OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

Armand Paauw 0 Hein Trip 0 Marcin Niemcewicz 2 Ricela Sellek 1 4 Jonathan ME Heng 0 Roos H Mars-Groenendijk 0 Ad L de Jong 0 Joanna A Majchrzykiewicz-Koehorst 0 Jaran S Olsen 3 Evgeni Tsivtsivadze 0 0 Department of CBRN Protection, TNO , P.O. Box 45, Rijswijk 2280 AA , The Netherlands 1 Instituto Tecnologico La Maranosa, Spanish Ministry of Defence, San Martin de la Vega , Madrid , Spain 2 Military Institute of Hygiene and Epidemiology , 24-100 Pulawy, Lubelska 2 , Poland 3 Norwegian Defence Research Establishment , P. O. Box 25, Kjeller N-2027 , Norway 4 Radiological Environmental Recovery Program, Department of Environment, Centro de Investigaciones Energ eticas , Medioambientales y Tecnologicas (CIEMAT, Madrid , Spain Background: Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. Results: The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. Conclusions: The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved. - Background Cholera is an acute diarrheal disease caused by Vibrio cholerae that can be lethal within hours if left untreated. In 2011, a total of 589,854 cases were registered from 58 countries, including 7,816 deaths [1]. The severity, duration, and frequency of cholera epidemics appear to be increasing [2], indicating that cholera is a severe public health problem. In addition, V. cholerae is considered a category B bioterrorism agent by the CDC [3]. Infection usually occurs by consumption of contaminated water, the natural habitat of V. 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) 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 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 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 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 nonepidemic strains. Methods Bacterial strains In total, 48 clinical and environmental isolates of V. cholerae and Vibrio mimicus (Table 1) were obtained from Instituto Tecnolgico La Maraosa, Spanish Ministry of Defence, San Martn 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. From the isolates not belonging to serogroup O1 or O139, two isolates were of clinical origin and the 10 remaining isolates were of environmental origin. Confirmation of strain identification Identification of the isolates at species level was confirmed by MALDI-TOF MS using Biotyper 2.0 (Bruker Daltonics GmbH, Bremen, Germany) [11]. Serogroup and serotype were confirmed using the Vibrio cholerae E Agglutinating Sera kit containing specific antisera O1 polyvalent agglutination serum, Inaba agglutination serum, and Ogawa agglutination serum (Remel Europe Ltd. Darford, Kent, United Kingdom) according to the manufacturers guidelines. Genotyping of isolates with multilocus sequence typing (MLST) analysis MLST analysis was performed according to Teh et al. [21]. Internal gene fragments of dnaE, lap, recA, gyrB, and cat were PCR amplified and sequenced. The gmd gene was not included in the analysis due to low discriminatory power [21]. Each sequence variant of a locus was assigned a distinct allele number. In the case that no PCR product could be obtained for a specific allele, the number zero was assigned. The allele profiles were entered into BioNumerics version 6.6 software (Applied-Maths, Belgium) as character values, and the genetic relationship between isolates was constructed using the categorical coefficient and the Minim (...truncated)