[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Yersinia pestis, Bacillus anthracis and Francisella tularensis cause serious zoonotic diseases and have the potential to cause high morbidity and mortality in humans. In case of natural outbreaks and deliberate or accidental release of these pathogens rapid detection of the bacteria is crucial for limitation of negative effects of the release. In the present study we evaluated 11 commercially available rapid test kits for the detection of Y. pestis, B. anthracis and F. tularensis in terms of sensitivity, specificity and simplicity of the procedure. The results revealed that rapid and easy-to-perform lateral flow assays for detection of highly pathogenic bacteria have very limited sensitivity. In contrast, the immunofiltration assays showed high sensitivity but limited specificity and required a too complicated procedure to be applied in the field by non-laboratory workers (e.g. First Responders like fire, police and emergency medical personnel). Each sample - whether tested negative or positive by the rapid tests - should be retested in a reference laboratory using validated methods.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Public Health 11/2014; 2:199. DOI:10.3389/fpubh.2014.00199
[Show abstract][Hide abstract] ABSTRACT: AimsTwo independent trials investigated the decontamination of a BSL3 laboratory using vaporous hydrogen peroxide and compared the effect on spores of B. cereus, B. subtilis and B. thuringiensis as surrogates for Bacillus anthracis spores, while spores of Geobacillus stearothermophilus served as control.Methods and ResultsCarriers containing 1.0 x106 spores were placed at various locations within the laboratory before fumigation with hydrogen peroxide following a previously validated protocol. Afterwards, carriers were monitored by plating out samples on agar and observing enrichment in nutrtient medium for up to 14 days. Three months later the experiment was repeated and results were compared. On 98 out of 102 carriers no viable spores could be detected after decontamination, while the remaining four carriers exhibited growth of CFU only after enrichment for several days. Reduction factors between 4.0 and 6.0 log levels could be reached.ConclusionsA validated decontamination of a laboratory with hydrogen peroxide represents an effective alternative to fumigation with formaldehyde. Spores of B. cereus seem to be more resistant than those of G. stearothermophilus.Significance and Impact of StudyThe results of this study provide important results in the field of hydrogen peroxide decontamination when analyzing the effect on spores other than those of G. stearothermophilus.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial susceptibility testing (AST) and conclusive assessment of minimal inhibitory concentrations (MICs) of highly pathogenic bacteria is difficult due to a lack of (consensus) standards and incomplete breakpoint definitions. Standards are available from the Clinical and Laboratory Standards Institute (CLSI) and furthermore, from WHO only for Bacillus anthracis and Francisella tularensis. The CLSI M45‐A2 document provides recommendations for testing conditions (microdilution), but the available breakpoints are not for all relevant substances and often only for the category "susceptible". Furthermore, during external quality assurance (EQA) exercises of the European joint action Quality Assurance Exercises and Networking on the Detection of Highly Infectious Pathogens (QUANDHIP) several discrepancies in AST were observed among the participants. Therefore, a working group from six European laboratories was established to identify one AST method appropriate for all highly pathogenic bacteria to standardize this method and to evaluate it in consecutive QUANDHIP EQAs.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
PLoS ONE 09/2013; 8(9):e73052. DOI:10.1371/journal.pone.0073052 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The detection of airborne pathogens is becoming a subject of great concern in modern day society. Recent studies have shown that electrostatic samplers are suitable for collecting microorganisms as well as preserving their viability. In most of these studies, flow rates were lower than 12.5 L/min or required a concentration stage to increase the flow rate to 100 L/min. In the present study, a single stage electrostatic sampler was developed for an efficient collection of microorganisms at 100 L/min. The design is based on the positive DC corona between coaxial cylinders to continuously create charged particles as they pass through the sampler. The physical collection efficiency of the device was investigated by sampling the ambient air particles. The efficiency in collecting live biological samples was determined by sampling live bacterial spores with the collector situated inside an aerosol chamber. It is shown that particles of 0.3–0.35 μm are captured with an efficiency of about 86%, whereas cultivable spores of Bacillus thuringiensis are collected with an efficiency of about 94%. Results are compared with an analytical predictive model. The experimental results are similar to the efficiencies previously reported in the literature; however, the current sampler design features a higher flow rate which enables the device to be used as an alarm trigger in a shorter period of time.
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal of medical microbiology: IJMM 01/2013; DOI:10.1016/j.ijmm.2012.11.004 · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Deutsches Ärzteblatt International 12/2012; 109(49):843-8. DOI:10.3238/arztebl.2012.0843 · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY 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.
Epidemiology and Infection 07/2012; DOI:10.1017/S0950268812001008 · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 06/2012; 7(6):e39928. DOI:10.1371/journal.pone.0039928 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases.
Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 01/2012; 17(26). · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tularaemia, caused by Francisella tularensis, had not been registered in Kosovo before an outbreak in 1999 and 2000. A national surveillance system has been implemented in Kosovo since 2000 to monitor a number of diseases, including tularaemia. Antibody detection in human sera was used for laboratory diagnosis of tularaemia and F. tularensis lipopolysaccharide antigen was used as a marker of infection. The purpose of this study is to describe the incidence of tularaemia in Kosovo after the 1999-00 outbreak. In 2001 and 2002, a second outbreak occurred, with 327 serologically confirmed cases. From 2001 to 2010, 25-327 cases were registered per year, giving a mean annual incidence of 5.2 per 100,000 population. The most likely sources of infection were contaminated drinking water and food. The dominant clinical manifestations were the glandular (79%) and ulcero-glandular (21%) forms. By 2010, the disease had spread throughout Kosovo. Presumably as a result of war and subsequent environmental disruption, mass population displacement and breakdown of sanitation and hygiene, the two major outbreaks of tularaemia resulted in the establishment of an active endemic area of tularaemia in Kosovo.
Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 01/2012; 17(28). · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.