the gap through the construction and use of a versatile room-sized (25 m3 ) chamber to study airborne pathogen survival and inactivation. METHODS AND RESULTS: Here, we report on the quantitative recovery and detection of an enveloped (Phi6) and a non-enveloped bacteriophage (MS2). The two phages, respectively, acted as surrogates for airborne human pathogenic enveloped (e.g., influenza, Ebola and coronavirus SARS-CoV-2) and non-enveloped (e.g., norovirus) viruses from indoor air deposited directly on the lawns of their respective host bacteria using a programmable slit-to-agar air sampler. Using this technique, two different devices based on HEPA filtration and UV light were tested for their ability to decontaminate indoor air. This safe, relatively simple and inexpensive procedure augments the use of phages as surrogates for the study of airborne human and animal pathogenic viruses. CONCLUSIONS: This simple, safe and relatively inexpensive method of direct recovery and quantitative detection of viable airborne phage particles can greatly enhance their applicattion as surrogates for the study of vertebrate virus survival in indoor air and assessment of technologies for their decontamination. SIGNIFICANCE AND IMPACT OF THE STUDY: The safe, economical and simple technique reported here can be applied widely to investigate the role of indoor air for virus survival and transmission and also to assess the potential of air decontaminating technologies.

A review of Cryptosporidium spp. and their detection in water.

Cryptosporidium spp. are one of the most important waterborne pathogens worldwide and a leading cause of mortality from waterborne gastrointestinal diseases. Detection of Cryptosporidium spp. in water can be very challenging due to their low numbers and the complexity of the water matrix. This review describes the biology of Cryptosporidium spp. and current methods used in their detection with a focus on C. parvum and C. hominis. Among the methods discussed and compared are microscopy, immunology-based methods using monoclonal antibodies, molecular methods including PCR (polymerase chain reaction)-based assays, and emerging aptamer-based methods. These methods have different capabilities and limitations, but one common challenge is the need for better sensitivity and specificity, particularly in the presence of contaminants. The application of DNA aptamers in the detection of Cryptosporidium spp. oocysts shows promise in overcoming these challenges, and there will likely be significant developments in aptamer-based sensors in the near future.

Highly sensitive magnetic-microparticle-based aptasensor for Cryptosporidium parvum oocyst detection in river water and wastewater: Effect of truncation on aptamer affinity.

Many methods have been reported to detect Cryptosporidium parvum (C. parvum) oocysts in the water environment using monoclonal antibodies. Herein, we report the use of DNA aptamers as an alternative ligand. We present the highly sensitive detection of C. parvum oocysts in wastewater samples based on aptamer-conjugated magnetic beads. A previously selected DNA aptamer (R4-6) that binds to C. parvum oocysts with high affinity and selectivity was rationally truncated into two minimer aptamers (Min_Crypto1 and Min_Crypto2), and conjugated to micro-magnetic beads. In flow cytometry tests with phosphate buffer, river water, and wastewater samples, both the minimers showed improved affinity and specificity toward C. parvum oocysts than the parent R4-6. Moreover, Min_Crypto2 showed higher affinity to its target than the parent aptamer when testing in wastewater, indicating superior binding properties in a complex matrix. Using a fluorescence microplate-based assay, and when incubated with different numbers of oocysts, Min_Crypto2 showed a limit of detection as low as 5 C. parvum oocysts in 300 µL of wastewater. Results described here indicate that Min_Crypto2 has superior specificity and sensitivity for the detection of C. parvum oocysts, and has a strong potential to be used successfully in a sensor.

Combating SARS-CoV-2: leveraging microbicidal experiences with other emerging/re-emerging viruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan City, China, late in December 2019 is an example of an emerging zoonotic virus that threatens public health and international travel and commerce. When such a virus emerges, there is often insufficient specific information available on mechanisms of virus dissemination from animal-to-human or from person-to-person, on the level or route of infection transmissibility or of viral release in body secretions/excretions, and on the survival of virus in aerosols or on surfaces. The effectiveness of available virucidal agents and hygiene practices as interventions for disrupting the spread of infection and the associated diseases may not be clear for the emerging virus. In the present review, we suggest that approaches for infection prevention and control (IPAC) for SARS-CoV-2 and future emerging/re-emerging viruses can be invoked based on pre-existing data on microbicidal and hygiene effectiveness for related and unrelated enveloped viruses.

Potential Role of Oral Rinses Targeting the Viral Lipid Envelope in SARS-CoV-2 Infection.

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

Development and application of DNA-aptamer-coupled magnetic beads and aptasensors for the detection of Cryptosporidium parvum oocysts in drinking and recreational water resources.

