Characterization of Bacteria in Ballast Water Using MALDI-TOF Mass Spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time

Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams

Vibrio parahaemolyticus is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers (tlh species-specific gene and tdh virulence gene) of V. parahaemolyticus in bivalve mollusc. The analyses were performed on clams inoculated with V. parahaemolyticus ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other Vibrio species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for tdh and tlh genes, showed a detection limit of 104 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by tdh gene in real-time PCR reached the definitely highest sensitivity in shorter times, 101 CFU/g after 3 h of pre-enrichment, while the sensitivity for the tlh gene was not promising, detecting between 105and 106 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of V. parahaemolyticus based on tdh gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases