The Resistance of Bacillus Spores: Implications for the Strain-Specific Response to High-Performance Disinfectants.

Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.

In vitro acaricidal activity of several natural products against ibex-derived Sarcoptes scabiei.

In this study we analysed the effect of the temperature, diverse strains of Bacillus thuringiensis, Lysinibacillus sphaericus and nanoformulations with essential plant oils (EONP) on the survival of Sarcoptes scabiei mites derived from naturally-infested Iberian ibex (Capra pyrenaica). In general, mites maintained at 12ºC survived more than those maintained at 35ºC (40.7 hr and 31.2 hr, respectively). Mites with no treatment survived 27.6 h on average. Mites treated with B. thuringiensis serovar. konkukian and geranium EONP showed significant reduction in their survival. Despite the fact that these agents seem to be promising candidates for controlling sarcoptic mange in the field, further research is still needed to get stable, efficient and eco-friendly acaricides.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

Anticoagulation and antibacterial properties of heparinized nanosilver with different morphologies.

Synthesis and characterization of nanoparticles with different morphologies coupled to minimal chemical interventions for sustainable applications is one of the contemporary topics in the field of nanotechnology. In the current study, heparinized silver nanoparticles were synthesized using a chemical reduction method. Different concentrations of heparin were used to investigate its role in the stability and morphological properties of silver nanoparticles. Interestingly, it has been observed that the concentration of the stabilizing agent heparin plays a pivotal role in dictating the size and shape of the nanosilver. As visualized under a transmission electron microscope, nanosilver with different morphological states such as triangles, truncated triangles, hexagon, and spheres has been experimentally trapped. Such modular property of heparin coated nanosilver has also exhibited substantial differences in their anticoagulation and antimicrobial activities.

New chitosan derivatives inspired on heterocyclic anhydride of potential bioactive for medical applications.

In the present work new chitosan derivatives inspired heterocyclic anhydride were prepared to improve the biological activities of chitosan via imidization reaction of chitosan (CS) and N-(1,3-dioxoisoindolin-2-yl)-1,3-dioxo-1,3-dihydroiso-benzofuran-5-carboxamide (5) to yield amic acid CS-6 at room temperature and imide CS-8 thermally. However, the reaction between (CS) and anhydride (5) in presence of sodium tripolyphosphate (TPP) or Poly (ethylene glycol) diglycidyl ether (PEGDG) at room temperature yielded CS-6 NPs and CS-7 respectively. The structure of new chitosan derivatives was characterized using morphological and spectroscopic analyses. From evaluation of the biological activities, the greatest enzymatic inhibitory for trypsin and α-chymotrypsin revealed by CS-7 at 88.33 ± 2.27 and 79.63 ± 3.16% respectively. Furthermore, the highest inhibition zones, (MIC) and (MBC) against S. aureus and B. subtilis recorded by CS-6 NPs at 21 ± 0.75, 22 ± 0.98 mm, 19.5, 19.5, 38 and 38 ppm respectively. Additionally, CS-8 displayed the best cell growth inhibition against vero cell line at 93.17 ± 0.29%.

Compatibility of Bt biopesticides and adjuvants for Spodoptera frugiperda control.

Spodoptera frugiperda is a pest of economic importance for several crops with resistance reports to Bt crops and pesticides. Eco-friendly Bt biopesticides may be an alternative to chemical insecticides due to their selectivity and specificity. However, the efficacy of Bt biopesticides may be influenced by the association with other chemicals, such as adjuvants. This study evaluated the compatibility and toxicity of Bt biopesticides mixed with adjuvants for the control of S. frugiperda. The treatments included the association of Dipel SC and Dipel PM with adjuvants. Compatibility tests were used to evaluate the Bt mixture. Bt suspensions obtained from mixtures of Bt and adjuvants at 106 and 3 × 108 spores/mL-1 were used to evaluate S. frugiperda mortality and distilled water was used as the control. The addition of the adjuvant LI increased growth and sporulation, indicating compatibility with Bt biopesticides. The other adjuvants were toxic to reducing Bt growth and sporulation. Only the mixture of Bt with LI and Bt alone was effective to S. frugiperda. The addition of adjuvants to Bt biopesticide affect the Bt sporulation, growth and mortality.

The Penicillin-Binding Protein PbpP Is a Sensor of ß-Lactams and Is Required for Activation of the Extracytoplasmic Function σ Factor σP in Bacillus thuringiensis.

