Bacillus anthracis in South Africa, 1975-2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

Genetic Homogeneity of Francisella tularensis subsp. mediasiatica Strains in Kazakhstan.

Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation.

Avirulence of a spontaneous Francisella tularensis subsp. mediasiatica prmA mutant.

Francisella tularensis, the causative agent of tularemia, is divided into three subspecies. Two of these, subspecies holarctica and tularensis, are highly pathogenic to humans and consequently relatively well studied. The third subspecies, mediasiatica, is rarely isolated and remains poorly studied. It is distributed in the sparsely populated regions of Central Asia and Siberia. Curently this subspecies is not known to have been responsible for human infections in spite of its high virulence in laboratory animals. Subspecies mediasiatica is currently divided into three subgroups-MI, present in Central Asia, MII, present in southern Siberia, and MIII represented by a unique strain, 60(B)57, isolated in Uzbekistan in 1960. We describe here the unexpected observation that MIII strain 60(B)57 is avirulent and immunogenic. We observed that infection with this strain protected mice from challenge 21 days later with a virulent subsp. mediasiatica strain. With an increase of this interval, the protection for mice was significantly reduced. In contrast, guinea pigs were protected from challenge with strains of the subspecies holarctica and mediasiatica (but not subsp. tularensis) 90 days after infection with 60(B)57. We performed genome assembly based on whole genome sequencing data obtained using the Nanopore MinION for strain 60(B)57 and two subsp. mediasiatica strains representing the Central Asian MI and Siberian MII phylogenetic subgroups. The prmA gene is truncated due to a nonsense mutation in strain 60(B)57. The deletion of gene prmA has previously been shown to induce a loss of virulence in Francisella novicida the closest model organism suggesting that the observed mutation might the cause of the avirulence of strain 60(B)57.

Genomic Diversity and Zoonotic Potential of Brucella neotomae.

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955-1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

New Research on the Bacillus anthracis Genetic Diversity in Siberia.

Anthrax is a particularly dangerous infection of humans and ungulates caused by the Gram-positive spore-forming bacterium Bacillus anthracis. The highly monomorphic and clonal species B. anthracis is commonly divided into three main lineages, A, B, and C, which in turn are divided into several canSNP groups. We report here a phylogenetic analysis based on the whole-genome sequence (WGS) data of fifteen strains isolated predominantly in Siberia or Central and Southern Russia. We confirm the wide distribution of the cluster of strains of the B.Br.001/002 group, endemic to the Russian Arctic, which is also present in the steppe zone of Southern Siberia. We characterize additional branches within the major A.Br.001/002 polytomy comprising the A.Br.Ames and A.Br.Sterne lineages, one of which is identified in the Arctic.

Brucella abortus in Kazakhstan, population structure and comparison with worldwide genetic diversity.

Brucella abortus is the main causative agent of brucellosis in cattle, leading to severe economic consequences in agriculture and affecting public health. The zoonotic nature of the infection increases the need to control the spread and dynamics of outbreaks in animals with the incorporation of high resolution genotyping techniques. Based on such methods, B. abortus is currently divided into three clades, A, B, and C. The latter includes subclades C1 and C2. This study presents the results of whole-genome sequencing of 49 B. abortus strains isolated in Kazakhstan between 1947 and 2015 and of 36 B. abortus strains of various geographic origins isolated from 1940 to 2004. In silico Multiple Locus Sequence Typing (MLST) allowed to assign strains from Kazakhstan to subclades C1 and to a much lower extend C2. Whole-genome Single-Nucleotide Polymorphism (wgSNP) analysis of the 46 strains of subclade C1 with strains of worldwide origins showed clustering with strains from neighboring countries, mostly North Caucasia, Western Russia, but also Siberia, China, and Mongolia. One of the three Kazakhstan strains assigned to subclade C2 matched the B. abortus S19 vaccine strain used in cattle, the other two were genetically close to the 104 M vaccine strain. Bayesian phylodynamic analysis dated the introduction of B. abortus subclade C1 into Kazakhstan to the 19th and early 20th centuries. We discuss this observation in view of the history of population migrations from Russia to the Kazakhstan steppes.

Whole genome sequence analysis of Neisseria meningitidis strains circulating in Kazakhstan, 2017-2018.

