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.

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.

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%.

MALDI-TOF mass spectrometry compatible inactivation method for highly pathogenic microbial cells and spores.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Effects of ethanol on cytokine production after surgery in a murine model of gram-negative pneumonia.

BACKGROUND: Both alcohol abuse and surgery have been shown to impair immune function. The frequency of postoperative infectious complications is 2- to 5-fold increased in long-term alcoholic patients, leading to prolonged hospital stay. Following surgery, an increase in interleukin (IL)-6 has been shown to be associated with increased tissue injury and interleukin 1-(IL-10) is known to represent an anti-inflammatory signal. The purpose of this study was to test the hypothesis that several days of excess alcohol consumption results in more pronounced immunosuppression. We assume that alcoholic animals show increased levels of IL-10 in response to infection and increased IL-6 due to a more pronounced lung pathology. METHODS: Thirty-two female Balb/c mice were pretreated with ethanol (EtOH) at a dose of (3.8 mg/g body weight) or saline (NaCl) for 8 days. At day 8 of the experiment all mice underwent a median laparotomy. Two days postsurgery mice were either applicated 10(4) CFU Klebsiella pneumoniae or received sham-infection with saline. A total number of 4 groups (EtOH/K. pneumoniae; NaCl/K. pneumoniae; EtOH/Sham-infection, NaCl/Sham-infection) was investigated and a clinical score evaluated. Twenty-four hours later mice were killed; lung, spleen, and liver were excised for protein isolation and histological assessment. IL-6 and IL-10 levels were detected by ELISA. RESULTS: Alcohol-exposed mice exhibited a worsened clinical appearance. The histological assessment demonstrated a distinct deterioration of the pulmonary structure in alcohol-treated animals. In the lung, IL-6 and IL-10 was significantly increased in alcohol-exposed infected mice compared to saline-treated infected mice. The clinical score correlated significantly with IL-6 (r = 0.71; p < 0.01) and IL-10 levels (r = 0.64; p < 0.01) in the lung. CONCLUSIONS: Ethanol treatment in this surgical model led to a more severe pulmonary infection with K. pneumoniae which was associated with more tissue destruction and increased levels of IL-6 and IL-10 and a worsened clinical score.

Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon.

We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR.

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.