Herbert Nattermann

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

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”.

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

ABSTRACT This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.

Characterization of Yersinia Using MALDI-TOF Mass Spectrometry and Chemometrics

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.

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.

Efficient killing of anthrax spores using aqueous and alcoholic peracetic acid solutions

ZusammenfassungMit Suspensions- und Keimträger versuchen wurde die sporizide Wirkung von unterschiedlichen Konzentrationen wässriger und alkoholischer Peressigsäure- (PES-)Lösungen auf Milzbrandsporen untersucht. Während in Suspensionsversuchen alle Sporen mit einer 1%igen PES-Lösung schon in weniger als 2 Minuten und mit einer 0,5%igen PES-Lösung in weniger als 3 Minuten abgetötet werden konnten, überlebten die Sporen im Keimträger versuch – einer Prüfung unter praxisnahen Bedingungen – bei 38% der Keimträger eine Behandlung mit einer 1%igen wässrigen PES-Lösung für 15 Minuten. Die PES in 80%iger ethanolischer Lösung war sowohl im Suspensions- als auch im Keimträger versuch der wässrigen Lösung hinsichtlich ihrer sporiziden Wirkung deutlich überlegen. Eine 30-minütige Behandlung mit einer 1%igen wässrigen PES-Lösung überlebten Milzbrandsporen auf 14% der Keimträger. Im Gegensatz dazu konnten mit einer 30-minütigen Einwirkzeit einer 1%igen alkoholischen PES-Lösung Milzbrandsporen unter den Bedingungen dieses praxisnahen Prüfverfahrens sicher abgetötet werden. Die nachgewiesene Verbesserung der sporiziden Wirkung der PES in alkoholischer Lösung sollte in der Desinfektionspraxis Berücksichtigung finden. In weiteren Untersuchungen gilt es, die Anwendungsbedingungen zu optimieren.AbstractWe analysed the sporicidal effect of different concentrations of aqueous and alcoholic peracetic acid (PAA) solutions on anthrax spores in suspension and germ carrier tests. In activation of anthrax spores in suspension assays was achieved in less than 2 min using 1% PAA solution and in less than 3 min using 0.5% PAA solution, respectively. In contrast, in germ carrier as says, a test under practical conditions, spores on 38% of the germ carriers survived treatment with 1% PAA solution for 15 min. The use of PAA in 80% ethyl alcohol outclassed the sporicidal effect of aqueous PAA solutions in both suspension and germ carrier assays. Anthrax spores on 14% of germ carriers tested survived 30 min of treatment with a 1% aqueous PAA solution. In contrast anthrax spores were reliably inactivated under the same test procedure using a 1% alcoholic PAA solution for 30 min. The proven enhancement of the sporicidal effect of alcoholic PAA solutions should be kept in mind when using disinfectants in practice. In further surveys we will optimise the test conditions.

Effiziente Abtötung von Milzbrandsporen durch wässrige und alkoholische Peressigsäure–Lösungen

ZusammenfassungMit Suspensions- und Keimträger versuchen wurde die sporizide Wirkung von unterschiedlichen Konzentrationen wässriger und alkoholischer Peressigsäure- (PES-)Lösungen auf Milzbrandsporen untersucht. Während in Suspensionsversuchen alle Sporen mit einer 1%igen PES-Lösung schon in weniger als 2 Minuten und mit einer 0,5%igen PES-Lösung in weniger als 3 Minuten abgetötet werden konnten, überlebten die Sporen im Keimträger versuch – einer Prüfung unter praxisnahen Bedingungen – bei 38% der Keimträger eine Behandlung mit einer 1%igen wässrigen PES-Lösung für 15 Minuten. Die PES in 80%iger ethanolischer Lösung war sowohl im Suspensions- als auch im Keimträger versuch der wässrigen Lösung hinsichtlich ihrer sporiziden Wirkung deutlich überlegen. Eine 30-minütige Behandlung mit einer 1%igen wässrigen PES-Lösung überlebten Milzbrandsporen auf 14% der Keimträger. Im Gegensatz dazu konnten mit einer 30-minütigen Einwirkzeit einer 1%igen alkoholischen PES-Lösung Milzbrandsporen unter den Bedingungen dieses praxisnahen Prüfverfahrens sicher abgetötet werden. Die nachgewiesene Verbesserung der sporiziden Wirkung der PES in alkoholischer Lösung sollte in der Desinfektionspraxis Berücksichtigung finden. In weiteren Untersuchungen gilt es, die Anwendungsbedingungen zu optimieren.AbstractWe analysed the sporicidal effect of different concentrations of aqueous and alcoholic peracetic acid (PAA) solutions on anthrax spores in suspension and germ carrier tests. In activation of anthrax spores in suspension assays was achieved in less than 2 min using 1% PAA solution and in less than 3 min using 0.5% PAA solution, respectively. In contrast, in germ carrier as says, a test under practical conditions, spores on 38% of the germ carriers survived treatment with 1% PAA solution for 15 min. The use of PAA in 80% ethyl alcohol outclassed the sporicidal effect of aqueous PAA solutions in both suspension and germ carrier assays. Anthrax spores on 14% of germ carriers tested survived 30 min of treatment with a 1% aqueous PAA solution. In contrast anthrax spores were reliably inactivated under the same test procedure using a 1% alcoholic PAA solution for 30 min. The proven enhancement of the sporicidal effect of alcoholic PAA solutions should be kept in mind when using disinfectants in practice. In further surveys we will optimise the test conditions.

