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In November 2018, an outbreak of tularemia occurred among hare hunters in Bavaria, Germany. At least one infected hare was confirmed as the source of infection. A number of hunting dogs showed elevated antibody titers to Francisella tularensis , but the absence of titer increases in subsequent samples did not point to acute infections in dogs. Altogether, 12 persons associated with this hare hunt could be diagnosed with acute tularemia by detection of specific antibodies. In nine patients, the antibody and cytokine responses could be monitored over time. Eight out of these nine patients had developed detectable antibodies three weeks after exposure; in one individual the antibody response was delayed. All patients showed an increase in various cytokines and chemokines with a peak for most mediators in the first week after exposure. Cytokine levels showed individual variations, with high and low responders. The kinetics of seroconversion has implications on serological diagnoses of tularemia.

Tularemia is a zoonotic disease caused by Francisella tularensis a small, pleomorphic, facultative intracellular bacterium. In Europe, infections in animals and humans are caused mainly by Francisella tularensis subspecies holarctica . Humans can be exposed to the pathogen directly and indirectly through contact with sick animals, carcasses, mosquitoes and ticks, environmental sources such as contaminated water or soil, and food. So far, F. tularensis subsp. holarctica is the only Francisella species known to cause tularemia in Germany. On the basis of surveillance data, outbreak investigations, and literature, we review herein the epidemiological situation-noteworthy clinical cases next to genetic diversity of F. tularensis subsp. holarctica strains isolated from patients. In the last 15 years, the yearly number of notified cases of tularemia has increased steadily in Germany, suggesting that the disease is re-emerging. By sequencing F. tularensis subsp. holarctica genomes, knowledge has been added to recent findings, completing the picture of genotypic diversity and geographical segregation of Francisella clades in Germany. Here, we also shortly summarize the current knowledge about a new Francisella species ( Francisella sp. strain W12-1067) that has been recently identified in Germany. This species is the second Francisella species discovered in Germany.

Europe-wide activities to improve biosafety and biosecurity performed within the frameworks of the European Union (EU)-funded Joint Actions EMERGE and QUANDHIP led to the development of an Integrated European Checklist for Laboratory Biorisk Management (ECL).To better understand different approaches shaping biorisk management (BRM) systems on an operational level in high containment laboratories, the ECL was used to map the implementation of BRM in 32 high containment laboratories in 18 countries in Europe. The results suggest that the BRM elements referring to standard microbiological working practices and the handling of infectious material were fulfilled particularly well. The elements safety exercises involving internal and external emergency responders, and appropriate decommissioning plans were not fulfilled particularly well. BRM in Biosafety Level (BSL) 4 laboratories handling Risk Group (RG) 4 viruses appear to vary among each other less than BSL3 laboratories handling RG 3 bacteria. It is important to agree on comparable regulations in Europe as high containment laboratories are indispensable for a safe, quick and effective response to public health threats. As high containment laboratories may also present a public health risk it is crucial to have robust BRM on organisational and operational levels.

Objectives: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms., Methods: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs., Results: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28)., Conclusions: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacillus cereus biovar anthracis (Bcbva) is an untypical anthrax-causing pathogen responsible for high wildlife mortality in Taï National Park (TNP), Côte d'Ivoire. However, nothing is known about its effect on the rural population living in the region bordering TNP. Contact to bushmeat is a known risk factor for exposure to a variety of zoonotic pathogens, but no human infections with Bcbva were noted so far. Therefore, we performed a retrospective seroprevalence analysis with sera from 1,386 study volunteers. We used assays which detect antibodies against the protective antigen PA, which is synthesized by both Bcbva and classic B. anthracis, and against the recently described antigen pXO2-60, a 35-kDa protein only produced by Bcbva. We found a high seroprevalence (22.37%) of antibodies against PA, and approximately half of those sera (10.46%) were also positive for the Bcbva-specific antigen pXO2-60. All sera negative for PA were also negative for antibodies against pXO2-60, confirming specificity and suitability of the PA/pXO2-60 combined serological assay. The fact that a large fraction of sera was positive for PA but negative for pXO2-60 can most likely be explained by lower immunogenicity of pXO2-60, but exposure to classic B. anthracis cannot be excluded. As only Bcbva has been detected in the TNP area so far, exposure to Bcbva can be suspected from the presence of antibodies against PA alone. In a questionnaire, most study participants reported contact to bushmeat and livestock carcasses. Unfortunately, risk factor analysis indicated that neither animal contacts, sex, age, nor country of origin were significant predictors of Bcbva seroprevalence. Nevertheless, our study added to an assessment of the distribution of Bcbva and its impact on the human population, and our data can serve to raise awareness of anthrax in the affected regions., Competing Interests: The authors have declared that no competing interests exist.

A fast, effective, and safe disinfection of personal protective equipment (PPE) is vitally important for emergency forces involved in biological hazards. This study aimed to investigate a broad range of disinfectants to improve the established disinfection procedure. We analyzed the efficacy of chlorine-, peracetic acid-, and oxygen-based disinfectants against Bacillus spores on PPE. Therefore, spores of different Bacillus species were exposed to disinfectants on PPE material by using a standardized procedure covering the dried spores with disinfectants and applying mechanical distribution. Efficacy of disinfectants was quantified by determining the reduction factor (log 10 levels) and number of viable spores left afterward. The chlorine-based granulate Hypochlorit CA G (2% chlorine) sufficiently inactivated Bacillus spores of risk groups 1 and 2, even with temperatures ranging from -20 to 35°C. Wofasteril ® SC super (1.75% peracetic acid) achieved a reliable reduction of risk groups 1 and 2 and even fully virulent Bacillus spores by ≥5 log 10 levels on PPE. With this, Hypochlorit-CA G and Wofasteril ® SC super proved to be promising alternatives to the previously proven and widely used peracetic acid compound Wofasteril ® (2% peracetic acid) for the disinfection of PPE when bacterial spores are known to be the contaminating agent. These results will help to improve the disinfection of PPE during biological hazards by providing new data on promising alternative compounds.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients ( n = 25) and free-living animals ( n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%., (Copyright © 2019 Appelt, Köppen, Radonić, Drechsel, Jacob, Grunow and Heuner.)

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The Ebola virus disease outbreak in West Africa in 2014/2015 was by far the biggest, most prolonged, and geographically most widespread outbreak of this disease since the discovery of the Ebola virus in 1976. Although no cases of Ebola virus disease were confirmed in Germany, a number of crisis management activities were initiated.Based on a combination of local, national, and international lessons learned, literature research, and a large number of discussions among German colleagues as well as German and foreign colleagues, the experiences of selected German public health actors as well as implications for health protection activities in Germany are presented.On the one hand, preparedness for managing unusual high consequence health events-caused by rare, highly pathogenic biological agents-including the provision of adequate material and personnel resources remains important in Germany. On the other hand, more German engagement in global health is necessary, because the dividing line between global health and local health is increasingly disappearing.