ß-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. ß-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to ß-lactams complicates the treatment of bacterial infections. In recent years, the spread of ß-lactam resistance has increased with growing intensity. Resistance is often conferred by ß-lactamases, which inactivate ß-lactams, or the expression of alternative ß-lactam-resistant PBPs. σP is an extracytoplasmic function (ECF) σ factor that controls ß-lactam resistance in the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis σP is normally held inactive by the anti-σ factor RsiP. σP is activated by ß-lactams that trigger the proteolytic destruction of RsiP. Here, we identify the penicillin-binding protein PbpP and demonstrate its essential role in the activation of σP Our data show that PbpP is required for σP activation and RsiP degradation. Our data suggest that PbpP acts as a ß-lactam sensor since the binding of a subset of ß-lactams to PbpP is required for σP activation. We find that PbpP likely directly or indirectly controls site 1 cleavage of RsiP, which results in the degradation of RsiP and, thus, σP activation. σP activation results in increased expression of ß-lactamases and, thus, increased ß-lactam resistance. This work is the first report of a PBP acting as a sensor for ß-lactams and controlling the activation of an ECF σ factor.IMPORTANCE The bacterial cell envelope is the target for numerous antibiotics. Many antibiotics target the synthesis of peptidoglycan, which is a central metabolic pathway essential for bacterial survival. One of the most important classes of antibiotics has been ß-lactams, which inhibit the transpeptidase activity of penicillin-binding proteins to decrease the cross-linking of peptidoglycan and the strength of the cell wall. While ß-lactam antibiotics have historically proven to be effective, resistance to ß-lactams is a growing problem. The ECF σ factor σP is required for ß-lactam resistance in B. thuringiensis and close relatives, including B. anthracis Here, we provide insight into the mechanism of activation of σP by ß-lactams.

Ultraviolet protection of Bacillus thuringiensis through microencapsulation with Pickering emulsion method.

An encapsulated formulation of Bacillus thuringiensis (Bt) was produced by the Pickering emulsion technique to improve its activity and stability under UV-A radiation. In this technique latex particles, GO nanosheets, olive oil, ethanol, and water were used to encapsulate Bt in colloidosomes. The protective efficacy of this formulation in protecting Bt subsp. Kurstaki against deactivation by UV-A irradiation was measured, so that spore viability and mortality on Ephestia kuehniella (E. kuehniella) Zeller larvae under UV-A radiation are investigated. According to the results of both tests, encapsulated formulation at a concentration of 0.045% has the highest protection of viability. Hence, colloidosome microcapsule formulations successfully provide good protection against UV radiation.

Evaluation of the Effectiveness of Metabolites of Bacterial Strains Bacillus thuringiensis against Human Influenza Virus A/Aichi/2/68 (H3N2) In Vitro and In Vivo.

The morphological and physiological characteristics of Bacillus thuringiensis strains were analyzed and conditions for obtaining culture fluid with maximum yield of secreted RNases were determined. Zymographic analysis showed that culture fluid of B. thuringiensis strains along with low-molecular-weight (15-20 kDa) RNases contained enzymes with a molecular weight ~55 kDa and their content depended on the duration and conditions of culturing. Preparations based on B. thuringiensis culture fluid were effective against human influenza virus A/Aichi/2/68 (H3N2). In experiments on mice infected with 10 LD50 influenza virus strain A/Aichi/2/68 (H3N2), we selected effective variants of preparations based on culture fluid of B. thuringiensi strains for preventive administration that provided reliable protection of infected animals (protection coefficient 50%), close to that of the reference drug Tamiflu.

Respiration, predatory behavior and prey consumption by Podisus nigrispinus (Heteroptera: Pentatomidae) nymphs exposed to some insecticides.

