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Fast turnaround times are of utmost importance for biomedical reconnaissance, particularly regarding dangerous pathogens. Recent advances in sequencing technology and its devices allow sequencing within a short time frame outside stationary laboratories close to the epicenter of the outbreak. In our study, we evaluated the portable sequencing device MinION as part of a rapidly deployable laboratory specialized in identification of highly pathogenic agents. We tested the device in the course of a NATO live agent exercise in a deployable field laboratory in hot climate conditions. The samples were obtained from bio-terroristic scenarios that formed part of the exercise and contained unknown bacterial agents. To simulate conditions of a resource-limited remote deployment site, we operated the sequencer without internet access. Using a metagenomic approach, we were able to identify the causative agent in the analyzed samples. Furthermore, depending on the obtained data, we were able to perform molecular typing down to strain level. In our study we challenged the device and discuss advances as well as remaining limitations for sequencing biological samples outside of stationary laboratories. Nevertheless, massive parallel sequencing as a non-selective methodology yields important information and is able to support outbreak investigation − even in the field.

Clostridium species cause several local and systemic diseases. Conventional identification of these microorganisms is in part laborious, not always reliable, time consuming or does not always distinguish different species, i.e., C. botulinum and C. sporogenes. All in, there is a high interest to find out a reliable, powerful and rapid method to identify Clostridium spp. not only on genus but also on species level. The aim of the present study was to identify Clostridium spp. strains and also to find differences and metabolic groups of C. botulinum by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). A total of 123 strains of Clostridium spp. (C. botulinum, n = 40, C. difficile, n = 11, C. tetani, n = 11, C. sordellii, n = 20, C. sporogenes, n = 18, C. innocuum, n = 10, C. perfringens, n = 13) were analyzed by MALDI-TOF MS in combination with methods of multivariate statistical analysis. MALDI-TOF MS analysis in combination with methods of multivariate statistical analysis was able to discriminate between the different tested Clostridium spp., even between species which are closely related and difficult to differentiate by traditional methods, i.e., C. sporogenes and C. botulinum. Furthermore, the method was able to separate the different metabolic groups of C. botulinum. Especially, E gene-positive C. botulinum strains are clearly distinguishable from the other species but also from those producing other toxin types. Thus, MALDI-TOF MS represents a reliable and above all quick method for identification of cultivated Clostridium species.

Since the 1990s, a broad spectrum of regional conflicts and crises have evolved that have been accompanied by a growing threat of international terrorism. How vulnerable our modern societies would be towards a covert biological attack became evident in the 2001 anthrax letters attack in the United States. Biothreats are currently associated with asymmetric warfare scenarios and non-state actors rather than with state-driven biowarfare facilities. Against this backdrop, NATO has to consider biological warfare and bioterrorism as a serious threat to its forces. In bioterroristic scenarios the deliberate release of a biological agent will most probably remain undetected until a cluster of cases will suggest an unusual outbreak of disease. In military settings it is primarily the responsibility of the Medical Services to recognize the outbreak and to launch an appropriate outbreak investigation. Major goals of a medical bio-reconnaissance mission are to rapidly identify the causative agent of the outbreak and to differentiate between natural and deliberate outbreaks. In contrast to the investigation of overt natural outbreaks, forensic aspects have to be considered and appropriate procedures have to be implemented quite from the beginning when unusual outbreaks are to be investigated. If a biothreat agent is detected, it may be necessary to enter further genetic analysis in order to differentiate between natural and intentional outbreaks and to trace back the origin of the agent. Microbial forensics is mainly concerned with taking molecular fingerprints of biothreat agents by means of molecular typing techniques enabling the investigator to identify and trace back a particular strain by comparing it with the fingerprints stored in a typing database. The bioforensic approach may well be capable of elucidating the source of an outbreak as has been evidenced in the Amerithrax case in 2001. In order to detect molecular differences of microbial strains, a number of sophisticated typing techniques are currently employed, the most recent of which is whole genome sequencing, which has even entered the field laboratories by means of portable next generation sequencing devices like the MinION™.

The activity of finafloxacin against 73 strains of the Bacteroides fragilis group, 10 other Gram-negative anaerobic rods and 31 Clostridium difficile strains was determined by the broth microdilution technique. The activity was compared with that of moxifloxacin, levofloxacin, ciprofloxacin, clindamycin, imipenem, piperacillin/tazobactam and metronidazole. MIC(50/90) values (minimum inhibitory concentration, in μg/mL, at which 50% and 90% of the isolates tested are inhibited, respectively) for finafloxacin for the different species were determined: B. fragilis group, 0.5/2; other Gram-negative rods, 0.06/0.25; and C. difficile, 4/16. Furthermore, the MICs against 11 selected B. fragilis strains were determined under acidic conditions and resulted in MIC(50/90) values for finafloxacin of 0.25/4 μg/mL. Thus, finafloxacin shows promising activity against several pathogenic species of anaerobes. Furthermore, finafloxacin has increased activity against selected B. fragilis strains under acidic conditions compared with activity at neutral pH.

Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli.

This study was designed to investigate the killing activity of levofloxacin, gatifloxacin, moxifloxacin and garenoxacin against 12 Bacteroides fragilis strains by kill kinetics over time. MIC values were determined by Etest and by agar dilution. B. fragilis strains were divided according to their MIC values into two groups: one group with strains with MIC8.0 µg ml(-1) and one group with strains with MIC ≥ 8.0 µg ml(-1). For kill kinetics over time, the strains with MIC8.0 µg ml(-1) were incubated with the antibiotics at 0.5, 1, 2 and 4 times their MIC values. The strains with MIC ≥ 8.0 µg ml(-1) were incubated with 0.5, 1, 2, and 4 times the maximum achievable concentrations of the antibiotics in human plasma (Cmax). Among the strains with MIC8.0 µg ml(-1) levofloxacin and gatifloxacin showed equal efficacy. The growth of the strains with MIC ≥ 8.0 µg ml(-1) was barely affected by levofloxacin, while gatifloxacin had bactericidal action when concentrations of 4 × C max were used. Moxifloxacin was more effective against both groups of strains compared with levofloxacin and gatifloxacin. Garenoxacin was the most active agent against all strains investigated. Due to the varying in vitro activity of the quinolones against obligate anaerobes the treatment with quinolones of patients with intra-abdominal infections needs intensive scrutiny.

We established a modular, rapidly deployable laboratory system that provides diagnostic support in resource-limited, remote areas. Developed as a quick response asset to unusual outbreaks of infectious diseases worldwide, several of these laboratories have been used as part of the World Health Organization response to the Ebola virus outbreaks by teams of the ‘European Mobile Lab’ project in West Africa since March 2014. Within three days from deployment, the first European mobile laboratory became operational at the Ebola Treatment Unit (ETU) in Guéckédou, southern Guinea. Deployment in close proximity to the ETU decreased the turnaround time to an average of 4 h instead of several days in many cases. Between March 2014 and May 2015, more than 5,800 samples were tested in this field laboratory. Further EMLab units were deployed to Nigeria, Liberia and Sierra Leone in the following months of the Ebola outbreak. The technical concept of the EMLab units served as a blueprint for other mobile Ebola laboratories which have been set up in Mali, Côte d’Ivoire, Sierra Leone and other countries in West Africa. Here, we describe design, capabilities and utility of this deployable laboratory system for use in response to disease outbreaks, epidemiological surveillance and patient management.

Summary Background Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. Material/Methods We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. Results Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. Conclusions This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics.