Epidemiology of Pathogens Listed as Potential Bioterrorism Agents, the Netherlands, 2009‒2019.

We provide incidences (cases/10 million persons) in the Netherlands during 2009-2019 for pathogens listed as potential bioterrorism agents. We included pathogens from the highest categories of the European Medicines Agency or the US Centers for Disease Control and Prevention. Notifiable diseases and recently published data were used to calculate the average annual incidence. Coxiella burnetii had the highest incidence because of a Q fever epidemic during 2007-2010. Incidence then decreased to 10.8 cases/. Pathogens with an incidence >1 were Brucella spp. (2.5 cases), Francisella tularensis (1.3 cases), and Burkholderia pseudomallei (1.1 cases). Pathogens with an incidence <1 were hemorrhagic fever viruses (0.3 cases), Clostridium botulinum (0.2 cases), and Bacillus anthracis (0.1 cases). Variola major and Yersinia pestis were absent. The generally low incidences make it unlikely that ill-meaning persons can isolate these pathogens from natural sources in the Netherlands. However, the pathogens are stored in laboratories, underscoring the need for biosecurity measures.

Beyond the Dirty Dozen: A Proposed Methodology for Assessing Future Bioweapon Threats.

Background: Defense policy planners and countermeasure developers are often faced with vexing problems involving the prioritization of resources and efforts. This is especially true in the area of Biodefense, where each new emerging infectious disease outbreak brings with it questions regarding the causative agent's potential for weaponization. Recent experience with West Nile Virus, Severe Acute Respiratory Syndrome, Monkeypox, and H1N1 Influenza highlights this problem. Appropriately, in each of these cases, the possibility of bioterrorism was raised, although each outbreak ultimately proved to have a natural origin. In fact, determining whether an outbreak has an unnatural origin can be quite difficult. Thus, the questions remain: could the causative agents of these and other emerging infectious disease outbreaks pose a future weaponization threat? And how great is that threat? Should precious resources be diverted from other defense efforts in order to prepare for possible hostile employment of novel diseases by belligerents? Answering such critical questions requires some form of systematic threat assessment. Methods: Through extensive collaborative work conducted within NATO's Biomedical Advisory Council, we developed a scoring matrix for evaluating the weaponization potential of the causative agents of such diseases and attempted to validate our matrix by examining the reproducibility of data using known threat agents. Our matrix included 12 attributes of a potential weapon and was provided, along with detailed scoring instructions, to 12 groups of biodefense experts in 6 NATO nations. Study participants were asked to score each of these 12 attributes on a scale of 0-3: Infectivity, Infection-to-Disease Ratio (Reliability), Predictability (& Incubation Period), Morbidity & Mortality (Virulence), Ease of Large-Scale Production & Storage, Aerosol Stability, Atmospheric Stability, Ease of Dispersal, Communicability, Prophylactic Countermeasure Availability, Therapeutic Countermeasure Availability, and Ease of Detection. Reproducibility of scoring data was assessed by examining the standard deviations (SD) of mean scores. Results: Our results were unexpected. Several familiar biothreat diseases such as anthrax and tularemia were judged, by our experts, to be less threatening than many others owing to a number of factors including ease of detection, lack of communicability, and the ready availability of countermeasures. Conversely, several toxins were judged by experts to have very high potential as threat agents owing, in part, to their reliability, virulence, and a lack of available countermeasures. Agreement among experts, as determined by lower SD about a mean score, was greater for more familiar threats. Discussion: Our study was designed to provide a concise and east-to-apply set of criteria that could be used by NATO nations to evaluate emerging infectious disease threats with respect to their weaponization potential. Our results were unexpected. We believe that a lack of appropriate weighting factors may explain these results and suggest that future studies weigh each of the 12 proposed criteria based on the intended use of the assessment data and other situational factors. We believe that the greatest value of our study lies in a codification of the attributes of a biological weapon.

Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.

Simultaneous Identification of Multiple ß-Lactamases in Acinetobacter baumannii in Relation to Carbapenem and Ceftazidime Resistance, Using Liquid Chromatography-Tandem Mass Spectrometry.

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect ß-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of ß-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing.

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS.

BACKGROUND: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. RESULTS: In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. CONCLUSIONS: MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

A novel mucin-sulphatase activity found in Burkholderia cepacia and Pseudomonas aeruginosa.

Lung infections due to Burkholderia cepacia and Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are common, are associated with respiratory morbidity and are a cause of mortality. Respiratory mucin in CF patients is highly sulphated, which increases its resistance to bacterial degradation. Desulphation increases the susceptibility of mucin to degradation by bacterial glycosidases and proteinases, and subsequent deglycosylation may facilitate bacterial colonisation by increasing available substrates and binding sites. This study determined whether clinical and environmental strains of B. cepacia and P. aeruginosa had the ability to desulphate mucin. Mucin-sulphatase activity was tested by incubating bacterial cell suspensions with 35S-sulphated mucins purified from LS174T and HT29-MTX human colon carcinoma cell lines. These mucins were also used to test for differences in substrate specificities. Mucin-sulphatase activity was detected in all nine B. cepacia strains and in four of six P. aeruginosa strains. There was strain variability in the level of mucin-sulphatase activity. Aryl-sulphatase activities of Pseudomonas isolates (determined with methylumbelliferyl sulphate) were c. 20-fold higher than those of B. cepacia strains, and were independent of mucin-sulphatase activity. This is the first report to demonstrate desulphation of mucin by B. cepacia and P. aeruginosa. It is concluded that B. cepacia and P. aeruginosa produce one or more cell-bound glycosulphatase(s), in addition to aryl-sulphatase activity. Mucin-sulphatase activity of B. cepacia and P. aeruginosa may contribute to their association with airway infections in patients with cystic fibrosis.