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13 C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: Tularemia is caused by Francisella tularensis and can occasionally establish foodborne transmission., Methods: Patients were identified by active case detection through contact with the treating physicians and consent for publication was obtained. Clinical data were accumulated through a review of the patient charts. Serology, culture, and PCR methods were performed for confirmation of the diagnosis., Case Cluster: A 46-year-old patient was hospitalised in the University Hospital Frankfurt (a tertiary care hospital) for pharyngitis and cervical lymphadenitis with abscess. A diagnosis of tularemia was made serologically, but treatment with ciprofloxacin initially failed. F. tularensis was detected in pus from the lymph node using a specific real-time PCR. The use of RD1 PCR led to the identification of the subspecies holarctica. Antibiotic therapy with high-dose ciprofloxacin and gentamicin was administered and was subsequently changed to ciprofloxacin and rifampicin. During a must-tasting, five other individuals became infected with tularemia by ingestion of contaminated must. All patients required treatment durations of more than 14 days., Conclusions: Mechanically harvested agricultural products, such as wine must, can be a source of infection, probably due to contamination with animal carcasses. The clinical course of tularemia can be complicated and prolonged and requires differentiated antibiotic treatment., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Exploratory field analyses of the inactivation capacity of disinfectants on contaminated personal protective equipment (PPE) are required to select a suitable surrogate for biohazardous agents like spores of Bacillus anthracis . The objectives of our study were (1) the determination of an appropriate surrogate for the inactivation of spores of B. anthracis with peracetic acid (PAA), and (2) application of optimized inactivation conditions for an effective decontamination of PPE with PAA under field conditions. For inactivation studies, B. anthracis spores from different strains and B. thuringiensis spores were fixed by air drying on carriers prepared from PPE fabric. Time and concentration studies with PAA-based disinfectants revealed that the spores of the B. thuringiensis strain DSM 350 showed an inactivation profile comparable to that of the spores of the B. anthracis strain with the highest stability, implying that B. thuringiensis can serve as an appropriate surrogate. Rapid (3 to 5 minutes) and effective surface decontamination was achieved with 2% PAA/0.2% surfactant. In field studies, PPE contaminated with spores of B. thuringiensis was treated with the disinfectant. Optimizing the decontamination technique revealed that spraying in combination with brushing was effective within 5 minutes of exposure.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica , belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging infectious disease threat in the European Union. Since 2000, the incidence and geographic range of confirmed CCHF cases have markedly increased, following changes in the distribution of its main vector, Hyalomma ticks.AimsTo review scientific literature and collect experts' opinion to analyse relevant aspects of the laboratory management of human CCHF cases and any exposed contacts, as well as identify areas for advancement of international collaborative preparedness and laboratory response plans.MethodsWe conducted a literature review on CCHF molecular diagnostics through an online search. Further, we obtained expert opinions on the key laboratory aspects of CCHF diagnosis. Consulted experts were members of two European projects, EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level) and EVD-LabNet (Emerging Viral Diseases-Expert Laboratory Network).ResultsConsensus was reached on relevant and controversial aspects of CCHF disease with implications for laboratory management of human CCHF cases, including biosafety, diagnostic algorithm and advice to improve lab capabilities. Knowledge on the diffusion of CCHF can be obtained by promoting syndromic approach to infectious diseases diagnosis and by including CCHFV infection in the diagnostic algorithm of severe fevers of unknown origin.ConclusionNo effective vaccine and/or therapeutics are available at present so outbreak response relies on rapid identification and appropriate infection control measures. Frontline hospitals and reference laboratories have a crucial role in the response to a CCHF outbreak, which should integrate laboratory, clinical and public health responses.

A Bacillus anthracis vaccine strain (Sterne), used as an attenuated laboratory comparative strain, was sequenced and analyzed. A comparison to assemblies of B. anthracis strain Sterne (NZ_CP009541 and NZ_CP009540) was performed. The lack of the pX02 plasmid and pX01 in approximately five copies was confirmed.

From December 2013 to March 2016, West Africa experienced the largest Ebola virus (EBOV) outbreak to date, leading to a European-wide activation of laboratory preparedness and response. At the end of the outbreak, laboratories associated with the two European preparedness networks of expert laboratories EMERGE JA and EVD-LabNet were invited to participate in an assessment of the response of European laboratories to the EBOV outbreak, to identify learning points and training needs to strengthen future outbreak responses. Response aspects assessed included diagnostics, biorisk management and quality assurance. The overall coverage of EBOV diagnostics in the European Union/European Economic Area (EU/EEA) was found to be adequate although some points for quality improvement were identified. These included the need for relevant International Organization for Standardization (ISO) accreditation, the provision of EBOV external quality assessments (EQA) in periods where there is no emergency, facilitating access to controls and knowledge, biorisk management without compromising biosafety and a rapid public health response, and the need for both sustained and contingency funding for preparedness and response activities.

