Martin A. Page

Inactivation of bacteriophage MS2 with potassium ferrate(VI)

Ferrate [Fe(VI); FeO(4)(2-)] is an emerging oxidizing agent capable of controlling chemical and microbial water contaminants. Here, inactivation of MS2 coliphage by Fe(VI) was examined. The inactivation kinetics observed in individual batch experiments was well described by a Chick-Watson model with first-order dependences on disinfectant and infective phage concentrations. The inactivation rate constant k(i) at a Fe(VI) dose of 1.23 mgFe/L (pH 7.0, 25 °C) was 2.27(±0.05) L/(mgFe × min), corresponding to 99.99% inactivation at a Ct of ~4 (mgFe × min)/L. Measured k(i) values were found to increase with increasing applied Fe(VI) dose (0.56-2.24 mgFe/L), increasing temperature (5-30 °C), and decreasing pH conditions (pH 6-11). The Fe(VI) dose effect suggested that an unidentified Fe byproduct also contributed to inactivation. Temperature dependence was characterized by an activation energy of 39(±6) kJ mol(-1), and k(i) increased >50-fold when pH decreased from 11 to 6. The pH effect was quantitatively described by parallel reactions with HFeO(4)(-) and FeO(4)(2-). Mass spectrometry and qRT-PCR analyses demonstrated that both capsid protein and genome damage increased with the extent of inactivation, suggesting that both may contribute to phage inactivation. Capsid protein damage, localized in the two regions containing oxidant-sensitive cysteine residues, and protein cleavage in one of the two regions may facilitate genome damage by increasing Fe(VI) access to the interior of the virion.

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine.

ABSTRACT Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

Kinetics of adenovirus type 2 inactivation with free chlorine

The objective of this study was to elucidate the effects of pH, temperature, and other relevant water quality parameters on the kinetics of adenovirus serotype 2 inactivation with free chlorine. Over a pH range of 6.5-10, a temperature range of 1-30 degrees C, and in a variety of water types, free chlorine was highly effective against adenovirus type 2. Its disinfection efficacy decreased with increasing pH and decreasing temperature, yet was unaffected by hardness and buffering species. Under the most challenging conditions investigated in this study (pH 10, 1 degrees C), a four-log reduction of adenovirus viability would be achieved at a CT value of 2.6mgCl(2)min/L. The inactivation kinetics was characterized by three phases of inactivation under most conditions. The first phase resulted from a reaction involving primarily the hypochlorous acid species and was characterized by rapid inactivation of viruses to a limit that increased with decreasing pH and increasing temperature. After reaching this limit, adenovirus exhibited two subsequent phases of inactivation at lesser rates that were not affected by temperature or pH. As with the first phase of kinetics, a limit of inactivation was approached in the second phase that decreased with increasing pH, and after which the kinetics was characterized by a third and final phase. An inactivation model consistent with these observations was found to provide adequate representation for the free chlorine inactivation of adenovirus serotype 2 as well as that reported in the literature for other adenovirus serotypes.

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

ABSTRACT There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages

ABSTRACT Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viable B. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxAB in an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 105 CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 104 CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Characterization of the activity of the spore cortex lytic enzyme CwlJ1

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography‐mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N‐acetylmuramoyl‐l‐alanine amidase. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid‐δ‐lactam‐containing glycan chains and preference for the tetrapeptide side chain. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria. Biotechnol. Bioeng. 2015;112: 1365–1375.

Nanoscale Ion Pump Derived from a Biological Water Channel

Biological molecular machines perform the work of supporting life at the smallest of scales, including the work of shuttling ions across cell boundaries and against chemical gradients. Systems of artificial channels at the nanoscale can likewise control ionic concentration by way of ionic current rectification, species selectivity, and voltage gating mechanisms. Here, we theoretically show that a voltage-gated, ion species-selective, and rectifying ion channel can be built using the components of a biological water channel aquaporin. Through all-atom molecular dynamics simulations, we show that the ionic conductance of a truncated aquaporin channel nonlinearly increases with the bias magnitude, depends on the channels orientation, and is highly cation specific but only for one polarity of the transmembrane bias. Further, we show that such an unusually complex response of the channel to transmembrane bias arises from mechanical motion of a positively charged gate that blocks cation transport. By combining two truncated aquaporins, we demonstrate a molecular system that pumps ions against their chemical gradients when subject to an alternating transmembrane bias. Our work sets the stage for future biomimicry efforts directed toward reproducing the function of biological ion pumps using synthetic components.

Bench-scale aerosol filtration test system and evaluation of an acoustic bioaerosol removal device for indoor air streams

It is important to have well-defined, reproducible methods to evaluate and compare newly developed air filtration equipment. To facilitate accurate assessment of air purification devices at the bench scale, an experimental system was designed, built, and documented to evaluate particulate removal efficiency (PRE) of air filtration devices based on principles used in ASHRAE standards. The system was then carefully characterized and used to evaluate PRE and total energy consumption of a novel acoustically enhanced impaction (AEI) air purification device. The AEI device demonstrated 99.998% PRE of 0.5–1.5 μm diameter KCl particles while causing a 120 Pa pressure drop and requiring a total of 3.0 W/l of air treated at indoor ambient conditions. A single element of the AEI device operated in a biological safety level 2 facility was then used to evaluate PRE of bioaerosol consisting of Bacillus cereus (BC) spores. PRE of BC was 99.86 ± 0.05% at indoor ambient temperature and pressure. This research describes the use of the Bench-scale Air Purification Testing and Evaluation Chamber (BAP-TEC) to experimentally evaluate and compare PRE and total energy requirements of novel air purification devices at the bench scale (280–1400 alpm). Further, an AEI device containing a fibrous filter media and high intensity sound field in the same control volume is evaluated using the BAP-TEC. Temporally resolved PRE of a bioaerosol by the AEI is also presented. Copyright 2013 American Association for Aerosol Research

Thiol Reactivity Analyses To Predict Mammalian Cell Cytotoxicity of Water Samples

An in chemico high throughput assay based on N-acetylcysteine was developed and used in conjunction with previous and new mammalian cell cytotoxicity data. Our objective was to derive an empirical equation with confidence levels for mammalian cell cytotoxicity prediction. Modeling data included 16 unique sources of waters and wastewaters of distinct water qualities to encompass a wide range of real environmental samples. This approach provides a quick screen to identify those water and wastewaters that could be prioritized for in depth analytical biological analyses and toxicity. The resulting model can serve as a preliminary convenient tool to screen for potential mammalian cell cytotoxicity in organic extracts of a wide variety of water samples.