cholerae, or contaminated food. Within the V. cholerae species, over 200 serogroups have been identified but only serogroup O1 and O139 strains that

are able to produce cholera enterotoxin (CT) and toxin-coregulated pilus (TCP) Caspase inhibitor in vivo can cause epidemics. The toxigenicity of a V. cholerae strain depends on its ability to produce the CT, encoded by the ctxAB genes, and TCP, encoded by the Vibrio pathogenicity island (VPI) [4]. However, these virulence factors are also described in non-O1/O139 V. cholerae isolates without causing an epidemic threat [5]. Next, occasionally, other strains of V. cholerae may cause diarrhea, but they do not have epidemic potential [6]. Rapid detection and identification of threatening microorganisms is essential for an effective response to an infectious disease outbreak. Therefore, rapid discrimination between epidemic V. cholerae O1/O139 strains and other V. cholerae strains is crucial. Matrix-assisted CT99021 cost laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for quick identification of bacteria and possesses advantages over

conventional techniques in that it is fast, accurate, cheap and suitable for high-throughput identification [7–10]. The discriminatory power of MALDI-TOF MS in analysis of whole bacterial cell lysates overlaid with α-cyano-4-hydroxycinnamic acid as a matrix is usually sufficient to identify bacteria to the species level but may also be used to differentiate between

strains belonging to one species if adequate protein extraction procedures are performed [11–15]. The aim of this Thymidylate synthase study was to develop a MALDI-TOF MS assay able to discriminate between toxigenic and epidemic V. cholerae O1/O139 strains and other mostly non-O1/O139 isolates. To extend the measurable range of the MALDI-TOF MS and thereby increase the discriminatory power of the MS spectra, ferulic acid was used as a matrix [16, 17]. The outer membrane protein OmpU was identified as a suitable biomarker for discriminating between toxigenic and epidemic strains and non-epidemic strains. Methods Bacterial strains In total, 48 clinical and environmental isolates of V. cholerae and Vibrio mimicus (Table 1) were obtained from Instituto Tecnológico La Marañosa, Spanish Ministry of Defence, San Martín de la Vega, Madrid, Spain, Norwegian Defence Research Establishment, Kjeller, Norway, and Military Institute of Hygiene and Epidemiology, Pulawy, Poland (Table 1) [18–20]. The human isolates were all collected as part of standard patient care. The isolates were collected from different areas of the world. Thirty-three, three, and twelve isolates were serotyped as O1, O139, and non-O1/O139 serogroups, respectively. From the 33 serogroup O1 isolates, 18 were clinical isolates, 10 were environmental isolates, and five isolates were from an unknown source. Two serogroup O139 isolates were clinical isolates and one was of unknown origin.