Enhanced production yields of rVSV-SARS-CoV-2 vaccine using Fibra-Cel® macrocarriers.

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.

Rapid Phenotypic Antibiotic Susceptibility Profiling of Clinical Escherichia coli and Klebsiella pneumoniae Blood Cultures.

Bloodstream infections (BSI) are defined by the presence of viable bacteria or fungi, accompanied by systemic signs of infection. Choosing empirical therapy based solely on patient risk factors and prior antibiotic susceptibility test (AST) may lead to either ineffective treatment or unnecessarily broad-spectrum antibiotic exposure. In general, Clinical & Laboratory Standards Institute guideline-approved ASTs have a turnaround time of 48-72 h from sample to answer, a period that may result in a critical delay in the appropriate selection of therapy. Therefore, reducing the time required for AST is highly advantageous. We have previously shown that our novel rapid AST method, MAPt (Micro-Agar-PCR-test), accurately identifies susceptibility profiles for spiked bioterrorism agents like Bacillus anthracis, Yersinia pestis and Francisella tularensis directly from whole-blood and blood culture samples, even at low bacterial levels (500 CFU/mL). This study evaluated the performance of MAPt on routine bloodstream infection (BSI), focusing on Escherichia coli and Klebsiella pneumoniae isolates from clinical cultures, including resistant strains to some of the six tested antibiotics. Notably, MAPt yielded results exceeding 95% agreement with the standard hospital method within a significantly shorter timeframe of 6 h. These findings suggest significant potential for MAPt as a rapid and reliable BSI management tool.

Global transcriptomic analysis of Francisella tularensis SchuS4 differentially expressed genes in response to doxycycline or ciprofloxacin exposure.

OBJECTIVE: As part of a research aiming at presenting an alternative approach for rapid determination of antimicrobial susceptibility by quantification of changes in expression levels of specific marker genes and gene sets, cultures of the virulent bacterial strain Francisella tularensis SchuS4 were grown in the presence of inhibitory/sub-inhibitory concentrations of either ciprofloxacin or doxycycline and their transcriptomic profiles were elucidated using differential expression analysis followed by functional annotation. DATA DESCRIPTION: RNA sequencing was performed to identify differentially expressed genes (DEGs) in response to exposure of F. tularensis SchuS4 to either ciprofloxacin or doxycycline, the antibiotics of choice for Tularemia therapy. Accordingly, RNA samples were collected 2 h post antibiotic exposure and subjected to RNA sequence analysis. Transcriptomic quantification of RNA representing duplicated samples generated highly similar gene expression data. Exposure to sub-inhibitory concentration [0.5 x MIC (minimal inhibitory concentration)] of doxycycline or ciprofloxacin modulated the expression of 237 or 8 genes, respectively, while exposure to an inhibitory concentration (1 x MIC) resulted in the modulation of 583 or 234 genes, respectively. Amongst the genes modulated upon doxycycline exposure upregulation of 31 genes encoding for translation-functions could be distinguished, as well as downregulation of 14 genes encoding for functions involved in DNA transcription and repair. Ciprofloxacin exposure impacted differently the RNA sequence profile of the pathogen, resulting in upregulation of 27 genes encoding mainly DNA replication and repair functions, transmembrane transporters and molecular chaperons. In addition, 15 downregulated genes were involved in translation processes.

Monkeypox DNA levels correlate with virus infectivity in clinical samples, Israel, 2022.

The current monkeypox virus global spread and lack of data regarding clinical specimens' infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value (Cq ≥ 35, ≤ 4,300 DNA copies/mL), corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox.

Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens.

As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.

Evaluation of a Frozen Micro-Agar Plates of MAPt Antibiotic Susceptibility Test for Enhanced Bioterror Preparedness.

There is an urgent need for rapid antibiotic susceptibility tests to improve clinical treatment and to support antibiotic stewardship, especially concerning the emergence of multi-drug-resistant bacteria. Nowadays this need is even more profound due to progress in synthetic biology procedures that may facilitate the malicious preparation of engineered antibiotic-resistant pathogens. We recently described a novel, rapid, simple, specific, and sensitive method named a Micro-Agar-PCR-test (MAPt) and showed its performance on clinical as well as environmental samples. The method does not require any isolation or purification steps and is applicable to a wide range of bacterial concentrations, thus allowing a short time to respond within a bioterror event (5-7 h for B. anthracis, 10-12 h for Y. pestis, and 16 h for F. tularensis). Ready-to-use reagents for this assay may add a level of preparedness. We examined the option of freezing pre-prepared MAPt agar plates and thawing them upon need. Our results show that adequate minimal inhibitory concentration (MIC) values are obtained with the use of thawed 6- and 12-month frozen agar plates. The ability to store MAPt micro-agar plates at -70 °C for a year, together with all other reagents required for MAPt, holds a great advantage for bioterror preparedness.

Whole genome sequencing and taxonomic profiling of two Pantoea sp. isolated from environmental samples in Israel.