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica , the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks ( Ixodes ricinus) , animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 ( F. tularensis subsp. novicida -like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val ( Francisella Integration Vector-tRNA Val -specific), using the attL/R- sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli . The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp . where FIV-Val stably integrated site specifically into the tRNA Val gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Tularemia, also known as "rabbit fever," is a zoonosis caused by the facultative intracellular, gram-negative bacterium Francisella tularensis . Infection occurs through contact with infected animals (often hares), arthropod vectors (such as ticks or deer flies), inhalation of contaminated dust or through contaminated food and water. In this review, we would like to provide an overview of the current epidemiological situation in Germany using published studies and case reports, an analysis of recent surveillance data and our own experience from the laboratory diagnostics, and investigation of cases. While in Germany tularemia is a rarely reported disease, there is evidence of recent re-emergence. We also describe some peculiarities that were observed in Germany, such as a broad genetic diversity, and a recently discovered new genus of Francisella and protracted or severe clinical courses of infections with the subspecies holarctica . Because tularemia is a zoonosis, we also touch upon the situation in the animal reservoir and one-health aspects of this disease. Apparently, many pieces of the puzzle need to be found and put into place before the complex interaction between wildlife, the environment and humans are fully understood. Funding for investigations into rare diseases is scarce. Therefore, combining efforts in several countries in the framework of international projects may be necessary to advance further our understanding of this serious but also scientifically interesting disease.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Bacillus cereus biovar anthracis (Bcbva) is a member of the B. cereus group which carries both B. anthracis virulence plasmids, causes anthrax-like disease in various wildlife species and was described in several sub-Saharan African rainforests. Long-term monitoring of carcasses in Taï National Park, Côte d'Ivoire, revealed continuous wildlife mortality due to Bcbva in a broad range of mammalian species. While non-lethal anthrax infections in wildlife have been described for B. anthracis, nothing is known about the odds of survival following an anthrax infection caused by Bcbva. To address this gap, we present the results of a serological study of anthrax in five wildlife species known to succumb to Bcbva in this ecosystem. Specific antibodies were only detected in two out of 15 wild red colobus monkeys (Procolobus badius) and one out of 10 black-and-white colobus monkeys (Colobus polykomos), but in none of 16 sooty mangabeys (Cercocebus atys), 9 chimpanzees (Pan troglodytes verus) and 9 Maxwell's duikers (Cephalophus maxwellii). The combination of high mortality and low antibody detection rates indicates high virulence of this disease across these different mammalian species.

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation.

Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F. tularensis subsp. holarctica is highly pathogenic for humans, F. novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13 C-labeling experiments with F. tularensis subsp. holarctica wild type (beaver isolate), F. tularensis subsp. holarctica strain LVS, or F. novicida strain U112 in complex media containing either [U- 13 C 6 ]glucose, [1,2- 13 C 2 ]glucose, [U- 13 C 3 ]serine, or [U- 13 C 3 ]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F. tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F. novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F. tularensis subsp. holarctica or metabolized, with higher rates in F. novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F. tularensis and F. novicida . Glycerol efficiently served as a gluconeogenetic substrate in F. novicida , but only less in the F. tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., via acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the F. tularensis subsp. holarctica strains. In summary, the data reflect differential metabolite fluxes in F. tularensis subsp. holarctica and F. novicida suggesting that the different utilization of substrates could be related to host specificity and virulence of Francisella .

Highly infectious diseases can spread rapidly across borders through travel or trade, and international coordination is essential to a prompt and efficient response by public health laboratories. Therefore, developing strategies to identify priorities for a rational allocation of resources for research and surveillance has been the focus of a large body of research in recent years. This paper describes the activities and the strategy used by a European-wide consortium funded by the European Commission, named EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level), for the selection of high-threat pathogens with cross-border potential that will become the focus of its preparedness activities. The approach used is based on an objective scoring system, a close collaboration with other networks dealing with highly infection diseases, and a diagnostic gaps analysis. The result is a tool that is simple, objective and adaptable, which will be used periodically to re-evaluate activities and priorities, representing a step forward towards a better response to infectious disease emergencies.

In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

We examined clinical samples from Nigerian patients with acute watery diarrhea for Vibrio cholerae during the 2010 cholera outbreak. A total of 109 suspected isolates were characterized, but only 57 V. cholerae strains could be confirmed using multiplex real-time PCR as well as rpoB sequencing and typed as V. cholerae O:1 Ogawa biotype El Tor. This finding highlighted the need for accurate diagnosis of cholera in epidemic countries to implement life-saving interventions., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d'Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans., Competing Interests: The authors have declared that no competing interests exist.

A teenage woman migrating from Syria arrived in May 2015 in Germany. She gave birth to a healthy child in early 2016, but became febrile shortly after delivery. Blood cultures revealed Brucella melitensis. In retrospect, she reported contact with sheep in Syria and recurrent pain in the hip joints over about five months before diagnosis of brucellosis. We discuss consequences for adequate treatment of mother and child as well as for clinical and laboratory management., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

Pavlovich's medium T was compared with other broadly used media and extensively checked by growth of various subspecies of Francisellatularensis as well as other risk group 3 bacteria. The medium was successfully re-evaluated as an optimal liquid medium suitable for enrichment of fastidious and/or highly pathogenic bacteria., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A German businessman developed acute watery diarrhoea after a three-day trip to the Philippines. He was admitted with severe hypotension and acute renal failure, but recovered with rapid rehydration. Vibrio cholerae O1 serotype Ogawa was isolated. Physicians need to be aware of endemic cholera in Asia including the Philippines and consider this in their pre-travel advice.