Neisseria meningitidis (meningococcus) is a cosmopolitan bacterium that is often found in the upper respiratory tract of asymptomatic humans. However, N. meningitidis also causes meningeal inflammation and/or sepsis in humans with a periodic resurgence in incidence and high mortality rates. The pathogen is highly diverse genetically and antigenically, so that genotyping is considered important for vaccine matching to circulating strains. Annual incidence of meningococcal disease in Kazakhstan ranges between 0.2 and 2.5 cases per 100 thousand population. In total, 78 strains of N. meningitidis were isolated from clinical patients and contact persons during the years 2017-2018 in Kazakhstan. Of these, 41 strains including four from the patients and 37 from contacts, were sequenced using Illumina MiSeq. In silico typing was completed using the Neisseria pipeline 1.2 on the Galaxy Workflow Management System and PubMLST. Whole genome SNP (single nucleotide polymorphisms) trees were built using BioNumerics 8. Seven-gene multilocus sequence typing (MLST) identified ten sequence types (ST), two of which have not been previously described (ST-16025; ST-16027). ST-16025 was detected in two patients with invasive meningococcal disease in 2017 and 2018 in Akmola region and 16 contacts in 2017 in Turkistan region. This prevalent type ST-16025 demonstrates considerable intertypic diversity as it consists of three subcomplexes with a distance of more than 2000 SNPs. Invasive and carrier strains belong to different serogroups (MenB and MenC), PorA and FetA_VR. Two invasive strains were MenB, one MenC and one MenW (Hajj lineage). The strains from the contact persons were: MenC (n = 18), cnl (n = 9), MenY (n = 7), MenW (n = 1), MenB (n = 1) and one unidentifiable. Different numbers of alleles were present: 12, 11, 7, and 7 alleles for PorA, FetA, fHbp, and NHBA, respectively. This study is the first report of the genetic diversity of N. meningitidis strains in Kazakhstan. Despite limitations with the studied sample size, important conclusions can be drawn based on data produced. This study provides evidence for regulatory authorities with regard to changing routine diagnostic protocols to increase the collecting of samples for WGS.

CoxBase: an Online Platform for Epidemiological Surveillance, Visualization, Analysis, and Typing of Coxiella burnetii Genomic Sequences.

Q (query) fever is an infectious zoonotic disease caused by the Gram-negative bacterium Coxiella burnetii. Although the disease has been studied for decades, it still represents a threat due to sporadic outbreaks across farms in Europe. The absence of a central platform for Coxiella typing data management is an important epidemiological gap that is relevant in the case of an outbreak. To fill this gap, we have designed and implemented an online, open-source, web-based platform called CoxBase (https://coxbase.q-gaps.de). This platform includes a database that holds genotyping information on more than 400 Coxiella isolates alongside metadata that annotate them. We have also implemented features for in silico genotyping of completely or minimally assembled Coxiella sequences using five different typing methods, querying of existing isolates, visualization of isolate geodata via aggregation on a world map, and submission of new isolates. We tested our in silico typing method on 50 Coxiella genomes downloaded from the RefSeq database, and we successfully genotyped all genomes except for cases where the sequence quality was poor. We identified new spacer sequences using our implementation of the multispacer sequence typing (MST) in silico typing method and established adaA gene phenotypes for all 50 genomes as well as their plasmid types. IMPORTANCE Q fever is a zoonotic disease that is a source of active epidemiological concern due to its persistent threat to public health. In this project, we have identified areas in the field of Coxiella research, especially regarding public health and genomic analysis, where there is an inadequacy of resources to monitor, organize, and analyze genomic data from C. burnetii. Subsequently, we have created an open, web-based platform that contains epidemiological information, genome typing functions comprising all the available Coxiella typing methods, and tools for isolate data discovery and visualization that could help address the above-mentioned challenges. This is the first platform to combine all disparate genotyping systems for Coxiella burnetii as well as metadata assets with tools for genomic comparison and analyses. This platform is a valuable resource for laboratory researchers as well as research epidemiologists interested in investigating the relatedness or dissimilarity among C. burnetii strains.

Bacillus anthracis Phylogeography: New Clues From Kazakhstan, Central Asia.

