Miniaturizing Wet Scrubbers for Aerosolized Droplet Capture.

Aerosols dispersed and transmitted through the air (e.g., particulate matter pollution and bioaerosols) are ubiquitous and one of the leading causes of adverse health effects and disease transmission. A variety of sampling methods (e.g., filters, cyclones, and impactors) have been developed to assess personal exposures. However, a gap still remains in the accessibility and ease-of-use of these technologies for people without experience or training in collecting airborne samples. Additionally, wet scrubbers (large non-portable industrial systems) utilize liquid sprays to remove aerosols from the air; the goal is to "scrub" (i.e., clean) the exhaust of industrial smokestacks, not collect the aerosols for analysis. Inspired by wet scrubbers, we developed a device fundamentally different from existing portable air samplers by using aerosolized microdroplets to capture aerosols in personal spaces (e.g., homes, offices, and schools). Our aerosol-sampling device is the size of a small teapot, can be operated without specialized training, and features a winding flow path in a supersaturated relative humidity environment, enabling droplet growth. The integrated open mesofluidic channels shuttle coalesced droplets to a collection chamber for subsequent sample analysis. Here, we present the experimental demonstration of aerosol capture in water droplets. An iterative study optimized the non-linear flow manipulating baffles and enabled an 83% retention of the aerosolized microdroplets in the confined volume of our device. As a proof-of-concept for aerosol capture into a liquid medium, 0.5-3 µm model particles were used to evaluate aerosol capture efficiency. Finally, we demonstrate that the device can capture and keep a bioaerosol (bacteriophage MS2) viable for downstream analysis.

Modifications of IL-6 by Hypochlorous Acids: Effects on Receptor Binding.

Interleukin-6 (IL-6) has been implicated in the pathogenesis of inflammatory events including those seen with COVID-19 patients. Positive clinical responses to monoclonal antibodies directed against IL-6 receptors (IL-6Rs) suggest that interference with IL-6-dependent activation of pro-inflammatory pathways offers a useful approach to therapy. We exposed IL-6 to hypochlorous acid (HOCl) in vitro at concentrations reported to develop in vivo. After HOCl treatment, binding of IL-6 to IL-6R was reduced in a dose-dependent manner using a bioassay with human cells engineered to provide a luminescence response to signal transduction upon receptor activation. Similar results followed the exposure of IL-6 to N-chlorotaurine (NCT) and hypobromous acid (HOBr), two other reactive species produced in vivo. SDS-PAGE analysis of HOCl-treated IL-6 showed little to no fragmentation or aggregation up to 1.75 mM HOCl, suggesting that the modifications induced at concentrations below 1.75 mM took place on the intact protein. Mass spectrometry of trypsin-digested fragments identified oxidative changes to two amino acid residues, methionine 161 and tryptophan 157, both of which have been implicated in receptor binding of the cytokine. Our findings suggest that exogenous HOCl and NCT might bring about beneficial effects in the treatment of COVID-19. Further studies on how HOCl and HOBr and their halogenated amine derivatives interact with IL-6 and related cytokines in vivo may open up alternative therapeutic interventions with these compounds in COVID-19 and other hyperinflammatory diseases.

Modification of Major Contributors Responsible for Latrine Malodor on Exposure to Hypochlorous Acid: The Potential for Simultaneously Impacting Odor and Infection Hazards to Encourage Latrine Use.

Open defecation remains a common practice in developing countries and leads to high incidence and prevalence of acute gastroenteritis, which is most often caused by human noroviruses (human NoV). Encouraging the use of toilets and pit latrines is one method of improving sanitation; however, it is often hindered by not only cultural traditions but also from a reluctance to use latrines and toilets due to their odor and impression of uncleanliness. In an effort to establish new means to encourage toilet and latrine use, laboratory experiments tested the ability of hypochlorous acid (HOCl) to modify the malodorous compounds identified in the air in latrines in developing countries (indole, p-cresol, dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and butyric acid) and inactivate MS2 bacteriophage, a surrogate for human NoV. After 5 minutes, > 94% of indole, p-cresol, DMDS, and DMTS was modified as determined by high-pressure liquid chromatography in the presence of 100 ppm HOCl. A log10 reduction value (LRV) greater than 6 was seen for MS2 bacteriophage after 5 minutes of exposure to 100 ppm HOCl in solution. Sensory studies indicated that there was a significant difference (P ≤ 0.05) between the untreated and HOCl-treated samples for all five malodorous compounds tested. The findings suggest that introduction of HOCl into the headspace air could encourage latrine and toilet use. Optimization of HOCl dosing in air to accomplish both odor control and reduction of infectious hazards is worthy of further study.

