Heterologous expression of NoxA confers aerotolerance in Clostridium sporogenes.

Clostridium is a genus of gram-positive obligate anaerobic bacteria. Some species of Clostridium, including Clostridium sporogenes, may be of use in bacteria-mediated cancer therapy. Spores of Clostridium are inert in healthy normoxic tissue but germinate when in the hypoxic regions of solid tumors, causing tumor regression. However, such treatments fail to completely eradicate tumors partly because of higher oxygen levels at the tumor's outer rim. In this study, we demonstrate that a degree of aerotolerance can be introduced to C. sporogenes by transfer of the noxA gene from Clostridium aminovalericum. NoxA is a water-forming NADH oxidase enzyme, and so has no detrimental effect on cell viability. In addition to its potential in cancer treatment, the noxA-expressing strain described here could be used to alleviate challenges related to oxygen sensitivity of C. sporogenes in biomanufacturing.

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.

Generalizable epileptic seizures prediction based on deep transfer learning.

Predicting seizures before they happen can help prevent them through medication. In this research, first, a total of 22 features were extracted from 5-s segmented EEG signals. Second, tensors were developed as inputs for different deep transfer learning models to find the best model for predicting epileptic seizures. The effect of Pre-ictal state duration was also investigated by selecting four different intervals of 10, 20, 30, and 40 min. Then, nine models were created by combining three ImageNet convolutional networks with three classifiers and were examined for predicting seizures patient-dependently. The Xception convolutional network with a Fully Connected (FC) classifier achieved an average sensitivity of 98.47% and a False Prediction Rate (FPR) of 0.031 h-1 in a 40-min Pre-ictal state for ten patients from the European database. The most promising result of this study was the patient-independent prediction of epileptic seizures; the MobileNet-V2 model with an FC classifier was trained with one patient's data and tested on six other patients, achieving a sensitivity rate of 98.39% and an FPR of 0.029 h-1 for a 40-min Pre-ictal scheme.

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.

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.

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.

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.

Airborne Infectious Agents and Other Pollutants in Automobiles for Domestic Use: Potential Health Impacts and Approaches to Risk Mitigation.

The world total of passenger cars is expected to go from the current one billion to >2.5 billion by 2050. Cars for domestic use account for ~74% of the world's yearly production of motorized vehicles. In North America, ~80% of the commuters use their own car with another 5.6% travelling as passengers. With the current life-expectancy of 78.6 years, the average North American spends 4.3 years driving a car! This equates to driving 101 minutes/day with a lifetime driving distance of nearly 1.3 million km inside the confined and often shared space of the car with exposure to a mix of potentially harmful pathogens, allergens, endotoxins, particulates, and volatile organics. Such risks may increase in proportion to the unprecedented upsurge in the numbers of family cars globally. Though new technologies may reduce the levels of air pollution from car exhausts and other sources, they are unlikely to impact our in-car exposure to pathogens. Can commercial in-car air decontamination devices reduce the risk from airborne infections and other pollutants? We lack scientifically rigorous protocols to verify the claims of such devices. Here we discuss the essentials of a customized aerobiology facility and test protocols to assess such devices under field-relevant conditions.