Recently, we identified a putative prophage on a genomic island (GI) within the genome sequence of Francisella hispaniensis isolate AS0-814 (Francisella tularensis subsp. novicida-like 3523) by the analysis of the CRISPR-Cas systems of Francisella. Various spacer DNAs within the CRISPR region of different F. tularensis subsp. novicida strains were found to be homologous to the putative prophage (Schunder et al., 2013, Int. J. Med. Microbiol. 303:51-60). Now we identified the GI (FhaGI-1) as a mobile element which is able to form a circular episomal structure. The circular episomal form of FhaGI-1 is generated by F. hispaniensis, and the excision of the island is an integrase-dependent and site-specific process. Furthermore, we could demonstrate that the excision of the island is also possible in other bacterial species (Escherichia coli). In addition, we could show that a genetically generated small variant of the island is also functional and, after its electroporation into strain F. tularensis subsp. holarctica LVS, the GI was stable and site-specifically integrated into the genome of the transformants. The integrase is sufficient for the integration and excision of the small variant into and from the DNA backbone, respectively. Thus, the element may be suitable to be used as a genetic tool in F. tularensis research. Furthermore, we identified the tRNA(Val) gene of Francisella as an integration site for GIs. Genomic island FphGI-1 was identified in Francisella philomiragia ATCC 25016. We were not able to detect the episomal form of this GI, probably due to a mutated attR site. However, we could demonstrate that integrative GIs are present in Francisella and that they may allow horizontal gene transfer between different Francisella species., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Anthrax is a rare disease in humans but elicits great public fear because of its past use as an agent of bioterrorism. Injectional anthrax has been occurring sporadically for more than ten years in heroin consumers across multiple European countries and this outbreak has been difficult to trace back to a source., Methods: We took a molecular epidemiological approach in understanding this disease outbreak, including whole genome sequencing of Bacillus anthracis isolates from the anthrax victims. We also screened two large strain repositories for closely related strains to provide context to the outbreak., Findings: Analyzing 60 Bacillus anthracis isolates associated with injectional anthrax cases and closely related reference strains, we identified 1071 Single Nucleotide Polymorphisms (SNPs). The synapomorphic SNPs (350) were used to reconstruct phylogenetic relationships, infer likely epidemiological sources and explore the dynamics of evolving pathogen populations. Injectional anthrax genomes separated into two tight clusters: one group was exclusively associated with the 2009-10 outbreak and located primarily in Scotland, whereas the second comprised more recent (2012-13) cases but also a single Norwegian case from 2000., Interpretation: Genome-based differentiation of injectional anthrax isolates argues for at least two separate disease events spanning > 12 years. The genomic similarity of the two clusters makes it likely that they are caused by separate contamination events originating from the same geographic region and perhaps the same site of drug manufacturing or processing. Pathogen diversity within single patients challenges assumptions concerning population dynamics of infecting B. anthracis and host defensive barriers for injectional anthrax., Funding: This work was supported by the United States Department of Homeland Security grant no. HSHQDC-10-C-00,139 and via a binational cooperative agreement between the United States Government and the Government of Germany. This work was supported by funds from the German Ministry of Defense (Sonderforschungsprojekt 25Z1-S-431,214). Support for sequencing was also obtained from Illumina, Inc. These sources had no role in the data generation or interpretation, and had not role in the manuscript preparation., Panel 1 Research in Context Systematic Review: We searched PubMed for any article published before Jun. 17, 2015, with the terms "Bacillus anthracis" and "heroin", or "injectional anthrax". Other than our previously published work (Price et al., 2012), we found no other relevant studies on elucidating the global phylogenetic relationships of B. anthracis strains associated with injectional anthrax caused by recreational heroin consumption of spore-contaminated drug. There were, however, publically available genome sequences of two strains involved (Price et al., 2012, Grunow et al., 2013) and the draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user (Ruckert et al., 2012) with only limited information on the genotyping of closely related strains (Price et al., 2012, Grunow et al., 2013)., Lay Person Interpretation: Injectional anthrax has been plaguing heroin drug users across Europe for more than 10 years. In order to better understand this outbreak, we assessed genomic relationships of all available injectional anthrax strains from four countries spanning a > 12 year period. Very few differences were identified using genome-based analysis, but these differentiated the isolates into two distinct clusters. This strongly supports a hypothesis of at least two separate anthrax spore contamination events perhaps during the drug production processes. Identification of two events would not have been possible from standard epidemiological analysis. These comprehensive data will be invaluable for classifying future injectional anthrax isolates and for future geographic attribution.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

Quality assurance exercises and networking on the detection of highly infectious pathogens (QUANDHIP) is a joint action initiative set up in 2011 that has successfully unified the primary objectives of the European Network on Highly Pathogenic Bacteria (ENHPB) and of P4-laboratories (ENP4-Lab) both of which aimed to improve the efficiency, effectiveness, and response capabilities of laboratories directed at protecting the health of European citizens against high consequence bacteria and viruses of significant public health concern. Both networks have established a common collaborative consortium of 37 nationally and internationally recognized institutions with laboratory facilities from 22 European countries. The specific objectives and achievements include the initiation and establishment of a recognized and acceptable quality assurance scheme, including practical external quality assurance exercises, comprising living agents, that aims to improve laboratory performance, accuracy, and detection capabilities in support of patient management and public health responses; recognized training schemes for diagnostics and handling of highly pathogenic agents; international repositories comprising highly pathogenic bacteria and viruses for the development of standardized reference material; a standardized and transparent Biosafety and Biosecurity strategy protecting healthcare personnel and the community in dealing with high consequence pathogens; the design and organization of response capabilities dealing with cross-border events with highly infectious pathogens including the consideration of diagnostic capabilities of individual European laboratories. The project tackled several sensitive issues regarding Biosafety, Biosecurity and "dual use" concerns. The article will give an overview of the project outcomes and discuss the assessment of potential "dual use" issues.

