Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream

Abstract

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48–72 hours.

Rapid Identification of Vibrio Species of the Harveyi Clade Using MALDI-TOF MS Profiling With Main Spectral Profile Database Implemented With an In-House Database: Luvibase

“…For instance, the use of different culture media for Yersinia enterocolitica , Bacillus subtilis , and Escherichia coli identification by MALDI-TOF MS profiling showed an unambiguous core set of proteins, ensuring reliable identification regardless the culture media ( Valentine et al, 2005 ). Recently, the identification of V. anguillarum pathogens isolated from seabass and seabream ( Sparus aurata ) revealed that differences in culture media and incubation period have no effect on MALDI-TOF MS profiles but divergences in growth temperature may affect MALDI-TOF MS profiles ( Kazazić et al, 2019 ). Importantly, because the spectra of different species may not be affected by the same parameters, the effect of altering procedures should be studied for each single species.…”

Mass Spectrometry in Food Analysis

“…Proteomics has been applied to the detection and identification of bacterial species from aquaculture products both after their direct detection in fish products, or after their isolation and growing in different culture media. For instance, MALDI-ToF-MS techniques enabled us to achieve mass spectral fingerprints of Vibrio spp., a Gram-negative bacteria that cause gastrointestinal diseases in humans after ingestion of poorly cooked infected seafood, such as seabream and mollusks [79,80]. A study by Li and coworkers reported that the exhaustive proteome and transcriptome data analysis obtained by iTRAQ coupled with MRM provided some critical protein signatures for the study of the regulatory mechanisms of the intestinal mucosal immunity in grass carp (Ctenopharyngodon idella) against Vibrio mimicus [81].…”

Sample preparation and culture condition effects on MALDI‐TOF MS identification of bacteria: A review

“…), further processing is needed (Anderson et al, 2012). Each colony is overlaid by (mostly) 70% FA and after drying, by MALDI matrix (Kazazić, Topić Popović, Strunjak‐Perović, Babić, et al, 2019; Kazazić, Topić Popović, Strunjak‐Perović, Florio, et al, 2019). The direct deposition of bacteria often involves the treatment of cells with 40% ethanol to eliminate the formation of cell clusters and enhance sample homogeneity and shot‐to‐shot reproducibility (Madonna et al, 2000; Šedo et al, 2011).…”

“…Although MALDI‐TOF MS is a reliable and rapid technique for phenotypic identification, some barriers remain unresolved. Variability was noted in reproducibility and accuracy related to various bacterial culture media and culture conditions of certain strains (Goldstein et al, 2013; Kazazić, Topić Popović, Strunjak‐Perović, Babić, et al, 2019; Kazazić, Topić Popović, Strunjak‐Perović, Florio, et al, 2019; Sandrin et al, 2013). At this time, there is no optimal standardized protocol for sample preparation to profile bacteria, but attempts have been made (Cuénod et al, 2021; Drevinek et al, 2012; Liu et al, 2007; Wang et al, 2021).…”