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Article ; Online: Alternate atxA and acpA dependent response of Bacillus anthracis to serum, HCO3- and CO2.

Itai Glinert / Elad Bar-David / Amir Ben-Shmuel / Assa Sittner / Reut Puni / Shira Laredo / David Kobiler / Shay Weiss / Haim Levy

PLoS ONE, Vol 18, Iss 2, p e

2023 Volume 0281879

Abstract: Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be ...

Abstract	Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is independently mediated by two regulators; acpA and acpB. In addition, it was demonstrated that acpA has at least two promotors, one of them shared with atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works utilizing NBY, CA or R-HCO3- medium under CO2 enriched conditions, we used a sDMEM-based medium. Thus, toxin and capsule production can be induced in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA based response in an atxA-independent manner, with little to no toxin (protective antigen PA) production. atxA based response is activated in response to serum independently of CO2, inducing toxin and capsule production in an acpA or acpB dependent manner. HCO3- was also found to activate atxA based response, but in non-physiological concentrations. Our findings may help explain the first stages of inhalational infection, in which spores germinating in dendritic cells require protection (by encapsulation) without affecting cell migration to the draining lymph-node by toxin secretion.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples

Efi Makdasi / Orly Laskar / Itai Glinert / Ron Alcalay / Adva Mechaly / Haim Levy

Pathogens, Vol 9, Iss 3, p

2020 Volume 164

Abstract: Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both ... ...

Abstract	: Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both challenging and time-consuming. This is due to the high homology with other Bacillus species as well as the complex composition of environmental samples, which further impedes assay sensitivity. Previously, we showed that a short incubation of B.anthracis spores in a defined growth medium results in rapid germination, bacterial growth, and secretion of toxins, including protective antigen. In this work, we tested whether coupling the incubation process to a newly developed immune-assay will enable the detection of secreted toxins as markers for the presence of spores in environmental samples. The new immune assay is a flow cytometry-based multiplex that simultaneously detects a protective antigen, lethal factor, and edema factor. Our combined assay detects 1 × 10 3 −1 × 10 4 /mL spores after a 2 h incubation followed by the ~80 min immune-multiplex detection. Extending the incubation step to 5 h increased assay sensitivity to 1 × 10 2 /mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent B. anthracis spores. There was no substantial influence of contaminants derived from real environmental samples on the performance of the assay compared to clean samples, which allow the unequivocal detection of 3 × 10 3 /mL and 3 × 10 2 /mL spores following 2 and 5 hour’s incubation, respectively. Overall, we propose this method as a rapid, sensitive, and specific procedure for the identification of B. anthracis spores in environmental samples.
Keywords	bacillus anthracis ; anthrax ; environment samples ; multiplex ; protective antigen ; lethal factor ; edema factor ; Medicine ; R
Subject code	333
Language	English
Publishing date	2020-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: An Improvement in Diagnostic Blood Culture Conditions Allows for the Rapid Detection and Isolation of the Slow Growing Pathogen Yersinia pestis

Efi Makdasi / Yafit Atiya-Nasagi / David Gur / Ayelet Zauberman / Ofir Schuster / Itai Glinert / Shlomo Shmaya / Elad Milrot / Haim Levy / Shay Weiss / Theodor Chitlaru / Emanuelle Mamroud / Orly Laskar

Pathogens, Vol 11, Iss 255, p

2022 Volume 255

Abstract: Plague, caused by the human pathogen Yersinia pestis , is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing ... ...

Abstract	Plague, caused by the human pathogen Yersinia pestis , is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca +2 )- and iron (Fe +2 )-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca +2 and Fe +2 . The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an ...
Keywords	Y. pestis ; plague ; blood culture ; F1 and V antigens ; bacterial diagnostics ; Medicine ; R
Subject code	630
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Using old antibiotics to treat ancient bacterium-β-lactams for Bacillus anthracis meningitis.

Assa Sittner / Amir Ben-Shmuel / Itai Glinert / Elad Bar-David / Josef Schlomovitz / David Kobiler / Shay Weiss / Haim Levy

PLoS ONE, Vol 15, Iss 2, p e

2020 Volume 0228917

Abstract: As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid ( ... ...

Abstract	As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid (oxazolidinone), for the treatment of anthrax, particularly for the late, meningeal stages of the disease. Previously we demonstrated that treatment with Meropenem or Linezolid, either as a single treatment or in combination with Ciprofloxacin, fails to protect rabbits from anthrax-meningitis. In addition, we showed that the failure of Meropenem was due to slow BBB penetration rather than low antibacterial activity. Herein, we tested the effect of increasing the dose of the antibiotic on treatment efficacy. We found that for full protection (88% cure rate) the dose should be increased four-fold from 40 mg/kg to 150 mg/kg. In addition, B. anthracis is a genetically stable bacterium and naturally occurring multidrug resistant B. anthracis strains have not been reported. In this manuscript, we report the efficacy of classical β-lactams as a single treatment or in combination with β-lactamase inhibitors in treating anthrax meningitis. We demonstrate that Ampicillin based treatment of anthrax meningitis is largely efficient (66%). The high efficacy (88-100%) of Augmentin (Amoxicillin and Clavulonic acid) and Unasyn (Ampicillin and Sulbactam) makes them a favorable choice due to reports of β-lactam resistant B. anthracis strains. Tazocin (Piperacillin and Tazobactam) proved inefficient compared to the highly efficient Augmentin and Unasyn.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2020-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Infection with a Nonencapsulated Bacillus anthracis Strain in Rabbits—The Role of Bacterial Adhesion and the Potential for a Safe Live Attenuated Vaccine

Itai Glinert / Shay Weiss / Assa Sittner / Elad Bar-David / Amir Ben-Shmuel / Josef Schlomovitz / David Kobiler / Haim Levy

Toxins, Vol 10, Iss 12, p

2018 Volume 506

Abstract: Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully ... ...

Abstract	Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine.
Keywords	Bacillus anthacis ; vaccine strain ; BslA ; cell adherence ; encephalitis ; CNS infection ; Medicine ; R
Subject code	572
Language	English
Publishing date	2018-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Neutralization of SARS-CoV-2 Variants by rVSV-ΔG-Spike-Elicited Human Sera

Vaccines, Vol 10, Iss 291, p

2022 Volume 291

Abstract: The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific ... ...

Abstract	The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife ® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants’ mutations. We show that human sera from BriLife ® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife ® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife ® -acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.
Keywords	COVID-19 ; SARS-CoV-2 ; variants ; VOC ; vaccine ; BriLife ® ; Medicine ; R
Subject code	333
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Pathology of wild-type and toxin-independent Bacillus anthracis meningitis in rabbits.

Assa Sittner / Elad Bar-David / Itai Glinert / Amir Ben-Shmuel / Shay Weiss / Josef Schlomovitz / David Kobiler / Haim Levy

PLoS ONE, Vol 12, Iss 10, p e

2017 Volume 0186613

Abstract: Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP). Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals ... ...

Abstract	Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP). Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals presented histopathology of meningitis at the time of death, some without any sign of hemorrhage. A similar pathology was observed in animals that succumbed following infection with the toxin deficient mutant, thus indicating that anthrax meningitis is a toxin-independent phenomenon. In this manuscript we describe a histopathological study of the B. anthracis infection of the central nervous system (CNS). Though we could find sporadic growth of the bacteria around blood vessels in the cortex, we report that the main infiltration route is the choroid plexus. We found massive destruction of entire sections of the choroid plexus coupled with massive aggregation of bacilli in the ventricles, in close proximity to the parenchyma. The choroid plexus also contained significant amounts of intravascular bacterial aggregates, often enclosed in what appear to be fibrin-like clots. The high concentration of these aggregates in areas of significant tissue destruction combined with the fact that capsular B. anthracis bacteria have a low tendency to adhere to endothelial cells, might suggest that these clots are used as an adherence mechanism by the bacteria. The major histopathological finding is meningitis. We find massive bacterial growth in the meninges without evidence of encephalitis, even when the bacteria emerge from a parenchymal blood vessel. Erythrocytes were present within the meningeal space but no clear vasculitis could be detected. Histology of the brain stem indicates meningitis, edema and hemorrhages that might explain death from suffocation due to direct damage to the respiratory center. All of these processes are toxin-independent, since they were observed following infection with either the wild type strain or the toxin-deficient mutant. Herein, we propose that the first step of anthrax-meningitis is bacterial adhesion to the blood vessels by manipulating coagulation, mainly in the choroid plexus. The trapped bacteria then destroy sections of the choroid plexus, resulting in penetration into the CSF, leading to meningitis and hemorrhage. Death could be the result of increased intracranial pressure and/or damage to the brain stem.
Keywords	Medicine ; R ; Science ; Q
Subject code	630
Language	English
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Toxin-independent virulence of Bacillus anthracis in rabbits.

Haim Levy / Itai Glinert / Shay Weiss / Assa Sittner / Josef Schlomovitz / Zeev Altboum / David Kobiler

PLoS ONE, Vol 9, Iss 1, p e

2014 Volume 84947

Abstract: The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a ... ...

Abstract	The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.
Keywords	Medicine ; R ; Science ; Q
Subject code	630
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

Haim Levy / Itai Glinert / Shay Weiss / Elad Bar-David / Assa Sittner / Josef Schlomovitz / Zeev Altboum / David Kobiler

PLoS ONE, Vol 9, Iss 11, p e

2014 Volume 112319

Abstract: Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is ... ...

Abstract	Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.
Keywords	Medicine ; R ; Science ; Q
Subject code	630
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Mice with induced pulmonary morbidities display severe lung inflammation and mortality following exposure to SARS-CoV-2

Reut Falach / Liat Bar-On / Shlomi Lazar / Tamar Kadar / Ohad Mazor / Moshe Aftalion / David Gur / Yentl Evgy / Ohad Shifman / Tamar Aminov / Ofir Israeli / Inbar Cohen-Gihon / Galia Zaide / Hila Gutman / Yaron Vagima / Efi Makdasi / Dana Stein / Ronit Rosenfeld / Ron Alcalay /

Eran Zahavy / Haim Levy / Itai Glinert / Amir Ben-Shmuel / Tomer Israely / Sharon Melamed / Boaz Politi / Hagit Achdout / Shmuel Yitzhaki / Chanoch Kronman / Tamar Sabo

JCI Insight, Vol 6, Iss

2021 Volume 12

Abstract: Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary ... ...

Abstract	Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2–refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin–pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2–3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.
Keywords	COVID-19 ; Medicine ; R
Subject code	570
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	American Society for Clinical investigation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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