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

Autoři: Snježana P. Kazazić aff001;  Natalija Topić Popović aff002;  Ivančica Strunjak-Perović aff002;  Sanja Babić aff002;  Daniela Florio aff004;  Marialetizia Fioravanti aff004;  Krunoslav Bojanić aff002;  Rozelindra Čož-Rakovac aff002
Působiště autorů: Laboratory for Mass Spectrometry and Functional Proteomics, Rudjer Bošković Institute, Zagreb, Croatia aff001;  Laboratory for Aquaculture Biotechnology, Rudjer Bošković Institute, Zagreb, Croatia aff002;  Centre of Excellence for Marine Bioprospecting-BioProCro, Rudjer Bošković Institute, Zagreb, Croatia aff003;  Department of Veterinary Medical Sciences, Alma Mater Studiorum Università di Bologna, Ozzano Emilia, Italy aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225343


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.

Klíčová slova:

Bacterial pathogens – Marine bacteria – Marine fish – Matrix-assisted laser desorption ionization time-of-flight mass spectrometry – Vibrio – Mass spectra – Septicemia


1. Hickey ME, Lee JL (2017) A comprehensive review of Vibrio (Listonella) anguillarum: ecology, pathology and prevention. Rev Aquac 0: 1–26.

2. Yanagihara Y, Shimizu T, Takagi A, Mifuchi I (1984) Cellular sugar pattern of Vibrio anguillarum. Microbiol Immunol 28: 499–502. doi: 10.1111/j.1348-0421.1984.tb00701.x 6748970

3. Pedersen K, Grisez L, van Houdt R, Tiainen T, Ollevier F, Larsen JL (1999) Extended serotyping scheme for Vibrio anguillarum with the definition and characterization of seven provisional O-serogroups. Curr Microbiol 38: 183–189. doi: 10.1007/pl00006784 9922470

4. Alsina M, Martínez-Picado J, Jofre J, Blanch AR (1994) A medium for presumptive identification of Vibrio anguillarum. Appl Environ Microbiol 60: 1681–1683. 8017947

5. Austin B, Austin D (2007) Bacterial fish pathogens: Disease in farmed and wild fish (4th ed.) Laird L. (ed), Springer-Praxis series in aquaculture and fisheries. Praxis Publishing Ltd, Chichester UK.

6. Topić Popović N, Barišić J, Čož-Rakovac R, Strunjak-Perović I (2014) A tool for rapid identification of Vibrio anguillarum: Managing a field outbreak. Fresen Environ Bull 23 (1): 151–153.

7. Topić Popović N, Čož-Rakovac R, Strunjak-Perović I (2007) Commercial phenotypic tests (API 20E) in diagnosis of fish bacteria: a review. Vet Med (Praha) 52 (2): 49–53.

8. Martínez-Picado J, Alsina M, Blanch AR, Cerdà M, Jofre J (1996) Species-specific detection of Vibrio anguillarum in marine aquaculture environments by selective culture and DNA hybridization. Appl Environ Microbiol 62 (2): 443–449. 16535233

9. Frans I, Michiels CW, Bossier P, Willems KA, Lievens B, Rediers H (2011) Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. J Fish Dis 34: 643–661. doi: 10.1111/j.1365-2761.2011.01279.x 21838709

10. Lay JO Jr (2001) MALDI-TOF mass spectrometry of bacteria. Mass Spectrom Rev 20 (4): 172–194. doi: 10.1002/mas.10003 11835305

11. Topić Popović N, Kazazić PS, Strunjak-Perović I, Čož-Rakovac R (2017) Differentiation of environmental aquatic bacterial isolates by MALDI-TOF MS. Environ Res 152: 7–16. doi: 10.1016/j.envres.2016.09.020 27741451

12. Crisafi F, Denaro R, Yakimov M, Felice MR, Giuliano L, Genovese L (2015) NaCl concentration in the medium modulates the secretion of active EmpA protease in Vibrio anguillarum at post-transcriptional level. J Appl Microbiol 119: 1494–1501. doi: 10.1111/jam.12957 26394191

13. Piccininno G, Ciuchini F, Adone R, Ceschia G, Giorgetti G (1996) Morphological, physico-chemical and biological variations in Vibrio anguillarum cultured at low osmolarity. New Microbiol 19 (4): 321–326. 8914133

14. Veloo ACM, Elgersma PE, Friedrich AW, Nagy E, van Winkelhoff AJ (2014) The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria. Clin Microbiol Infect 20: 1091–1097.

15. Emami K, Askari V, Ullrich M, Mohinudeen K, Anil C, Khandeparker L et al. (2012) Characterization of bacteria in ballast water using MALDI-TOF Mass Spectrometry. PLoS ONE. 7 (6): e38515 doi: 10.1371/journal.pone.0038515 22685576

16. Valentine N, Wunschel S, Wunschel D, Petersen C, Wahl K (2005) Effect of culture conditions on microorganism identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 71 (1) 58–64. doi: 10.1128/AEM.71.1.58-64.2005 15640170

17. Pérez-Sancho M, Vela Ana I, Awad M, Kostrzewa M, Dominguez L, Fernández-Garayzábal JF (2016) Differentiation of Photobacterium damselae subspecies using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in fish isolates. Aquaculture 464: 159–164.

18. Balážova T, Makovcová J, Šedo O, Slaný M, Faldyna M, Zdráhal Z (2014) The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling. FEMS Microbiol Lett 353 (1) 77–84. doi: 10.1111/1574-6968.12408 24571790

19. Zhang Y, Liu Y, Ma Q, Song Y, Zhang Q, Wang X et al. (2014) Identification of Lactobacillus from the saliva of adult patients with caries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PloS ONE 9 (8): e106185. doi: 10.1371/journal.pone.0106185 25166027

20. Šalplachta J, Horká M, Ružička F, Šlais K (2018) Identification of bacterial uropathogens by preparative isolelectric focusing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Chromatogr A 1532: 232–237. doi: 10.1016/j.chroma.2017.11.072 29217114

21. Azrad M, Keness Y, Nitzan O, Pastukh N, Tkhawkho L, Freidus V et al. (2019) Cheap and rapid in-house method for direct identification of positive blood cultures by MALDI-TOF MS technology. BMC Infect Dis. doi: 10.1186/s12879-019-3709-9 30658585

Článek vyšel v časopise


2019 Číslo 11