Podisus nigrispinus Dallas (Heteroptera: Pentatomidae) preys on insect pests in eucalyptus plantations where it can be exposed to insecticides used in pest control. The effect of insecticides on non-target natural enemies requires further study. The objective of the present study was to evaluate the side-effects of Bacillus thuringiensis (Bt), permethrin, tebufenozide and thiamethoxam on third instar nymphs of the predator P. nigrispinus in the laboratory. The toxicity of insecticides for this insect was determined by estimating their lethal concentrations. Podisus nigrispinus behavior after exposure to insecticides was analyzed using a video tracking system and the respiratory rate with a respirometer. Prey/nymph consumption was assessed after 24 h of starvation. The preference of P. nigrispinus nymphs, for prey treated or not with the insecticides, was evaluated in free choice tests. The insecticides Bt [LC50 = 1.10(0.83-1.46) mg mL-1], permethrin [LC50 = 0.25(0.17-0.34) mg mL-1], tebufenozide [LC50 = 5.71(4.17-7.57) mg mL-1] and thiamethoxam [LC50 = 0.04(0.02-0.06) mg mL-1] are toxic to P. nigrispinus nymphs. Bt and the insecticides tebufenozide, permethrin and thiamethoxam reduced the respiratory rate of P. nigrispinus. The insecticides permethrin, tebufenozide and thiamethoxam affect the locomotion of this insect's nymphs. Prey treated with Bt, permethrin and thiamethoxam are less preferred by P. nigrispinus. The survival of the nymphs of this predator was 93.3%, 66.7%, 56.6%, 0% and 0% in the control, tebufenozide, Bt, permethrin and thiamethoxam treatments, respectively. In addition, the reduction of prey consumption, treated with neurotoxic insecticides, reduces the predatory potential of this natural enemy. Bt and tefubenozide present low toxicity for P. nigrispinus, but the neurotoxic products have low compatibility with this natural enemy and, therefore, are not recommended, with this predator in the management of forest insect pests.

Mechanisms of killing of Bacillus thuringiensis Al Hakam spores in a blast environment with and without iodic acid.

AIMS: To determine the mechanism of killing of spores of Bacillus thuringiensis Al Hakam, a Bacillus anthracis spore surrogate, in a blast environment with or without HIO3 and whether the spores are truly dead. METHODS AND RESULTS: Spores exposed to an aluminium-based blast environment with or without HIO3 with dynamic peak gas phase temperatures near 1000°C persisting for 10's of ms, were killed 97 and 99·99% without and with HIO3 respectively and the spores were truly dead. The survivors of the detonations did not acquire mutations, did not become wet heat sensitive, became sensitive to elevated NaCl but not lack of glucose in recovery media, and many dead spores remained phase bright and retained their Ca-dipicolinic acid. A large fraction of the dead spores could germinate, but most of these germinated spores were dead. CONCLUSIONS: Most spores exposed to a blast environment are truly dead, and HIO3 increases spore death. The likely mechanism of spore killing in these blast environments is damage to some essential spore protein, although spore inner membrane damage could contribute. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that spores of a surrogate for B. anthracis spores are killed in a blast environment without or with HIO3 present, this approach could inactivate up to 99·99% of dry B. anthracis spores, and the spores are likely killed by damage to some essential spore protein.

Synthesis, Characterization and Antimicrobial Evaluation of Some N-Substituted Benzimidazole Derivatives.

BACKGROUND: Due to the appearance of communicable microbial diseases and the toxicity related with presently used several antimicrobials such as ß-lactam antibiotics, tetracyclines, quinolones, macrolides, glycopeptides (vancomycin) etc, demand for new antimicrobial agents has become a great concern in new technologies to improve efficacy and safety. METHODS: In search of new antimicrobial agents with higher potency, some N-substituted benzimidazole derivatives (4, 5a-5h & 6) were obtained by chloroacetylation of benzimidazole followed by reaction with different amines, which were characterized by spectroscopic methods. All the target compounds were evaluated for their antibacterial and antifungal activity against microorganisms using two-fold serial dilution method. RESULTS: Among the compounds evaluated, compounds 4 and 5d exhibited potent activity against Bacillus thuringiensis and Candida albicans while showed moderate activity against Escherischia coli when compared to amoxicillin and fluconazole. Compound 5a showed significant activity against tested microorganisms. CONCLUSION: From the current study, it may be concluded that synthesized compounds are fulfilling in terms of their structural distinctiveness and marked biological properties. These compounds might be encouraged to initiate a new class of antimicrobial agents.

Antimicrobial tunicamycin derivatives from the deep sea-derived Streptomyces xinghaiensis SCSIO S15077.

Tunicamycin E (1), featuring a methyl substitution at C-10', was isolated from marine-derived Streptomyces xinghaiensis SCSIO S15077 originated from the South China Sea sediment together with six known compounds, tunicamycin B (2), tunicamycin X (3), tunicamycin A (4), streptovirudin D2 (5), tunicamycin C (6), and tunicamycin C3 (7). The structure of compound 1 was elucidated by detailed spectroscopic data analyses. All the compounds exhibited strong to moderate antibacterial activity against Gram-positive bacteria Bacillus thuringiensis BT01 and B. thuringiensis W102 with MIC values ranging from 0.008 to 2 µg/mL. Moreover, compounds 1-7 exhibited moderate antifungal activity against Candida albicans ATCC 96901 and C. albicans CMCC (F) 98001 with MIC values ranging from 2 to 32 µg/mL. This is the first report that tunicamycins exhibit antimicrobial activities against B. thuringiensis, C. albicans CMCC (F) 98001 and a fluconazole resistant strain C. albicans ATCC 96901.