Environmentally stable and disinfectant-resistant oocysts of Cryptosporidium spp. shed in the feces of infected humans and animals frequently contaminate water resources and are subsequently spread via potable and recreational waters. The current monoclonal-antibody-based methods for detecting them in water are slow, labor-intensive, and demand skills to interpret the results. We have developed DNA-aptamer-based aptasensors, coupled with magnetic beads, to detect and identify the oocysts of C. parvum for monitoring recreational and drinking water sources. A sensitive and specific electrochemical aptasensor (3'-biotinylated R4-6 aptamer) was used as a secondary ligand to bind the streptavidin-coated magnetic beads. This was incorporated into a probe using gold nanoparticle modified screen-printed carbon electrodes. Square wave voltammetry allowed for specific recognition of C. parvum oocysts. The aptamer-coated probes had an oocyst detection limit of 50. It did not bind to the cysts of Giardia duodenalis, another common waterborne pathogen, thus indicating its high specificity for the target pathogen. The system could successfully detect C. parvum oocysts in spiked samples of the raw lake and river waters. Therefore, the combined use of the aptasensor and magnetic beads has the potential to monitor water quality for C. parvum oocysts in field samples without relying on monoclonal antibodies and skill-demanding microscopy.

Complete Genome Sequences of a Diverse Group of 13 Propionibacterium acnes Bacteriophages Isolated from Urban Raw Sewage.

We present complete genome sequences of 13 Propionibacterium acnes phages isolated from urban raw sewage. They belong to the family Siphoviridae, have genome sizes of 29,450.6 ± 256.5 nucleotides and G+C contents of 54.14% ± 0.22% and contain 42 to 45 coding DNA sequences (CDS). Genomic sequences of 9 of 13 phages were divergent by 6 to 10%, distinguishing them as species.

Airborne Pathogens inside Automobiles for Domestic Use: Assessing In-Car Air Decontamination Devices Using Staphylococcus aureus as the Challenge Bacterium.

Family cars represent ∼74% of the yearly global output of motorized vehicles. With a life expectancy of ∼8 decades in many countries, the average person spends >100 min daily inside the confined and often shared space of the car, with exposure to a mix of potentially harmful microbes. Can commercial in-car microbial air decontamination devices mitigate the risk? Three such devices (designated devices 1 to 3) with HEPA filters were tested in the modified passenger cabin (3.25 m3) of a four-door sedan housed within a biosafety level 3 containment facility. Staphylococcus aureus (ATCC 6538) was suspended in a soil load to simulate the presence of body fluids and aerosolized into the car's cabin with a 6-jet Collison nebulizer. A muffin fan (80 mm by 80 mm, with an output of 0.17 m3/min) circulated the air inside. Plates (150 mm diameter) of Trypticase soy agar (TSA), placed inside a programmable slit-to-agar sampler, were held at 36 ± 1°C for 18 to 24 h and examined for CFU. The input dose of the test bacterium, its rate of biological decay, and the log10 reductions by the test devices were analyzed. The arbitrarily set performance criterion was the time in hours a device took for a 3-log10 reduction in the level of airborne challenge bacterium. On average, the level of S. aureus challenge in the air varied between 4.2 log10 CFU/m3 and 5.5 log10 CFU/m3, and its rate of biological decay was -0.0213 ± 0.0021 log10 CFU/m3/min. Devices 1 to 3 took 2.3, 1.5, and 9.7 h, respectively, to meet the performance criterion. While the experimental setup was tested using S. aureus as an archetypical airborne pathogen, it can be readily adapted to test other types of pathogens and technologies.IMPORTANCE This study was designed to test the survival of airborne pathogens in the confined and shared space of a family automobile as well as to assess claims of devices marketed for in-car air decontamination. The basic experimental setup and the test protocols reported are versatile enough for work with all major types of airborne human pathogens and for testing a wide variety of air decontamination technologies. This study could also lay the foundation for a standardized test protocol for use by device makers as well as regulators for the registration of such devices.

Generic aspects of the airborne spread of human pathogens indoors and emerging air decontamination technologies.

