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.
Highlights
Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms, namely fish, crustaceans and bivalves
V. anguillarum isolates vary by cellular sugar compositions [2], and consist of at least 23 O-serogroups [3], of which serotypes O1-O5 are reported as virulent [1]
Its recovery by use of culture-based methods is possible from various media, such as tryptone soy agar (TSA), nutrient agar, brain heart infusion agar, thiosulphate citrate bile salts sucrose agar, and VAM medium, but neither is selectively specific for V. anguillarum exclusively [4,5]
Summary
Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia (vibriosis) in a wide range of marine organisms, namely fish, crustaceans and bivalves. A scheme comprising a combination of diagnostic and clinical methods was introduced by Topić Popović and co-workers [6] for the use of API 20E (Biomerieux, Marcy-l’Etoile, France) protocol, since biochemical tests, time consuming, were proven rather reliable for its identification [7]. Except from water samples, identification of uncultured fish tissue bacteria is a challenge [8]. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was developed to detect ribosomal proteins of cultured bacteria within minutes, with low costs, and high levels of sensitivity, specificity, and throughput. Most of the above mentioned tools and techniques are time-consuming and laborious, requiring highly trained operators, while MALDI-TOF MS, depending on the database, yields reliable identification results in several straight-forward steps [10,11]. The Bruker’s database version DB5989 currently includes 7 entries of V. anguillarum (02 EGS, 03 EGS, DSM 11323 DSM, DSM 21597T DSM, LMG 4437T HAM, serotype EGS, serotype EGS)
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