Purification of WAP domain-containing antimicrobial peptides from green tiger shrimp Peaneaus semisulcatus.

Antimicrobial peptide crustin was isolated and purified from Penaeus semisulcatus using Sephadox G-100 column gel filtration chromatography. P. semisulcatus crustins was observed as a single band with 14 kDa of molecular weight on SDS-PAGE and the retention time of 46 min in RP-HPLC. Circular dichroism spectra of P. semisulcatus crustin showed alpha helices in its secondary structure followed by random coils. Crystalline nature and functional groups arrangement were investigated by X-Ray Diffraction (XRD) and Fourier Transform Infra-Red spectroscopy (FTIR). P. semisulcatus crustin showed the effective antibacterial activity against Gram positive strains B. thuringienisis (4 µg/ml) and B. pumilis (6 µg/ml) when compare to Gram negative strains. Biofilm Inhibitory Concentration (BIC) were determined for these strains and percentage of biofilm inhibition was confirmed and visualized through in sit microscopic analysis. Hence, we reported the effect of crustin on biofilm inhibition and eradication at low concentrations by using crystal violet staining and confocal microscopic observations. In addition, haemolytic activity of this purified crustin also analysed using human RBCs. The results of this study, suggests that this bio peptide crustin is a potential and promising therapeutic agent to treat drug resistant bacteria and biofilm-related infections.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

Resistance to a chemical pesticide increases vulnerability to a biopesticide: Effects on direct mortality and mortality by predation.

Pesticide mixtures are increasingly used to fight pest species that developed resistance to pesticides. To assess the pesticide control efficiency and to reduce ecological damage to non-target species, it is important to quantify the effect of these mixtures and compare them with the effect of their single pesticides on pest species, non-target species and their predator-prey interactions. We studied the effects of the chemical pesticide chlorpyrifos (CPF), the biopesticide Bacillus thuringiensis israelensis (Bti) and their mixture both on the direct mortality and on the mortality by predation. We focused on larvae of a CPF-resistant and a non-resistant strain of the vector mosquito Culex quinquefasciatus and its predator, the pygmy backswimmer Plea minutissima. In the CPF-Bti mixture, both pesticides interacted antagonistically for direct mortality. Exposure to the mixture caused equal direct mortality and equal mortality by predation in both strains. As expected, exposure to CPF resulted in less direct mortality and less mortality by predation in the CPF-resistant mosquito strain compared to the non-resistant strain. Notably, Bti caused a higher mortality in the mosquito larvae of the CPF-resistant strain compared to the non-resistant strain. Furthermore, the predator killed more mosquito larvae of the resistant strain compared to the non-resistant strain when exposed before to Bti alone. These observations identify a novel cost of resistance to a chemical pesticide in terms of increased vulnerability to a biopesticide.

Conspecific and allospecific larval extracts entice mosquitoes to lay eggs and may be used in attract-and-kill control strategy.

One of the strategies of integrated vector management is to lure gravid mosquitoes for surveillance purposes or to entice them to lay eggs in water containing toxins that kill the offspring (attract-and-kill or trap-and-kill). Typically, the major challenge of this approach is the development of a lure that stimulates oviposition plus a toxin with no deterrent effect. Bacillus thuringiensis var. israelensis (Bti) satisfies the latter criterion, but lures for these autocidal gravid traps are sorely needed. We observed that gravid Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus laid significantly more eggs in cups with extracts from 4th-stage larvae (4 L) of the same or different species. No activity was found when 4 L were extracted with hexane, diethyl ether, methanol, or butanol, but activity was observed with dimethyl sulfoxide extracts. Larval extracts contained both oviposition stimulant(s)/attractant(s) and deterrent(s), which partitioned in the water and hexane phases, respectively. Lyophilized larval extracts were active after a month, but activity was reduced by keeping the sample at 4 °C. In the tested range of 0.1 to 1 larvae-equivalent per milliliter, oviposition activity increased in a dose-dependent manner. In field experiments, Ae. aegpti laid significantly more eggs in traps loaded with larval extracts plus Bti than in control traps with water plus Bti.