This article describes Bacillus anthracis strains isolated in Kazakhstan since the 1950s until year 2016 from sixty-one independent events associated with anthrax in humans and animals. One hundred and fifty-four strains were first genotyped by Multiple Locus VNTR (variable number of tandem repeats) Analysis (MLVA) using 31 VNTR loci. Thirty-five MLVA31 genotypes were resolved, 28 belong to the A1/TEA group, five to A3/Sterne-Ames group, one to A4/Vollum and one to the B clade. This is the first report of the presence of the B-clade in Kazakhstan. The MLVA31 results and epidemiological data were combined to select a subset of seventy-nine representative strains for draft whole genome sequencing (WGS). Strains from Kazakhstan significantly enrich the known phylogeny of the Ames group polytomy, including the description of a new branch closest to the Texas, United States A.Br.Ames sublineage stricto sensu. Three among the seven currently defined branches in the TEA polytomy are present in Kazakhstan, "Tsiankovskii", "Heroin", and "Sanitary Technical Institute (STI)". In particular, strains from the STI lineage are largely predominant in Kazakhstan and introduce numerous deep branching STI sublineages, demonstrating a high geographic correspondence between "STI" and Kazakhstan, Central Asia. This observation is a strong indication that the TEA polytomy emerged after the last political unification of Asian steppes in the fourteenth century of the Common Era. The phylogenetic analysis of the Kazakhstan data and of currently available WGS data of worldwide origin strengthens our understanding of B. anthracis geographic expansions in the past seven centuries.

Whole-Genome Sequence of a Brucella pinnipedialis Sequence Type 54 Strain Isolated from a Hooded Seal (Cystophora cristata) from the North Atlantic Ocean, Norway.

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammal species. We report the first complete genome sequence of a Brucella strain isolated from a hooded seal (Cystophora cristata), Brucella pinnipedialis strain 23a-1 of sequence type 54, found in the North Atlantic Ocean surrounding Norway.

Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan.

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.

Retrospective Analysis of the Relationship between Two Anthrax Outbreaks in Kazakhstan Based on Genomic Data.

We present a retrospective analysis of strains from two anthrax outbreaks in western Kazakhstan in 2009. The outbreaks occurred during the same period and in the same area located close to main roads, favoring a single source of infection. However, multilocus variable-number tandem-repeat analysis (MLVA), canonical single-nucleotide polymorphism (CanSNP) analysis, and genome-wide analysis demonstrated that the outbreaks were not connected.

Draft Genome Sequence of the Strain Francisella tularensis subsp. mediasiatica 240, Isolated in Kazakhstan.

Francisella tularensis subsp. mediasiatica is the least studied among the four F. tularensis subspecies. We present here the genome data of F. tularensis subsp. mediasiatica 240, isolated in the southern region of Kazakhstan.

Draft Genome Sequences of Three Pasteurella multocida Strains Isolated from Domestic Animals in Kazakhstan.

We report here the draft genome sequences of three strains of Pasteurella multocida isolated in Kazakhstan from domestic animals that died due to hemorrhagic septicemia.

Draft Genome Sequence of Moraxella bovoculi Strain KZ-1, Isolated from Cattle in North Kazakhstan.

Moraxella bovoculi strain KZ-1 was isolated from cattle that had symptoms of infectious bovine keratoconjunctivitis (IBK) in northern Kazakhstan. Here, we report the draft genome sequence of this strain.

The Basis for Natural Multiresistance to Phage in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is responsible for long-term infections and is particularly resistant to treatments when hiding inside the extracellular matrix or biofilms. Phage therapy might represent an alternative to antibiotic treatment, but up to 10% of clinical strains appear to resist multiple phages. We investigated the characteristics of P. aeruginosa clinical strains naturally resistant to phages and compared them to highly susceptible strains. The phage-resistant strains were defective in lipopolysaccharide (LPS) biosynthesis, were nonmotile and displayed an important degree of autolysis, releasing phages and pyocins. Complete genome sequencing of three resistant strains showed the existence of a large accessory genome made of multiple insertion elements, genomic islands, pyocins and prophages, including two phages performing lateral transduction. Mutations were found in genes responsible for the synthesis of LPS and/or type IV pilus, the major receptors for most phages. CRISPR-Cas systems appeared to be absent or inactive in phage-resistant strains, confirming that they do not play a role in the resistance to lytic phages but control the insertion of exogenous sequences. We show that, despite their apparent weakness, the multiphage-resistant strains described in this study displayed selective advantages through the possession of various functions, including weapons to eliminate other strains of the same or closely related species.