Association between Mycobacterium avium Complex Pulmonary Disease and Mycobacteria in Home Water and Soil.

Rationale: Nontuberculous mycobacteria (NTM), including Mycobacterium avium complex (MAC), are emerging pathogens that can opportunistically cause debilitating pulmonary disease in susceptible human hosts. Potential sources of exposure in homes include point-of-use water sources, such as taps and showerheads, as well as gardening soils. The relative human health impacts of NTM in these home environments remain poorly understood.Objectives: This study tested associations between MAC pulmonary disease and NTM colonization of five potential point-of-use sources of pathogen exposure in homes.Methods: A case-control study was conducted of Washington and Oregon residents who had been diagnosed with MAC pulmonary disease, and population controls were matched by age, sex, and geography. Samples were collected from bathroom faucets, kitchen faucets, shower aerosols, indoor soil, and outdoor soil. Mycobacteria in environmental samples were identified in a blinded fashion by using bacteriological culture combined with polymerase chain reaction. The isolation of NTM from case homes (n = 56) versus control homes (n = 51) was quantitatively compared using conditional logistic regression models with adjustment for potential confounding variables.Results: NTM were isolated from shower aerosols collected in case homes more often than in control homes. An adjusted conditional logistic regression analysis showed that NTM isolation from shower aerosols had a high odds ratio associated with disease (odds ratio, 4.0; 95% confidence interval, 1.2-13). Other home environmental samples (tap water, soils) did not exhibit this association.Conclusions: The results implicate shower aerosols as uniquely significant sources of NTM exposure in homes.

Avian influenza virus ecology and evolution through a climatic lens.

Avian influenza virus (AIV) is a major health threat to both avian and human populations. The ecology of the virus is driven by numerous factors, including climate and avian migration patterns, yet relatively little is known about these drivers. Long-distance transport of the virus is tied to inter- and intra-continental bird migration, while enhanced viral reassortment is linked to breeding habitats in Beringia shared by migrant species from North America and Asia. Furthermore, water temperature, pH, salinity, and co-existing biota all impact the viability and persistence of the virus in the environment. Changes in climate can potentially alter the ecology of AIV through multiple pathways. Warming temperatures can change the timing and patterns of bird migration, creating novel assemblages of species and new opportunities for viral transport and reassortment. Water temperature and chemistry may also be altered, resulting in changes in virus survival. In this review, we explain how these shifts have the potential to increase viral persistence, pathogenicity, and transmissibility and amplify the threat of pandemic disease in animal and human hosts. Better understanding of climatic influences on viral ecology is essential to developing strategies to limit adverse health effects in humans and animals.

Molecular Viability Testing of UV-Inactivated Bacteria.

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli, Aeromonas hydrophila, and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment.IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection.

Dead or alive: molecular assessment of microbial viability.

Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.

Biosynthetic enhancement of the detection of bacteria by the polymerase chain reaction.

Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices.

Enhanced inactivation of Bacillus subtilis spores during solar photolysis of free available chlorine.