Activation of the Extracytoplasmic Function σ Factor σP by ß-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP.

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σP is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Previous work showed that σP is induced by ampicillin, a ß-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σP is controlled or what other antibiotics may activate σP Here, we report that activation of σP is specific to a subset of ß-lactams and that σP is required for resistance to these ß-lactams. We demonstrate that activation of σP is controlled by the proteolytic destruction of the anti-σ factor RsiP and that degradation of RsiP requires multiple proteases. Upon exposure to ß-lactams, the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves at site-1. Following cleavage by the unknown protease, the N terminus of RsiP is further degraded by the site-2 intramembrane protease RasP. Our data indicate that RasP cleavage of RsiP is not the rate-limiting step in σP activation. This proteolytic cascade leads to activation of σP, which induces resistance to ß-lactams likely via increased expression of ß-lactamases.IMPORTANCE The discovery of antibiotics to treat bacterial infections has had a dramatic and positive impact on human health. However, shortly after the introduction of a new antibiotic, bacteria often develop resistance. The bacterial cell envelope is essential for cell viability and is the target of many of the most commonly used antibiotics, including ß-lactam antibiotics. Resistance to ß-lactams is often dependent upon ß-lactamases. In B. cereus, B. thuringiensis, and some B. anthracis strains, the expression of some ß-lactamases is inducible. This inducible ß-lactamase expression is controlled by activation of an alternative σ factor called σP Here, we show that ß-lactam antibiotics induce σP activation by degradation of the anti-σ factor RsiP.

Decontamination of Personal Protective Equipment.

Exploratory field analyses of the inactivation capacity of disinfectants on contaminated personal protective equipment (PPE) are required to select a suitable surrogate for biohazardous agents like spores of Bacillus anthracis. The objectives of our study were (1) the determination of an appropriate surrogate for the inactivation of spores of B. anthracis with peracetic acid (PAA), and (2) application of optimized inactivation conditions for an effective decontamination of PPE with PAA under field conditions. For inactivation studies, B. anthracis spores from different strains and B. thuringiensis spores were fixed by air drying on carriers prepared from PPE fabric. Time and concentration studies with PAA-based disinfectants revealed that the spores of the B. thuringiensis strain DSM 350 showed an inactivation profile comparable to that of the spores of the B. anthracis strain with the highest stability, implying that B. thuringiensis can serve as an appropriate surrogate. Rapid (3 to 5 minutes) and effective surface decontamination was achieved with 2% PAA/0.2% surfactant. In field studies, PPE contaminated with spores of B. thuringiensis was treated with the disinfectant. Optimizing the decontamination technique revealed that spraying in combination with brushing was effective within 5 minutes of exposure.

Inactivation of bacterial and fungal spores by UV irradiation and gaseous iodine treatment applied to air handling filters.

Exposure to viable bacterial and fungal spores re-aerosolized from air handling filters may create a major health risk. Assessing and controlling this exposure have been of interest to the bio-defense and indoor air quality communities. Methods are being developed for inactivating stress-resistant viable microorganisms collected on ventilation filters. Here we investigated the inactivation of spores of Bacillus thuringiensis var. kurstaki (Btk), a recognized simulant for B. antracis, and Aspergillus fumigatus, a common opportunistic pathogen used as an indicator for indoor air quality. The viability change was measured on filters treated with ultraviolet (UV) irradiation and gaseous iodine. The spores were collected on high-efficiency particulate air (HEPA) and non-HEPA filters, both flattened for testing purposes to represent "surface" filters. A mixed cellulose ester (MCE) membrane filter was also tested as a reference. Additionally, a commercial HEPA unit with a deep-bed (non-flattened) filter was tested. Combined treatments of Btk spores with UV and iodine on MCE filter produced a synergistic inactivation effect. No similar synergy was observed for A. fumigatus. For spores collected on an MCE filter, the inactivation effect was about an order of magnitude greater for Btk compared to A. fumigatus. The filter type was found to be an important factor affecting the inactivation of Btk spores while it was not as influential for A. fumigatus. Overall, the combined effect of UV irradiation and gaseous iodine on viable bacterial and fungal spores collected on flat filters was found to be potent. The benefit of either simultaneous or sequential treatment was much lower for Btk spores embedded inside the deep-bed (non-flattened) HEPA filter, but for A. fumigatus the inactivation on flattened and non-flattened HEPA filters was comparable. For both species, applying UV first and gaseous iodine second produced significantly higher inactivation than when applying them simultaneously or in an opposite sequence.