Background: Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany., Results: We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4-5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067., Conclusions: Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany.

Introduction: The largest known outbreak caused by a rare hybrid strain of Shiga toxin-producing E.coli (STEC) and enteroaggregative E. coli (EAEC) (E.coli O104:H4) of serotype O104:H4 occurred in Germany in 2011. Fenugreek sprouts acted as a transmission vehicle and were widely consumed in the outbreak area at the time of the epidemic. In total 3,842 people developed a clinical illness caused by this strain; however the rates of asymptomatic infections remain unclear. We aimed to develop a serological assay for detection of E.coli O104 LPS specific antibodies and to establish the post-outbreak levels of seropositivity among people with documented exposure to contaminated sprouts., Results and Discussion: Developed serological assays (ELISA with 84% sensitivity, 63% specificity and Western Blot with 100% sensitivity, 82.5% specificity) identified 33% (16/49) level of asymptomatic infection. Relatively small sample size and a significant time- lapse between the onset of symptoms and serum samples collection (appr. 8 weeks) might explain the assay variability. No association was found between clinical or demographic characteristics and assay positivity. Larger studies are needed to understand the complexity of human immune response and factors influencing development of clinical symptoms. Development of intra-outbreak research plans will substantially aid the conduct of more thorough scientific investigation during an outbreak period.

A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Injection anthrax is a rare disease that affects heroin users and is caused by Bacillus anthracis. In 2012, there were four cases in Germany, one of which was fatal, as well as a small number of cases in other European countries, including Denmark, France, and the United Kingdom. Three cases among drug users occurred in Germany in 2009/2010, in the setting of a larger outbreak centered on Scotland, where there were 119 cases. CASE PRESENTATION AND CLINICAL COURSE: We present three cases of injection anthrax, two of which were treated in Regensburg and one in Berlin. One patient died of multi-organ-system failure on the day of admission to the hospital. The others were treated with antibiotics, one of them also with surgical wound debridement. The laboratory diagnosis of injection anthrax is based on the demonstration of the pathogen, generally by culture and/or by polymerase chain reaction, in material removed directly from the patient's wound. The diagnosis is additionally supported by the detection of specific antibodies., Conclusion: Injection anthrax may be viewed either as an independent disease entity or as a special type of cutaneous anthrax with massive edema, necrotizing fasciitis in many cases, and about 30% mortality. It has appeared in recent years among heroin users in various European countries. In patients with suggestive clinical presentation and a history of heroin use, anthrax infection must be suspected early, so that the appropriate diagnostic tests can be performed without delay. Timely treatment can be life-saving. It is therefore important that physicians--and the individuals at risk--should be well-informed about this disease.

Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity.

In this manuscript, we describe the identification of highly pathogenic bacteria using an assay coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS) run on an Ibis PLEX-ID high-throughput platform. The biothreat cluster assay identifies most of the potential bioterrorism-relevant microorganisms including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia mallei and pseudomallei, Brucella species, and Coxiella burnetii. DNA from 45 different reference materials with different formulations and different concentrations were chosen and sent to a service screening laboratory that uses the PCR/ESI-MS platform to provide a microbial identification service. The standard reference materials were produced out of a repository built up in the framework of the EU funded project "Establishment of Quality Assurances for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk" (EQADeBa). All samples were correctly identified at least to the genus level.