Aqueous free available chlorine (FAC) can be photolyzed by sunlight and/or artificial UV light to generate various reactive oxygen species, including HO(•) and O((3)P). The influence of this chemistry on inactivation of chlorine-resistant microorganisms was investigated using Bacillus subtilis endospores as model microbial agents and simulated and natural solar radiation as light sources. Irradiation of FAC solutions markedly enhanced inactivation of B. subtilis spores in 10 mM phosphate buffer; increasing inactivation rate constants by as much as 600%, shortening inactivation curve lag phase by up to 73% and lowering CTs required for 2 log10 inactivation by as much as 71% at pH 8.0 and 10 °C. Similar results were observed at pH 7.4 and 10 °C in two drinking water samples with respective DOC concentrations and alkalinities of 0.6 and 1.2 mg C/L and 81.8 and 17.1 mg/L as CaCO3. Solar radiation alone did not inactivate B. subtilis spores under the conditions investigated. A variety of experimental data indicate that the observed enhancements in spore inactivation can be attributed to the concomitant attack of spores by HO(•) and O3, the latter of which was found to accumulate to micromolar concentrations during simulated solar irradiation of 10 mM phosphate buffer (pH 8, 10 °C) containing [FAC]0 = 8 mg/L as Cl2.

Isolation and characterization of methicillin-resistant Staphylococcus aureus from fire stations in two northwest fire districts.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated and characterized from environmental surfaces of two fire stations from two independent districts in the northwestern United States. After the first sampling and before the second sampling, education was provided, additional signage was added, and changes in disinfection protocols were put in place. Nasal carriage of MRSA was determined at the second sampling. METHODS: Environmental samples were collected using SANICULT swabs and RODAC plates. Biochemical tests and 16S rRNA sequencing confirmed MRSA isolates. Antimicrobial susceptibility testing was performed, and the mecA gene, multilocus sequence typing, and SCCmec typing were determined by polymerase chain reaction, sequencing, and pulsed-field gel electrophoresis analysis. RESULTS: MRSA was isolated from 44 of 1,064 samples examined (4.1%) and included USA300 isolates. The same strains of MRSA were found in both the garage (ie, medic and fire trucks and protective clothing) and the living quarters. Nasal carriage of MRSA from one fire district was 22.5%. CONCLUSION: Community-like and hospital-like MRSA were isolated from the environmental samples. The majority of the nasal MRSA/S aureus isolates were genetically related to the environmental MRSA strains, suggesting possible transmission between personnel and the environmental surfaces. Further research is needed to verify this hypothesis.

Molecular detection of viable bacterial pathogens in water by ratiometric pre-rRNA analysis.

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples.

Characterization of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococcus spp. isolated from US West Coast public marine beaches.

OBJECTIVES: The aim of this study was to isolate and characterize methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus spp. (MRCoNS) from marine water and intertidal beach sand from public beaches in Washington State, USA. METHODS: Fifty-one staphylococci from Washington State beaches were characterized using antimicrobial susceptibility testing, carriage of acquired tetracycline and/or macrolide resistance genes, staphylococcal cassette chromosome mec (SCCmec) typing, the BBL Crystal Gram-Positive ID System and/or 16S rRNA sequencing, coagulase test and multilocus sequence typing (MLST) for MRSA. RESULTS: Five multidrug-resistant MRSA SCCmec type I, of which three were MLST type ST45, one ST59 and one a new MLST type, ST1405, plus one susceptible non-typeable (NT) MRSA ST30 were characterized. Thirty-three MRCoNS isolates, representing 21 strains from 9 Staphylococcus spp., carried a range of SCCmec types [I (2), II (6), III (3), V (2), I/II (1) and NT (7)] and varied in their antibiotic susceptibility to other antibiotic classes and carriage of acquired tetracycline/macrolide resistance gene(s). MRSA and MRCoNS donors co-transferred tet(M) and erm(A) genes to an Enterococcus faecalis recipient at a frequency of 10(-8). CONCLUSIONS: This is the first report of MRSA and MRCoNS isolated from marine water and intertidal beach sand. The MLST types and antibiotic carriage of five MRSA isolates were similar to hospital MRSA isolates rather than US community-acquired MRSA isolates. Our results suggest that public marine beaches may be a reservoir for transmission of MRSA to beach visitors as well as an ecosystem for exchange of antibiotic resistance genes among staphylococci and related genera.

Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RT-PCR and TaqMan RT-PCR.

Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry.