Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods.

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Insights into the Antibacterial Mechanism of Action of Chelating Agents by Selective Deprivation of Iron, Manganese, and Zinc.

Bacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese, and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of 11 chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. The following four distinct effects were uncovered: (i) no apparent alteration in metal composition, (ii) depletion of manganese alongside reductions in iron and zinc levels, (iii) reduced zinc levels with a modest reduction in manganese, and (iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution; however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation. IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli. The chelators were studied both individually and in pairs, with the majority producing synergistic effects in combinations that maximize antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.

Contaminated hands or inanimate surfaces can act as a source of infection during outbreaks of human norovirus infection. We evaluated the virucidal efficacy of seven hand sanitizers containing various active ingredients, such as ethanol, triclosan, and chlorhexidine, and compared their effectiveness against feline calicivirus (FCV), murine norovirus (MNV), and a GII.4 norovirus fecal extract. We also tested the efficacy of 50, 70, and 90% of ethanol and isopropanol. Reduction of viral infectivity was measured by plaque assay, and the number of genomic copies was determined with a TaqMan real-time reverse transcription PCR assay. Based on the results of a quantitative suspension test, only one ethanol-based product (72% ethanol, pH 2.9) and one triclosan-based product (0.1% triclosan, pH 3.0) reduced the infectivity of both MNV and FCV (by >2.6 and ≥3.4 log units, respectively). Four of the seven products were effective against either MNV or FCV, whereas chlorhexidine was ineffective against both viruses. For these hand sanitizers, no correlation was found between reduced infectivity and decline of viral RNA. Ethanol and isopropanol concentrations ≥70% reduced the infectivity of MNV by ≥2.6 log units, whereas 50 and 70% ethanol reduced the infectivity of FCV by ≥2.2 log units after exposure for 5 min. The susceptibility of FCV to low pH and the relative high susceptibility of MNV to alcohols suggest that both surrogate viruses should be considered for in vitro testing of hand sanitizers.

Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm.

Many factors that are not fully understood may influence the effectiveness of sanitizer treatments for eliminating pathogens and spoilage microorganisms in food or detergent residues or in biofilms on food contact surfaces. This study was done to determine the sensitivities of Pseudomonas cells and Bacillus cereus cells and spores suspended in a liquid dishwashing detergent and inoculated onto the surface of stainless steel to treatment with chlorine, chlorine dioxide, and a commercial produce sanitizer (Fit). Cells and spores were incubated in a liquid dishwashing detergent for 16 to 18 h before treatment with sanitizers. At 50 microg/ml, chlorine dioxide killed a significantly higher number of Pseudomonas cells (3.82 log CFU/ml) than did chlorine (a reduction of 1.34 log CFU/ml). Stainless steel coupons were spot inoculated with Pseudomonas cells and B. cereus cells and spores, with water and 5% horse serum as carriers. Chlorine was more effective than chlorine dioxide in killing cells and spores of B. cereus suspended in horse serum. B. cereus biofilm on stainless steel coupons that were treated with chlorine dioxide or chlorine at 200 microg/ml had total population reductions (vegetative cells plus spores) of > or = 4.42 log CFU per coupon; the number of spores was reduced by > or = 3.80 log CFU per coupon. Fit (0.5%) was ineffective for killing spot-inoculated B. cereus and B. cereus in biofilm, but treatment with mixtures of Fit and chlorine dioxide caused greater reductions than did treatment with chlorine dioxide alone. In contrast, when chlorine was combined with Fit, the lethality of chlorine was completely lost. This study provides information on the survival and sanitizer sensitivity of Pseudomonas and B. cereus in a liquid dishwashing detergent, on the surface of stainless steel, and in a biofilm. This information will be useful for developing more effective strategies for cleaning and sanitizing contact surfaces in food preparation and processing environments.

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis.

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.

Influence of variations in methodology on populations of Listeria monocytogenes recovered from lettuce treated with sanitizers.

The elimination of Listeria monocytogenes inoculated onto a piece of cut iceberg lettuce (3.8 by 3.8 cm) by treatment with chlorinated water (200 micrograms/ml free chlorine) and a 0.5% (wt/vol) solution of FIT Professional Line Antibacterial Cleaner (FIT) was investigated. The efficacy of the two sanitizers was not influenced by the composition of the medium used to culture the L. monocytogenes used in the inocula, the number of strains in the inoculum, or the recovery medium used to enumerate the pathogen on lettuce after treatment. Drying inoculum on lettuce for 45 min at 37 degrees C caused more cells to die or not be retrieved compared with drying inoculum for 30 min at 25 degrees C. However, the percentage of cells in the inoculum recovered from lettuce treated with chlorine or FIT was not significantly different, regardless of the drying method. Stomaching, homogenizing, or stomaching followed by homogenizing lettuce treated with sanitizers resulted in recovery of similar numbers of L. monocytogenes, indicating that stomaching and homogenizing are equivalent in extracting cells; the sequential use of both processing methods did not substantially increase the efficiency of recovery. Washing lettuce with water or treating lettuce with 200 micrograms/ml chlorine or FIT resulted in decreases in populations of 0.60, 1.76, and 1.51 log CFU per lettuce piece, respectively, regardless of variations in test parameters. Reductions caused by sanitizers were significantly greater (alpha = 0.05) than that observed for water but not significantly different from each other. It is concluded that evaluation of sanitizers for their efficacy in killing L. monocytogenes on lettuce can be determined by spot inoculating 50 microliters of a five-strain mixture of cells from 24-h cultures suspended in 5% horse serum albumen, followed by drying the inoculum for 45 min at 37 degrees C, treatment by submerging in 50 ml of sanitizer for 5 min, stomaching samples in 50 ml of Dey-Engley neutralizing broth for 2 min, and enumerating survivors on modified Oxford medium.

Anaerobic biodegradation of aliphatic polyesters: poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) and poly(epsilon-caprolactone).

Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), PHBO, represents a class of PHA copolymers that contain both short-chain-length and medium-chain-length repeat units. Radiolabeled and cold PHBO, containing 90 mol % 3-hydroxybutyrate and 10 mol % 3-hydroxyoctanoate were chemically synthesized using a new difunctional alkoxyzinc initiator. (14)C-PHBO was incubated with samples of anaerobic digester sludge, septage, freshwater sediment, and marine sediment under conditions resembling those in situ. In addition, it was incubated in laboratory-scale landfill reactors. (14)C-PCL (poly-epsilon-caprolactone) was incubated with anaerobic digester sludge and in landfill reactors. Biodegradation was determined by measuring generation of (14)CO(2) and (14)CH(4) resulting from mineralization of the radiolabeled polymers. PHBO was extensively mineralized in digester sludge, septage sediments, and the landfill reactors, with half-lives less than 30 days. PCL was not significantly mineralized in digester sludge over 122 days. In the landfill reactors, PCL mineralization was slow and was preceded by a long lag period (>200 days), suggesting that PCL mineralization is limited by its rate of hydrolysis. The results indicate that PHBO is practically biodegradable in the major anaerobic habitats that it may enter. In contrast, anaerobic biodegradation of PCL is less ubiquitous and much slower.