Anthracis Spores Research Articles

Comparison of liquid and filter sampling techniques for recovery of Bacillus spores and Escherichia coli from environmental water

Historically, detecting water contamination has involved collecting and directly analyzing liquid samples, but recent advances in filter sampling methods offer numerous potential advantages. Emerging technologies, including environmental DNA (eDNA) samplers, could be used for remote microbial contamination sampling, but work is needed to determine if target microorganisms can be recovered from filters at comparable levels to traditional sampling methods. In this study, Escherichia coli and a surrogate for Bacillus anthracis spores were sampled from synthetic stormwater and quantified using both direct liquid and filter methods, and dwell time tests compared microorganism persistence in water and on filters. At nearly all tested timepoints, the recoveries of spores from membrane filters were within 0.5 log10 colony forming units per sample (CFU/sample) compared to the liquid-only samples, suggesting that the use of filter sampling is a feasible alternative to liquid-based sampling, and samples were held for up to 4 weeks without significant sample degradation. Recoveries for E. coli remained relatively consistent for ∼3 days in phosphate buffered saline (PBS), in synthetic stormwater, and on membrane filters, but decreases in recoveries were observed for samples held for >3 days. These results indicate that emerging water sampling technologies, which reduce logistical burdens and offer potential cost savings, can be leveraged to characterize biological contamination in water matrices with multiple types of microbiological agents.

Comparative analysis of the results of live anthrax vaccine identification by immunofluorescence and immunochromatography

INTRODUCTION. The quality evaluation of live anthrax vaccines will benefit from the implementation of alternative testing methods that are capable of specific identification of the Bacillus anthracis spore antigen using the appropriate diagnostic products that are authorised in the Russian Federation for the detection of B. anthracis spores.AIM. This study aimed to investigate the applicability of immunofluorescence analysis to the identification of live anthrax vaccines and compare this method with immunochromatography.MATERIALS AND METHODS. The study used commercial batches of a live anthrax vaccine and Russian diagnostic products, including diagnostic dry adsorbed fluorescent anti-anthrax spore immunoglobulins and an immunochromatographic assay (ICA) reagent kit for rapid detection and identification of B. anthracis spores (ICA system for B. anthracis). For smears and ICA system reactions, the authors prepared working solutions of bacterial suspensions at spore concentrations typical of the B. anthracis STI-1 vaccine strain. The spore concentrations were achieved using the pharmacopoeial reference standard (RS) for the opacity of bacterial suspensions of 10 international units (IU). Identification reactions involved the registration of immune complex formation.RESULTS. Immunofluorescence tests of the live anthrax vaccine demonstrated bright greenish-yellow envelope fluorescence with an intensity score of 3+ to 4+ for smears stained with diagnostic immunoglobulins at 1:32 and 1:64 dilutions. Immunochromatographic tests of the live anthrax vaccine detected the spore antigen at vaccine concentrations of 109 and 108 spores/mL, with the test strips showing two distinct dark-pink lines indicative of immunoprecipitation. According to the results obtained using the selected methods, the tested microbial culture was confirmed as B. anthracis.CONCLUSIONS. Immunochromatography and immunofluorescence tests with appropriate diagnostic preparations are convenient and reliable tools for the species-specific detection of B. anthracis STI-1 spores in the live anthrax vaccine. The results obtained in the anthrax vaccine identification tests provide a basis for recommending the above methods as supplementary alternatives to Ziehl–Neelsen bacteriological staining, which is currently prescribed by the State Pharmacopoeia of the Russian Federation.

Soil Sample Analysis of Bacillus anthracis Contaminated Animal Burial Sites.

Environmental contamination with Bacillus anthracis spores poses clear threats to livestock that play key roles in the economies of pastoral communities. Regular monitoring of contaminated sites is particularly important in anthrax-endemic parts of the world, such as Kars province in eastern Türkiye, where the Veterinary Microbiology Department of Kafkas University has conducted an anthrax surveillance programme for over 30 years. We reviewed the microbiological results of 232 soil samples collected during 2009-2023, from sites known to be contaminated with B. anthracis spores following burial or butchering of infected animal carcasses. Twenty-five contaminated sites in 16 villages were studied. Samples were taken from a total of 61 different positions within these sites and viable spores were detected in 136 (58.6%) of the samples examined. Of the 96 samples from which spores were not recovered, subsequent samples from the same positions proved positive on 21 occasions. Using a standardised sampling plan, it was discovered that samples taken 1-2 m on a downward slope from the centre-point of contamination had higher (p < 0.001) spore concentrations than those taken from other positions. Although spore concentrations at some sampling positions varied over time, the overall values remained stable. This finding contrasts with observations in other parts of the world where spore concentrations tend to decline with time and may reflect regional differences in soil composition that permit more prolonged spore persistence. Concentrations of >100 spores/g soil were found in 10 (66.7%) of the 15 samples taken 10-13 years following a contamination event. These results demonstrate the longevity of viable anthrax spores in the soil of agricultural environments following decomposition of infected animal carcasses, and therefore the need for prolonged bacteriological monitoring of contaminated sites. Furthermore, they underline the importance of appropriate decontamination, as burial on its own does not eliminate all spores.

Green Tea Catechin Epigallocatechin Gallate Inhibits Vegetative Cell Outgrowth and Expression of Beta-Lactamase Genes in Penicillin-Resistant Bacillus anthracis Strain PCr.

The green tea catechin epigallocatechin gallate (EGCg) has antimicrobial effects on many bacteria. In this study, we investigated the inhibitory effects of EGCg on Bacillus anthracis spores and vegetative cells. The B. anthracis spores were insensitive to EGCg, but the growth of vegetative cells derived from germinated spores was inhibited by EGCg. Moreover, EGCg decreased the minimum inhibitory concentration of penicillin and meropenem for penicillin-resistant B. anthracis. In the penicillin-resistant B. anthracis strain, the transcription levels of the beta-lactamase genes (bla1 and bla2) decreased significantly following the treatment with 50 µg/mL EGCg. These results suggest that the appropriate application of EGCg may effectively control the penicillin-resistant B. anthracis growth and beta-lactamase production.

Anthrax Epidemıc Management in an Eastern Turkey Region

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis. It has been known for centuries and remains hyperendemic in certain parts of the world, with occasional epidemic outbreaks. This study aimed to investigate an anthrax outbreak affecting a family and their close contacts in the Akçakiraz town of Elazığ province in eastern Turkey. Methods: Twenty-two individuals who had contact with a sick animal in Akçakiraz were included in the study. Weekly anamnesis, physical examinations, and follow-ups were conducted over three weeks. Swab samples were collected from the wound sites of hospitalized patients for culture and Gram-staining. Blood and wound samples were sent for polymerase chain reaction (PCR) analysis to detect B. anthracis. A sample was taken from the liver tissue of the infected animal. Statistical analysis was performed using SPSS 25. Results: Of the 22 individuals, 9 (40.9%) were hospitalized and treated, while 13 (59.1%) were monitored on an outpatient basis. B. anthracis spores were identified in Gram-stained samples from the liver of diseased animal. PCR positivity for B. anthracis was detected in the wound samples of two patients (22.2%), although the PCR results from the blood samples of these patients were negative. No bacterial growth was observed in wound cultures, and gram-staining did not reveal any notable features. All patients were successfully treated. Contacts, family members, and residents were informed and educated about the disease, and necessary public health measures were implemented through coordination with the Provincial Hygiene Board. Conclusion: Increasing awareness of zoonotic diseases, especially anthrax, is essential. It is crucial to implement societal prevention measures, including controlling animal production, educating at-risk populations in endemic areas, vaccinating animals, preventing contact with sick or deceased animals, and preventing environmental contamination by decontaminating affected areas.

Fate and transport of viable Bacillus anthracis simulant spores in ambient air during a large outdoor decontamination field exercise

ABSTRACT The Wide Area Demonstration (WAD) was a field exercise conducted under the U.S. EPA’s Analysis of Coastal Operational Resiliency program, in conjunction with the U.S. Department of Homeland Security and the U.S. Coast Guard. The purpose of the WAD was to operationalize at field scale aspects of remediation activities that would occur following an outdoor release of Bacillus anthracis spores, including sampling and analysis, decontamination, data management, and waste management. The WAD was conducted in May 2022 at Fort Walker (formerly known as Fort A.P. Hill) and utilized Bacillus atrophaeus as a benign simulant for B. anthracis. B. atrophaeus spores were inoculated onto the study area at the beginning of the study, and air samples were collected daily during each of the different phases of the WAD using Dry Filter Units (DFUs). Ten DFU air samplers were placed at the perimeter of the study area to collect bioaerosols onto two parallel 47-mm diameter polyester felt filters, which were then subsequently analyzed in a microbiological laboratory for the quantification of B. atrophaeus. The study demonstrated the use of DFUs as a rugged and robust bioaerosol collection device. The results indicated that the highest B. atrophaeus spore air concentrations (up to ~ 5 colony forming units/m3) occurred at the beginning of the demonstration (e.g. during inoculation and characterization sampling phases) and generally downwind from the test site, suggesting transport of the spores was occurring from the study area. Very few B. atrophaeus spores were detected in the air after several weeks and following decontamination of exterior surfaces, thus providing an indication of the site decontamination procedures’ effectiveness. No B. atrophaeus spores were detected in any of the blank or background samples. Implications: Following an incident involving a release of Bacillus anthracis spores or other biological threat agent into the outdoor environment, understanding the factors that may affect the bioagent’s fate and transport can help predict viable contaminant spread via the ambient air. This paper provides scientific data for the first time on ambient air concentrations of bacterial spores over time and location during different phases of a field test in which Bacillus atrophaeus (surrogate for B. anthracis) spores were released outdoors as part of a full-scale study on sampling and decontamination in an urban environment. This study advances the knowledge related to the fate and transport of bacterial spores (such as those causing anthrax disease) as an aerosol in the outdoor environment over the course of three weeks in a mock urban environment and has exposure and health risk implications. The highest spore air concentrations occurred at the beginning of the study (e.g. during inoculation of surfaces and characterization sampling), and in the downwind direction, but diminished over time; few B. atrophaeus spores were detected in the air after several weeks and following decontamination. Therefore, in an actual incident, potential reaerosolization of the microorganism and subsequent transport in the air during surface sampling and remediation efforts should be considered for determining exclusion zone locations and estimating potential risk to neighboring communities. The data also provide evidence suggesting that the large-scale decontamination of outdoor surfaces may reduce air concentrations of the bioagent, which is important since exposure of B. anthracis via inhalation is a primary concern.

Mapping high probability area for the Bacillus anthracis occurrence in wildlife protected area, South Omo, Ethiopia

Anthrax is a zoonotic disease caused by a spore-forming gram-positive bacterium, Bacillus anthracis. Increased anthropogenic factors inside wildlife-protected areas may worsen the spillover of the disease at the interface. Consequently, environmental suitability prediction for B. anthracis spore survival to locate a high-risk area is urgent. Here, we identified a potentially suitable habitat and a high-risk area for appropriate control measures. Our result revealed that a relatively largest segment of Omo National Park, about 23.7% (1,218 square kilometers) of the total area; 36.6% (711 square kilometers) of Mago National Park, and 29.4% (489 square kilometers) of Tama wildlife Reserve predicted as a high-risk area for the anthrax occurrence in the current situation. Therefore, the findings of this study provide the priority area to focus on and allocate resources for effective surveillance, prevention, and control of anthrax before it causes devastating effects on wildlife.

A Dual-Mode Fluorescent Nanoprobe for the Detection and Visual Screening of Pathogenic Bacterial Spores.

Bacterial vegetative cells turn into metabolically dormant spores in certain environmental situations. Once suitable conditions trigger the germination of spores belonging to the pathogenic bacterial category, public safety and environmental hygiene will be threatened, and lives will even be endangered when encountering fatal ones. Instant identification of pathogenic bacterial spores remains a challenging task, since most current approaches belonging to complicated biological methods unsuitable for onsite sensing or emerging alternative chemical techniques are still inseparable from professional instruments. Here we developed a polychromatic fluorescent nanoprobe for ratiometric detection and visual inspection of the pathogenic bacterial spore biomarker, dipicolinic acid (DPA), realizing rapidly accurate screening of pathogenic bacterial spores such as Bacillus anthracis spores. The nanoprobe is made of aminoclay-coated silicon nanoparticles and functionalized with europium ions, exhibiting selective and sensitive response toward DPA and Bacillus subtilis spores (simulants for Bacillus anthracis spores) with excellent linearity. The proposed sensing strategy allowing spore determination of as few as 0.3 × 105 CFU/mL within 10 s was further applied to real environmental sample detection with good accuracy and reliability. Visual quantitative determination can be achieved by analyzing the RGB values of the corresponding test solution color via a color recognition APP on a smartphone. Different test samples can be photographed at the same time, hence the efficient accomplishment of examining bulk samples within minutes. Potentially employed in various on-site sensing occasions, this strategy may develop into a powerful means for distinguishing hazardous pathogens to facilitate timely and proper actions of dealing with multifarious security issues.

Quantifying in vitro B. anthracis growth and PA production and decay: a mathematical modelling approach

Protective antigen (PA) is a protein produced by Bacillus anthracis. It forms part of the anthrax toxin and is a key immunogen in US and UK anthrax vaccines. In this study, we have conducted experiments to quantify PA in the supernatants of cultures of B. anthracis Sterne strain, which is the strain used in the manufacture of the UK anthrax vaccine. Then, for the first time, we quantify PA production and degradation via mathematical modelling and Bayesian statistical techniques, making use of this new experimental data as well as two other independent published data sets. We propose a single mathematical model, in terms of delay differential equations (DDEs), which can explain the in vitro dynamics of all three data sets. Since we did not heat activate the B. anthracis spores prior to inoculation, germination occurred much slower in our experiments, allowing us to calibrate two additional parameters with respect to the other data sets. Our model is able to distinguish between natural PA decay and that triggered by bacteria via proteases. There is promising consistency between the different independent data sets for most of the parameter estimates. The quantitative characterisation of B. anthracis PA production and degradation obtained here will contribute towards the ambition to include a realistic description of toxin dynamics, the host immune response, and anti-toxin treatments in future mechanistic models of anthrax infection.

Anthrax disease burden: Impact on animal and human health

Anthrax is a zoonotic infectious disease caused by Bacillus anthracis. According to current knowledge, the disease originates in sub-Saharan Africa, especially Egypt and Mesopotamia. Laboratory tests involving direct staining or culture of samples taken from malignant pustules, sputum, blood, or patient discharge must be performed to establish a diagnosis. B. anthracis infection can enter the body through the skin, mouth, or nose. Human infection is usually caused by contact with infected animals or animal products. Anthrax causes a reduction in resource efficiency and decreases livestock productivity. B. anthracis spores are resistant to extreme temperatures, pressure, pH, drying, solvents, and ultraviolet light. The biological weapon of this disease may be fatal if it is designed to spread B. anthracis spores by aerosols. In the past, the treatment of human anthrax with penicillin at a high dose was the preferred method. The public can take several measures to prevent anthrax infection, such as purchasing and consuming meat that has been legally certified to have been slaughtered in a slaughterhouse, consuming healthy and properly cooked animal meat, and washing hands with antiseptic soap after handling, processing, and cooking animal products. This review aimed to describe the etiology, pathogenesis, mechanism of infection, epidemiology, diagnosis, clinical symptoms, transmission, risk factors, public health importance, economic impact, potential as a bio-warfare agent, treatment, and control of anthrax. Keywords: animal health, anthrax, human health, infectious disease.

Molecular characterization of an outbreak-involved Bacillus anthracis strain confirms the spillover of anthrax from West Africa

BackgroundAnthrax, a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, remains a major global public health concern, especially in countries with limited resources. Sierra Leone, a West African country historically plagued by anthrax, has almost been out of report on this disease in recent decades. In this study, we described a large-scale anthrax outbreak affecting both animals and humans and attempted to characterize the pathogen using molecular techniques.MethodsThe causative agent of the animal outbreak in Port Loko District, Sierra Leone, between March and May 2022 was identified using the nanopore sequencing technique. A nationwide active surveillance was implemented from May 2022 to June 2023 to monitor the occurrence of anthrax-specific symptoms in humans. Suspected cases were subsequently verified using quantitative polymerase chain reaction. Full-genome sequencing was accomplished by combining long-read and short-read sequencing methods. Subsequent phylogenetic analysis was performed based on the full-chromosome single nucleotide polymorphisms.ResultsThe outbreak in Port Loko District, Sierra Leone, led to the death of 233 animals between March 26th and May 16th, 2022. We ruled out the initial suspicion of Anaplasma species and successfully identified B. anthracis as the causative agent of the outbreak. As a result of the government's prompt response, out of the 49 suspected human cases identified during the one-year active surveillance, only 6 human cases tested positive, all within the first month after the official declaration of the outbreak. The phylogenetic analysis indicated that the BaSL2022 isolate responsible for the outbreak was positioned in the A.Br.153 clade within the TransEuroAsian group of B. anthracis.ConclusionsWe successfully identified a large-scale anthrax outbreak in Sierra Leone. The causative isolate of B. anthracis, BaSL2022, phylogenetically bridged other lineages in A.Br.153 clade and neighboring genetic groups, A.Br.144 and A.Br.148, eventually confirming the spillover of anthrax from West Africa. Given the wide dissemination of B. anthracis spores, it is highly advisable to effectively monitor the potential reoccurrence of anthrax outbreaks and to launch campaigns to improve public awareness regarding anthrax in Sierra Leone.Graphical

Human Exposure to Naturally Occurring Bacillus anthracis in the Kars Region of Eastern Türkiye.

Environmental contamination with Bacillus anthracis spores poses uncertain threats to human health. We undertook a study to determine whether inhabitants of the anthrax-endemic region of Kars in eastern Türkiye could develop immune responses to anthrax toxins without recognised clinical infection. We measured anti-PA and anti-LF IgG antibody concentrations by ELISA in serum from 279 volunteers, 105 of whom had previously diagnosed anthrax infection (100 cutaneous, 5 gastrointestinal). Of the 174 without history of infection, 72 had prior contact with anthrax-contaminated material. Individuals were classified according to demographic parameters, daily working environment, and residence type. All villages in this study had recorded previous animal or human anthrax cases. Stepwise regression analyses showed that prior clinical infection correlated strongly with concentrations at the upper end of the ranges observed for both antibodies. For anti-PA, being a butcher and duration of continuous exposure risk correlated with high concentrations, while being a veterinarian or shepherd, time since infection, and town residence correlated with low concentrations. For anti-LF, village residence correlated with high concentrations, while infection limited to fingers or thumbs correlated with low concentrations. Linear discriminant analysis identified antibody concentration profiles associated with known prior infection. Profiles least typical of prior infection were observed in urban dwellers with known previous infection and in veterinarians without history of infection. Four individuals without history of infection (two butchers, two rural dwellers) had profiles suggesting unrecognised prior infection. Healthy humans therefore appear able to tolerate low-level exposure to environmental B. anthracis spores without ill effect, but it remains to be determined whether this exposure is protective. These findings have implications for authorities tasked with reducing the risk posed to human health by spore-contaminated materials and environments.

An overview of anthrax: a neglected zoonosis of the tropical region

Anthrax is a neglected bacterial zoonosis that can be highly contagious and potentially lethal. The disease, which mainly affects herbivorous ungulate animals, is caused by Bacillus anthracis, a rod-shaped, gram-positive bacterium. The pathogenicity depends on the production of toxins by Bacillus anthracis and the sensitivity of the host. The infection is transmitted to humans when they come in to contact with infected animals or their products. It is an important occupational risk to the abattoir workers. Clinical manifestations of disease in humans include different forms, including cutaneous, pulmonary and intestinal. The disease is widespread in African and Asian countries with frequent reports of cases. Lack of appropriate and effective control programs in these countries has led to increase in the number of human cases. Bacillus anthracis spores are extremely resistant to environmental conditions, and can persist for several decades in nature and, thus making the control or eradication of the disease difficult. Disease control involves managing contaminated animal products, avoiding contact with infected animals, livestock vaccination in endemic regions and routine surveillance of animals. The most effective and dependable way to control zoonotic diseases like anthrax is through collaborative monitoring programs including the "One Health" approach.

CDC Guidelines for the Prevention and Treatment of Anthrax, 2023.

Bacillus anthracis spores if resources become limited or a multidrug-resistant B. anthracis strain is used (Hendricks KA, Wright ME, Shadomy SV, et al.; Workgroup on Anthrax Clinical Guidelines. Centers for Disease Control and Prevention expert panel meetings on prevention and treatment of anthrax in adults. Emerg Infect Dis 2014;20:e130687; Meaney-Delman D, Rasmussen SA, Beigi RH, et al. Prophylaxis and treatment of anthrax in pregnant women. Obstet Gynecol 2013;122:885-900; Bradley JS, Peacock G, Krug SE, et al. Pediatric anthrax clinical management. Pediatrics 2014;133:e1411-36). Specifically, this report updates antimicrobial drug and antitoxin use for both postexposure prophylaxis (PEP) and treatment from these previous guidelines best practices and is based on systematic reviews of the literature regarding 1) in vitro antimicrobial drug activity against B. anthracis; 2) in vivo antimicrobial drug efficacy for PEP and treatment; 3) in vivo and human antitoxin efficacy for PEP, treatment, or both; and 4) human survival after antimicrobial drug PEP and treatment of localized anthrax, systemic anthrax, and anthrax meningitis. B. anthracis strain. In addition, these updated guidelines include new recommendations regarding special considerations for the diagnosis and treatment of anthrax meningitis, including comorbid, social, and clinical predictors of anthrax meningitis. The previously published CDC guidelines and recommendations described potentially beneficial critical care measures and clinical assessment tools and procedures for persons with anthrax, which have not changed and are not addressed in this update. In addition, no changes were made to the Advisory Committee on Immunization Practices recommendations for use of anthrax vaccine (Bower WA, Schiffer J, Atmar RL, et al. Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices, 2019. MMWR Recomm Rep 2019;68[No. RR-4]:1-14). The updated guidelines in this report can be used by health care providers to prevent and treat anthrax and guide emergency preparedness officials and planners as they develop and update plans for a wide-area aerosol release of B. anthracis.

Eu-doped ZIF-8 as a ratiometric fluorescence-scattering probe for the anthrax biomarker in food samples based on competitive coordination

Bacillus anthracis spores can cause contagious anthrax, so it is significant for the public safety to detect its biomarker dipicolinic acid (DPA). Ratiometric fluorescent probes for DPA have attracted great interest because of the self-calibration effect, but they suffer from the complicated construction of dual-emitting materials. With combining fluorescence and second-order scattering (SOS), Eu-doped Zn-based metal–organic framework (ZIF-8) was here designed as a ratiometric probe for DPA, avoiding the completed construction of dual-emitting materials. Eu-doped ZIF-8 exhibited no fluorescence but possessed strong SOS ascribed to the nanostructure. However, the competitive coordination of DPA not only triggered the antenna effect of Eu3+ but also caused the decomposition of Eu-doped ZIF-8. As a consequence, the fluorescence enhancement and the SOS weakness were observed upon the addition of DPA to Eu-doped ZIF-8. And thus, a ratiometric probe for DPA was constructed based on Eu-doped ZIF-8 by integrating fluorescence with SOS. The probe as-constructed could quantify DPA in a wide range of 0.1–150 μM with a rapid response (1 min) and a low detection limit (31 nM). It also displayed excellent manifestation for the analysis of food samples with reasonable accuracy (recoveries, 90.0%-101.5%) and satisfactory precision (RSDs, 0.6%-5.7%), offering a reliable tool for the supervision of the public safety.

Effect of the Bacteriocin Subtylosin P19 on &lt;i&gt;Bacillus Anthracis&lt;/i&gt; Spores

Abstract—This is the first report on electron microscopic investigation of the effect of bacteriocin on Bacillus anthracis dormant spores. The new bacteriocin, subtilosin P19, isolated from a natural Bacillus subtilis strain, effectively killed dormant B. anthracis spores at concentrations of 0.5‒35 µg/mL after 60-min exposure. Our data, combined with analysis of the known mechanisms of the action of bacteriocins, suggest that the mechanism of spore inactivation was apparently based on multiple disruptions of the integrity of the inner and outer membranes surrounding the cortex of the resting spore. It is also likely that the molecular configuration of subtilisin P19 allows it to penetrate through the outer integuments and to damage the vital membrane structures of the anthrax spores. Subtilosin P19 can serve as a promising basis for new antimicrobial preparations of natural origin.

Inactivating and damaging properties of the disinfectant “MultiDez” when exposed to bacteria and spores

Disinfectants play a crucial role in controlling the spread of infectious diseases caused by bacteria and spore-forming organisms. Bacteria and spores can persist on surfaces and in the environment for extended periods, posing a significant risk to public health. Disinfectants are designed to inactivate or kill these microorganisms by disrupting their cellular structures and functions. Effective disinfectants are essential for preventing the spread of infectious diseases in hospitals, laboratories, food processing facilities, and other settings where the risk of contamination is high.This study evaluated the effectiveness of a disinfectant called “MultiDez” on Y.pestis bacteria and Bacillus anthracis spores using microbiological and electron microscopic methods. Results showed that after exposure to a 0.5 % solution of the disinfectant, the death of all Y.pestis bacteria was achieved after 90 min, while the death of Bacillus anthracis spores was achieved after 240 min. Electron microscopy revealed that the disinfectant caused complete destruction of both bacterial cells and spores by enveloping their outer surfaces with polymer molecules, disrupting the structure and function of their membranes, and destroying their cytoplasm and nucleode. The mechanism of action of the disinfectant on bacteria and spores involved different processes, with the disinfectant causing rapid hydration of dehydrated spores and blocking the functions of spore membranes in the case of bacterial spores.

Assessment of the Effectiveness of Using Magnoimmunosorbents for the Selective Concentration of Anthrax Agent Spores

The aim of the study was to assess the effectiveness of the developed anthrax magnoimmunosorbents (MIS) for the selective concentration of Bacillus anthracis spores and to increase the sensitivity of anthrax agent detection techniques, including when testing soil samples.Materials and methods. We used 10 vaccine strains of B. anthracis and 30 strains of closely related bacilli of the genus Bacillus (B. cereus – 15, B. thuringiensis – 10, B. megaterium – 5) with typical species properties. The work was performed on three experimental batches of magnoimmunosorbents. DNA extraction and PCR setting was carried out in compliance with the instructions for reagent panel for B. anthracis DNA detection “ApliSens Bacillus anthracis-FRT”.Results and discussion. It is shown that when using MIS, the sensitivity of the cultural method is increased by at least 7 times (taking into account the possibility of sorption of 1–10 or more spores on a sorbent particle). The sensitivity of the PCR method is improved by 10 times and amounts to 50 B. anthracis spores per 1 ml for the samples concentrated with the help of MIS. The sensitivity of the bacteriological method using MIS increases by a factor of 7.5 when testing the artificially contaminated with B. anthracis soil samples. Hence, application of the developed MIS makes it possible to significantly enhance the sensitivity of anthrax agent detection methods and can be considered as an effective means of sample preparation for the investigation of environmental objects (soil).

The Detection of Anthrax Biomarker DPA by Ratiometric Fluorescence Probe of Carbon Quantum Dots and Europium Hybrid Material Based on Poly(ionic)- Liquid.

Bacillus anthracis has gained international attention as a deadly bacterium and a potentially deadly biological warfare agent. Dipicolinic acid (DPA) is the main component of the protective layer of anthracis spores, and is also an anthrax biomarker. Therefore, it is of great significance to explore an efficient and sensitive DPA detection method. Herein, a novel ratio hybrid probe (CQDs-PIL-Eu3+) was prepared by a simple one-step hydrothermal method using carbon quantum dots (CQDs) as an internal reference fluorescence and a covalent bond between CQDs and Eu3+ by using a polyionic liquid (PIL) as a bridge molecule. The ratiometric fluorescence probe was found to have the characteristics of sensitive fluorescence visual sensing in detecting DPA. The structure and the sensing properties of CQDs-PIL-Eu3+ were investigated in detail. In particular, the fluorescence intensity ratio of Eu3+ to CQDs (I616/I440) was linear with the concentration of DPA in the range of 0-50 μM, so the detection limit of the probe was as low as 32 nm, which was far lower than the DPA dose released by the number of anthrax spores in human body (60 μM) and, thus, can achieve sensitive detection. Therefore, the ratiometric fluorescence probe in this work has the characteristics of strong anti-interference, visual sensing, and high sensitivity, which provides a very promising scheme for the realization of anthrax biomarker DPA detection.

Bacillus anthracis spores are internalized in human lung epithelial cells by Rab GTPase-supported macropinocytosis

Inhalation anthrax, the deadliest form of the disease, requires inhaled B. anthracis spores to escape from the alveolar space and travel to the mediastinal lymph nodes, from where the vegetative form of the pathogen disseminates, resulting in a rapidly fatal outcome. The role of epithelia in alveolar escape is unclear, but previous work suggests these epithelial cells are involved in this process. Using confocal microscopy, we found that B. anthracis spores are internalized more rapidly by A549 type II alveolar epithelial cells compared to hAELVi type I alveolar epithelial cells. Internalization of spores by alveolar epithelial cells requires cytoskeletal rearrangement evidenced by significant inhibition by cytochalasin D, an actin inhibitor. Chemical inhibitors of macropinocytosis significantly downregulated B. anthracis spore internalization in human alveolar cells, while inhibitors of other endocytosis pathways had minimal effects. Additional studies using a macropinosome marker and electron microscopy confirmed the role of macropinocytosis in spore uptake. By colocalization of B. anthracis spores with four endocytic Rab proteins, we demonstrated that Rab31 played a role in B. anthracis spore macropinocytosis. Finally, we confirmed that Rab31 is involved in B. anthracis spore internalization by enhanced spore uptake in Rab31-overexpressing A549 cells. This is the first report that shows B. anthracis spore internalization by macropinocytosis in human epithelial cells. Several Rab GTPases are involved in the process.