Bioterroristisch relevante bakterielle Erreger

ZusammenfassungNach den bioterroristischen Anschlägen mit Milzbrandbriefen in den USA im Herbst 2001, den Tausenden daraufhin folgenden vermeintlichen Anschlägen auch in Deutschland und den vermuteten Waffenarsenalen in den so genannten „Schurkenstaaten“ rückte die Gefahr durch Biowaffen wieder in den Mittelpunkt des Interesses. Dabei wurde deutlich, wie wichtig eine schnelle und verlässliche Diagnostik bioterroristisch relevanter Erreger in allen möglichen Umweltproben sowie aus Patientenmaterial ist, um wirksam und angemessen auf eine mögliche Bedrohung reagieren zu können. Für die schnelle Diagnostik einer Vielzahl hochpathogener Erreger aus Umweltproben eignen sich am besten molekulargenetische Methoden wie die PCR, aber auch der direkte Nachweis spezifischer Antigene. Trotzdem sind auch die langjährig etablierten mikrobiologischen Methoden—Bakterioskopie und kulturelle Anzucht—unerlässlich, um die Lebensfähigkeit von Erregern zu bestätigen. Aufgrund ihrer möglicherweise starken mikrobiellen Verunreinigung und dem Vorhandensein von Inhibitoren stellen Umweltproben eine besondere Herausforderung für die Diagnostik dar, bei der auf Positivkontrollen großen Wert gelegt werden muss. Im Folgenden werden als bioterroristisch relevant eingestufte hochpathogene Erreger und die von ihnen hervorgerufenen Erkrankungen vorgestellt: Milzbrand, Pest, Tularämie, Rotz, Melioidose, Brucellose, Q-Fieber und Fleckfieber. Die diagnostischen Maßnahmen beim Verdacht auf Milzbranderreger werden ausführlich erläutert, und Methoden zur Diagnostik weiterer Erreger werden angesprochen.AbstractAfter the bioterrorist attacks with several anthrax-laden letters in the USA in autumn 2001, the subsequent thousands of pretended attacks also in Germany, and the assumed arsenals of the so-called rogue states, the threat of bioweapons became a focus of interest again. Thereby, it became clear just how important fast and reliable diagnostic methods are to detect bioterrorism-related agents in every possible environmental or clinical specimen to enable an effective and adequate reaction to a possible threat. For fast detection of a great number of highly pathogenic agents in environmental specimens, methods such as PCR or direct detection of specific antigens are most suitable. However, also the long-standing, well-established microbiological methods—bacterioscopy and cultivation—are indispensable to confirm the viability of bacterial agents. Due to their possibly strong microbial contamination and the presence of inhibitors, environmental specimens pose a special challenge for diagnostics,where great importance must be attached to positive controls. Highly pathogenic agents categorized as relevant for bioterrorism and the corresponding diseases are presented below: anthrax, plague, tularemia, glanders, melioidosis,brucellosis, Q fever, and typhus fever. The diagnostic measures that have to be taken in a case of suspicious anthrax are described in detail, and diagnostic methods for further bacterial agents are mentioned.

Decontamination of a BSL3 laboratory by hydrogen peroxide fumigation using three different surrogates for Bacillus anthracis spores

Two independent trials investigated the decontamination of a BSL3 laboratory using vaporous hydrogen peroxide and compared the effect on spores of Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis as surrogates for Bacillus anthracis spores, while spores of Geobacillus stearothermophilus served as control.

Bioterroristisch relevante bakterielle Erreger@@@Bioterrorism-related bacterial agents. Epidemiology, clinical picture, and diagnostics

ZusammenfassungNach den bioterroristischen Anschlägen mit Milzbrandbriefen in den USA im Herbst 2001, den Tausenden daraufhin folgenden vermeintlichen Anschlägen auch in Deutschland und den vermuteten Waffenarsenalen in den so genannten „Schurkenstaaten“ rückte die Gefahr durch Biowaffen wieder in den Mittelpunkt des Interesses. Dabei wurde deutlich, wie wichtig eine schnelle und verlässliche Diagnostik bioterroristisch relevanter Erreger in allen möglichen Umweltproben sowie aus Patientenmaterial ist, um wirksam und angemessen auf eine mögliche Bedrohung reagieren zu können. Für die schnelle Diagnostik einer Vielzahl hochpathogener Erreger aus Umweltproben eignen sich am besten molekulargenetische Methoden wie die PCR, aber auch der direkte Nachweis spezifischer Antigene. Trotzdem sind auch die langjährig etablierten mikrobiologischen Methoden—Bakterioskopie und kulturelle Anzucht—unerlässlich, um die Lebensfähigkeit von Erregern zu bestätigen. Aufgrund ihrer möglicherweise starken mikrobiellen Verunreinigung und dem Vorhandensein von Inhibitoren stellen Umweltproben eine besondere Herausforderung für die Diagnostik dar, bei der auf Positivkontrollen großen Wert gelegt werden muss. Im Folgenden werden als bioterroristisch relevant eingestufte hochpathogene Erreger und die von ihnen hervorgerufenen Erkrankungen vorgestellt: Milzbrand, Pest, Tularämie, Rotz, Melioidose, Brucellose, Q-Fieber und Fleckfieber. Die diagnostischen Maßnahmen beim Verdacht auf Milzbranderreger werden ausführlich erläutert, und Methoden zur Diagnostik weiterer Erreger werden angesprochen.AbstractAfter the bioterrorist attacks with several anthrax-laden letters in the USA in autumn 2001, the subsequent thousands of pretended attacks also in Germany, and the assumed arsenals of the so-called rogue states, the threat of bioweapons became a focus of interest again. Thereby, it became clear just how important fast and reliable diagnostic methods are to detect bioterrorism-related agents in every possible environmental or clinical specimen to enable an effective and adequate reaction to a possible threat. For fast detection of a great number of highly pathogenic agents in environmental specimens, methods such as PCR or direct detection of specific antigens are most suitable. However, also the long-standing, well-established microbiological methods—bacterioscopy and cultivation—are indispensable to confirm the viability of bacterial agents. Due to their possibly strong microbial contamination and the presence of inhibitors, environmental specimens pose a special challenge for diagnostics,where great importance must be attached to positive controls. Highly pathogenic agents categorized as relevant for bioterrorism and the corresponding diseases are presented below: anthrax, plague, tularemia, glanders, melioidosis,brucellosis, Q fever, and typhus fever. The diagnostic measures that have to be taken in a case of suspicious anthrax are described in detail, and diagnostic methods for further bacterial agents are mentioned.

Tularemia – A Disease with an Uncertain Impact on Public Health

Francisella tularensis is the causative agent of tularemia and is consi-dered as an agent with the potential to be used deliberately. The species includes subspecies and subtypes with different virulence for humans and occurs in certain areas of the Northern hemisphere. In North America, the virulence of F. tularensis subspecies holarctica is supposed to lie between the two subtypes A1 and A2 of F. tularensis subspecies tularensis. Clinical course and appearance depend on an early diagnosis and effective treatment. Detection and diagnosis require special laboratory tests including microbiological, molecular and immunological methods. During the last years, considerable substantial progress has been achieved to better understand the pathology and ecology of this zoonotic pathogen. However, the mechanisms of the obvious long-term persistence of this pathogen in the environment are not well known yet. In Germany, tularemia is a notifiable disease and occurs very rarely although it is probably underestimated. Interestingly, the cases are distributed almost over the entire territory of the country. Epidemiological studies will contribute to a better understanding of the reservoirs and ways of transmission of these bacteria. Outbreaks of tularemia are occasionally occurring in known and unknown endemic areas. The reasons and sources of such outbreaks are often not clarified. However, the intentional release of the agent must be excluded by all means. Therefore an algorithm has been developed to assess the probability of a biological attack and has come into use for tularemia outbreaks in Kosovo after the war in 1999. The results revealed that the unusual outbreak of tularemia in Kosovo most likely originated from a natural source supported by special ecological and hygienic conditions in a post-war situation.