A Review of Arguments for the Existence of Latent Infections of Bacillus anthracis, and Research Needed to Understand their Role in the Outbreaks of Anthrax.

Hugh-Jones and Blackburn and Turnbull's collective World Health Organization (WHO) report did literature reviews of the theories and the bases for causes of anthrax outbreaks. Both comment on an often-mentioned suspicion that, even though unproven, latent infections are likely involved. Hugh-Jones suggested Gainer do an updated review of our present-day knowledge of latent infections, which was the basis for Gainer's talk at the Biology of Anthrax Conference in Bari, Italy 2019. At the Conference Gainer met Vergnaud who presented anthrax genome studies that implied that the disease might have spread throughout Asia and from Europe to North America in a short time span of three or four centuries. Vergnaud wondered if latent infections might have played a role in the process. Several other presenters at the Conference also mentioned results that might suggest the existence of latent infections. Vergnaud subsequently looked into some of the old French literature about related observations, results, and discussions of early Pasteur vaccine usage (late 1800's) and found mentions of suspected latent infections. The first part of the paper is a focused summary and interpretation of Hugh-Jones and Blackburn's and Turnbull's reviews specifically looking for suggestions of latent infections, a few additional studies with slightly different approaches, and several mentions made of presentations and posters at the Conference in Italy. In general, many different investigators in different areas and aspects of the anthrax study at the Conference found reasons to suspect the existence of latent infections. The authors conclude that the affected species most studied, including Homo sapiens, provide circumstantial evidence of latent infections and modified host resistance. The last part of the review explores the research needed to prove or disprove the existence of latent infections.

Omp2b Porin Alteration in the Course of Evolution of Brucella spp.

The genus Brucella comprises major pathogenic species causing disease in livestock and humans, e.g. B. melitensis. In the past few years, the genus has been significantly expanded by the discovery of phylogenetically more distant lineages comprising strains from diverse wildlife animal species, including amphibians and fish. The strains represent several potential new species, with B. inopinata as solely named representative. Being genetically more distant between each other, relative to the "classical" Brucella species, they present distinct atypical phenotypes and surface antigens. Among surface protein antigens, the Omp2a and Omp2b porins display the highest diversity in the classical Brucella species. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. They share between 85 and 100% sequence identity depending on the Brucella species, biovar, or genotype. Only the omp2b gene copy has been shown to be expressed and genetic variation is extensively generated by gene conversion between the two copies. In this study, we analyzed the omp2 loci of the non-classical Brucella spp. Starting from two distinct ancestral genes, represented by Australian rodent strains and B. inopinata, a stepwise nucleotide reduction was observed in the omp2b gene copy. It consisted of a first reduction affecting the region encoding the surface L5 loop of the porin, previously shown to be critical in sugar permeability, followed by a nucleotide reduction in the surface L8 loop-encoding region. It resulted in a final omp2b gene size shared between two distinct clades of non-classical Brucella spp. (African bullfrog isolates) and the group of classical Brucella species. Further evolution led to complete homogenization of both omp2 gene copies in some Brucella species such as B. vulpis or B. papionis. The stepwise omp2b deletions seemed to be generated through recombination with the respective omp2a gene copy, presenting a conserved size among Brucella spp., and may involve short direct DNA repeats. Successive Omp2b porin alteration correlated with increasing porin permeability in the course of evolution of Brucella spp. They possibly have adapted their porin to survive environmental conditions encountered and to reach their final status as intracellular pathogen.

Characterization of sixteen Achromobacter xylosoxidans phages from Abidjan, Côte d'Ivoire, isolated on a single clinical strain.

Sixteen bacteriophages of Achromobacter xylosoxidans distributed into four genera have been isolated from sewage water in Abidjan, Côte d'Ivoire, using a single clinical strain, and their genomes have been sequenced. Three podoviruses belonged to the genus Phikmvvirus, and these represent the first A. xylosoxidans phages of this genus. Seven podoviruses, distributed into three groups, belonged to the genus Jwalphavirus. Among the siphoviruses, three revealed similarities to Pseudomonas phage 73 and members of the genus Septimatrevirus, and three were YuA-like phages. The virulence of these phages toward a panel of 10 genetically diverse strains was tested, with the phiKMV-like phages showing the broadest host range.

CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers.

In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palindromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.