Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis.

The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.

Development of a specific real-time PCR assay for simultaneous detection and differentiation of Coxiella burnetii strains from environmental soil samples.

Coxiella burnetii, the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii, based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible, and sensitive, allowing the detection of as few as 5 genome equivalents (GEs) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3 × 104 colony forming units per 0.5 g of soil (∼3 GEs per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples.

Phylogenetic Placement of Isolates Within the Trans-Eurasian Clade A.Br.008/009 of Bacillus anthracis.

The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis strains of this clade, derived from human infections, animal outbreaks or soil, mostly from European countries isolated between 1936 and 2008. The new data were subjected to comparative chromosomal analysis, together with 75 B. anthracis genomes available in public databases, and the relative placements of these isolates were determined within the global phylogeny of the A.Br.008/009 canSNP group. From this analysis, we have detected 3754 chromosomal SNPs, allowing the assignation of the new chromosomal sequences to established sub-clades, to define new sub-clades, such as two new Spanish, one Bulgarian or one German group(s), or to introduce orphan lineages. SNP-based results were compared with that of a multilocus variable number of tandem repeat analysis (MLVA). This analysis indicated that MLVA typing might provide additional information in cases when genomics yields identical genotypes or shows only minor differences. Introducing the delayed mismatch amplification assay (DMAA) PCR-analysis, we developed a cost-effective method to interrogate for a set of ten phylogenetically informative SNPs within genomes of A.Br.008/009 canSNP clade strains of B. anthracis. By this approach, additional 32 strains could be assigned to five of ten defined clades.

Extracción de ricina por procedimientos incluidos en publicaciones paramilitares y manuales relacionados con la red terrorista Al Qaeda / Extraction of ricin by procedures featured on paramilitary publications and manuals related to the Al Qaeda terrorist network

Antecedentes: La ricina es una fitotoxina con actividad citotóxica presente en las semillas de ricino (Ricinus communis L.). Algunas publicaciones paramilitares y manuales relacionados con la red terrorista Al Qaeda explican procedimientos para su extracción a partir de las semillas. Esto ha llevado a la actual preocupación por que esta toxina pueda ser empleada con fines terroristas. Objetivo: Determinar la viabilidad y eficacia de estas técnicas de extracción. Materiales y métodos: A partir de las semillas de Ricinus communis, variedades gibsonii y zanzibariensis, se siguieron dos procedimientos para la extracción de ricina incluidos en estas publicaciones y manuales. El primero de ellos se basa en la eliminación del aceite de la semilla mediante la extracción del mismo con disolventes orgánicos. El segundo consiste en la precipitación de la ricina, a partir del extracto acuoso, por modificación de la fuerza iónica mediante la adición de una sal. La detección cuantitativa de ricina en las muestras fue realizada por enzimoinmunoensayo de captura (ELISAc). Resultados: Los extractos de las semillas obtenidos por el primer procedimiento en las variedades zanzibariensis y gibsonii tenian un contenido en ricina de 0,33 por ciento y 0,01 por ciento (peso/peso), respectivamente. La muestra obtenida por la técnica de precipitación por adición de una sal no contenía ricina. Conclusiones: En este trabajo hemos podido constatar la viabilidad y la eficacia de varias técnicas de extracción para la ricina rápidas, baratas y a partir de productos de fácil adquisición. Su utilización como "arma de destrucción masiva", es decir, con el fin de causar un elevado número de intoxicados, estaría limitada por el bajo contenido en ricina de los extractos obtenidos y por la dificultad de diseminarlos de forma eficaz. Sin embargo, no se puede descartar su uso con fines terroristas selectivos, es decir, limitado a una o varias personas, así como el posible perfeccionamiento de los procedimientos incluidos en estas publicaciones (AU)