Indoor air can be an important vehicle for a variety of human pathogens. This review provides examples of airborne transmission of infectious agents from experimental and field studies and discusses how airborne pathogens can contaminate other parts of the environment to give rise to secondary vehicles leading air-surface-air nexus with possible transmission to susceptible hosts. The following groups of human pathogens are covered because of their known or potential airborne spread: vegetative bacteria (staphylococci and legionellae), fungi (Aspergillus, Penicillium, and Cladosporium spp and Stachybotrys chartarum), enteric viruses (noro- and rotaviruses), respiratory viruses (influenza and coronaviruses), mycobacteria (tuberculous and nontuberculous), and bacterial spore formers (Clostridium difficile and Bacillus anthracis). An overview of methods for experimentally generating and recovering airborne human pathogens is included, along with a discussion of factors that influence microbial survival in indoor air. Available guidelines from the U.S. Environmental Protection Agency and other global regulatory bodies for the study of airborne pathogens are critically reviewed with particular reference to microbial surrogates that are recommended. Recent developments in experimental facilities to contaminate indoor air with microbial aerosols are presented, along with emerging technologies to decontaminate indoor air under field-relevant conditions. Furthermore, the role that air decontamination may play in reducing the contamination of environmental surfaces and its combined impact on interrupting the risk of pathogen spread in both domestic and institutional settings is discussed.

Mathematical modeling and simulation of bacterial distribution in an aerobiology chamber using computational fluid dynamics.

BACKGROUND: Computer-aided design and draft, along with computer-aided engineering software, are used widely in different fields to create, modify, analyze, and optimize designs. METHODS: We used computer-aided design and draft software to create a 3-dimensional model of an aerobiology chamber built in accordance with the specifications of the 2012 guideline from the Environmental Protection Agency for studies on survival and inactivation of microbial pathogens in indoor air. The model was used to optimize the chamber's airflow design and the distribution of aerosolized bacteria inside it. RESULTS: The findings led to the identification of an appropriate fan and its location inside the chamber for uniform distribution of microbes introduced into the air, suitability of air sample collection from the center of the chamber alone as representative of its bacterial content, and determination of the influence of room furnishings on airflow patterns inside the chamber. CONCLUSIONS: The incorporation of this modeling study's findings could further improve the design of the chamber and the predictive value of the experimental data using it. Further, it could make data generation faster and more economical by eliminating the need for collecting air samples from multiple sites in the chamber.

Indoor air as a vehicle for human pathogens: Introduction, objectives, and expectation of outcome.

Airborne spread of pathogens can be rapid, widespread, and difficult to prevent. In this international workshop, a panel of 6 experts will expound on the following: (1) the potential for indoor air to spread a wide range of human pathogens, plus engineering controls to reduce the risk for exposure to airborne infectious agents; (2) the behavior of aerosolized infectious agents indoors and the use of emerging air decontamination technologies; (3) a survey of quantitative methods to recover infectious agents and their surrogates from indoor air with regard to survival and inactivation of airborne pathogens; (4) mathematical models to predict the movement of pathogens indoors and the use of such information to optimize the benefits of air decontamination technologies; and (5) synergy between different infectious agents, such as legionellae and fungi, in the built environment predisposing to possible transmission-related health impacts of aerosolized biofilm-based opportunistic pathogens. After the presentations, the panel will address a set of preformulated questions on selection criteria for surrogate microbes to study the survival and inactivation of airborne human pathogens, desirable features of technologies for microbial decontamination of indoor air, knowledge gaps, and research needs. It is anticipated that the deliberations of the workshop will provide the attendees with an update on the significance of indoor air as a vehicle for transmitting human pathogens with a brief on what is currently being done to mitigate the risks from airborne infectious agents.

Decontamination of indoor air to reduce the risk of airborne infections: Studies on survival and inactivation of airborne pathogens using an aerobiology chamber.

BACKGROUND: Although indoor air can spread many pathogens, information on the airborne survival and inactivation of such pathogens remains sparse. METHODS: Staphylococcus aureus and Klebsiella pneumoniae were nebulized separately into an aerobiology chamber (24.0 m3). The chamber's relative humidity and air temperature were at 50% ± 5% and 20°C ± 2°C, respectively. The air was sampled with a slit-to-agar sampler. Between tests, filtered air purged the chamber of any residual airborne microbes. RESULTS: The challenge in the air varied between 4.2 log10 colony forming units (CFU)/m3 and 5.0 log10 CFU/m3, sufficient to show a ≥3 log10 (≥99.9%) reduction in microbial viability in air over a given contact time by the technologies tested. The rates of biologic decay of S aureus and K pneumoniae were 0.0064 ± 0.00015 and 0.0244 ± 0.009 log10 CFU/m3/min, respectively. Three commercial devices, with ultraviolet light and HEPA (high-efficiency particulate air) filtration, met the product efficacy criterion in 45-210 minutes; these rates were statistically significant compared with the corresponding rates of biologic decay of the bacteria. One device was also tested with repeated challenges with aerosolized S aureus to simulate ongoing fluctuations in indoor air quality; it could reduce each such recontamination to an undetectable level in approximately 40 minutes. CONCLUSIONS: The setup described is suitable for work with all major classes of pathogens and also complies with the U.S. Environmental Protection Agency's guidelines (2012) for testing air decontamination technologies.