OBJECTIVE: As part of a research aiming at the isolation of bacteria secreting growth inhibiting compounds, cultures of Francisella tularensis were implanted in environmental samples and monitored for inhibition zones on agar. Two antibiotic-like secreting bacteria were isolated, their genomic sequence was deciphered and taxonomic profiling analysis classified them as belonging to the Pantoea genus. DATA DESCRIPTION: Two bacterial isolates exhibiting growth inhibition zones to F. tularensis (LVS) were analyzed using the Oxford Nanopore Technology (ONT). Preliminary de novo assembly of the reads was performed, followed by taxonomic profiling based on Multi Locus Sequence Analysis (MLSA) and implementation of the Average Nucleotide Identity (ANI) measure. The genomic sequences resulted in the identification of two different Pantoea species, denoted EnvD and EnvH. Subsequent de novo genome assembly generated 5 and 10 contigs for EnvD and EnvH, respectively. The largest contig (4,008,183 bps and 3,740,753 bps for EnvD and EnvH, respectively), overlaps to a major extent to the chromosome of closely related Pantoea species. ANI values calculated for both isolates revealed two apparently new species of the Pantoea genus. Our study deciphered the identity of two bacteria producing antibiotic-like compounds, and the genomic sequence revealed they represent distinct Pantoea species.

Exportation of Monkeypox Virus From the African Continent.

BACKGROUND: The largest West African monkeypox outbreak began September 2017, in Nigeria. Four individuals traveling from Nigeria to the United Kingdom (n = 2), Israel (n = 1), and Singapore (n = 1) became the first human monkeypox cases exported from Africa, and a related nosocomial transmission event in the United Kingdom became the first confirmed human-to-human monkeypox transmission event outside of Africa. METHODS: Epidemiological and molecular data for exported and Nigerian cases were analyzed jointly to better understand the exportations in the temporal and geographic context of the outbreak. RESULTS: Isolates from all travelers and a Bayelsa case shared a most recent common ancestor and traveled to Bayelsa, Delta, or Rivers states. Genetic variation for this cluster was lower than would be expected from a random sampling of genomes from this outbreak, but data did not support direct links between travelers. CONCLUSIONS: Monophyly of exportation cases and the Bayelsa sample, along with the intermediate levels of genetic variation, suggest a small pool of related isolates is the likely source for the exported infections. This may be the result of the level of genetic variation present in monkeypox isolates circulating within the contiguous region of Bayelsa, Delta, and Rivers states, or another more restricted, yet unidentified source pool.

Rapid Antibiotic Susceptibility Testing of Tier-1 Agents Bacillus anthracis, Yersinia pestis, and Francisella tularensis Directly From Whole Blood Samples.

Rapid antibiotic susceptibility tests, performed directly on whole blood samples, will offer great clinical advantages. This issue is of considerable importance when it comes to bioterror pathogens where prompt antibiotic treatment should be offered to infected patients as well as prophylaxis to suspected exposed individuals. Herein, we describe a novel and rapid method, named MAPt, that is based on the direct application of a blood sample onto solid agar that has been embedded with different concentrations of the tested antibiotic. Following a short incubation, bacterial growth is monitored by qPCR. The method was applied on blood cultures and whole blood samples inoculated with the Tier-1 pathogens Bacillus anthracis, Yersinia pestis, and Francisella tularensis. The use of agar medium, which better supports the growth of bacteria at low concentrations, together with the use of qPCR, which provides sensitivity and specificity, allowed minimal inhibitory concentration (MIC) determination to a wide range of bacterial concentrations, ranging from ∼5 × 102 cfu/ml up to 108 cfu/ml. The omission of the enrichment procedure in blood culture and the isolation step, both required in standard antibiotic susceptibility tests (ASTs), allowed a dramatic reduction in time to answer, from a few days to a few hours. The total time required for MIC determination was ∼6 h for fast-growing bacteria, such as B. anthracis, and 12-16 h for slow-growing bacteria, represented by Y. pestis and F. tularensis. Accordingly, MAPt may offer health authorities means for public preparedness in the case of a bioterror attack as well as prompt clinical treatment options in common blood stream infections.

Evaluation of the European Committee on Antimicrobial Susceptibility Testing Guidelines for Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis-, Yersinia pestis- and Francisella tularensis-Positive Blood Cultures.

Rapid determination of bacterial antibiotic susceptibility is important for proper treatment of infections. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently published guidelines for rapid antimicrobial susceptibility testing (RAST) performed directly from positive blood culture vials. These guidelines, however, were only published for a limited number of common pathogenic bacteria. In this study, we evaluated the applicability of these guidelines to three Tier 1 bioterror agents (Bacillus anthracis, Yersinia pestis and Francisella tularensis) that require prompt antibiotic treatment to mitigate morbidity and mortality. We used spiked-in human blood incubated in a BACTEC™ FX40 system to determine the proper conditions for RAST using disc-diffusion and Etest assays. We found that reliable disc-diffusion inhibition diameters and Etest MIC values could be obtained in remarkably short times. Compared to the EUCAST-recommended disc-diffusion assays that will require adjusted clinical breakpoint tables, Etest-based RAST was advantageous, as the obtained MIC values were similar to the standard MIC values, enabling the use of established category breakpoint tables. Our results demonstrate the promising applicability of the EUCAST RAST for B. anthracis-, Y. pestis- or F. tularensis-positive blood cultures, which can lead to shorter diagnostics and prompt antibiotic treatment of these dangerous pathogens.