Aims: To analyse the V1 region of the 16S rDNA gene by a universal pyrosequencing protocol to identify and subtype Francisella in 31 strains from a repository collection and 96 patient isolates., Methods and Results: Pyrosequencing was used to determine the nucleotide sequence of PCR amplification products of the variable region (V1) of the 16S rDNA from 31 repository strains and 96 isolates from Swedish patients with ulceroglandular tularaemia. Pyrosequencing resulted in a 37 nucleotide sequence, specific for Francisella sp., for all repository strains and patient samples analysed. In addition, the isolates could be divided into two groups based on the analysis of a single nucleotide polymorphism in the sequence: one group included Francisella tularensis ssp. tularensis, ssp. holarctica and ssp. mediasiatica, whereas the other group included Francisella tularensis ssp. novicida and other species of Francisella. The analysis of samples taken from patients suffering from ulceroglandular tularaemia revealed that all isolates belonged to the first group comprising subspecies of F. tularensis virulent for humans., Conclusions: The pyrosequencing analysis of the 16S rDNA V1 is a useful molecular tool for the rapid identification of suspected isolates of Francisella sp. in clinical or environmental samples., Significance and Impact of the Study: Virulent F. tularensis ssp. causing ulceroglandular tularaemia, or those with a potential to be used in a bioterrorism event, could rapidly be discriminated from subspecies less virulent for humans., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Yersinia are Gram-negative, rod-shaped facultative anaerobes, and some of them, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic in humans. Rapid and accurate identification of Yersinia strains is essential for appropriate therapeutic management and timely intervention for infection control. In the past decade matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with computer-aided pattern recognition has evolved as a rapid, objective, and reliable technique for microbial identification. In this comprehensive study a total of 146 strains of all currently known Yersinia species complemented by 35 strains of other relevant genera of the Enterobacteriaceae family were investigated by MALDI-TOF MS and chemometrics. Bacterial sample preparation included microbial inactivation according to a recently developed mass spectrometry compatible inactivation protocol. The mass spectral profiles were evaluated by supervised feature selection methods to identify family-, genus-, and species-specific biomarker proteins and--for classification purposes--by pattern recognition techniques. Unsupervised hierarchical cluster analysis revealed a high degree of correlation between bacterial taxonomy and subproteome-based MALDI-TOF MS classification. Furthermore, classification analysis by supervised artificial neural networks allowed identification of strains of Y. pestis with an accuracy of 100%. In-depth analysis of proteomic data demonstrated the existence of Yersinia-specific biomarkers at m/z 4350 and 6046. In addition, we could also identify species-specific biomarkers of Y. enterocolitica at m/z 7262, 9238, and 9608. For Y. pseudotuberculosis a combination of biomarkers at m/z 6474, 7274, and 9268 turned out to be specific, while a peak combination at m/z 3065, 6637, and 9659 was characteristic for strains of Y. pestis. Bioinformatic approaches and tandem mass spectrometry were employed to reveal the molecular identity of biomarker ions. In this way, the Y. pestis-specific biomarker at m/z 3065 could be identified as a fragment of the plasmid-encoded plasminogen activator, one of the major virulence factors in plague infections.

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var.) anthracis".

Methods for the immunological detection of Bacillus anthracis in various environmental samples and the discrimination of B. anthracis from other members of the B. cereus group are not yet well established. To generate specific discriminating antibodies, we immunized rabbits, mice, and chickens with inactivated B. anthracis spores and, additionally, immunized rabbits and mice with the tetrasaccharide beta-Ant-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-L-Rhap. It is a constituent of the exosporium glycoprotein BclA and contains the newly discovered sugar anthrose 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-D-glucose. The BclA protein is a major component of the exosporium of B. anthracis spores and is decorated by the tetrasaccharide indicated above. The anthrose-containing tetrasaccharide chain seems to be highly specific for B. anthracis, which makes it a key biomarker for the detection of these spores. The different immunizations led to anthrose-reactive polyclonal and monoclonal antibodies which were analyzed by various methods to characterize their ability to discriminate between B. anthracis and other Bacillus spp. Multiple applications, such as enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and electron microscopy, revealed the specificities of the polyclonal and monoclonal antibodies generated for B. anthracis spore detection. All polyclonal antibodies were able to correctly identify the B. anthracis strains tested and showed only minimal cross-reactivities with other Bacillus strains. Moreover, the antibodies generated proved functional in a new capture assay for B. anthracis spores and could therefore be useful for the detection of spores in complex samples.