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

Examples of in-house CCV analysis of <i>Vibrio</i> isolates S14 and S27 (A) and S14 and S32 (B).

<p>Panel C shows the spectrograms of <i>Vibrio</i> isolates S14, S27, S30, and S32.</p

In-house CCV analysis of MTB fingerprints of <i>Pseudoalteromonas</i> isolates from the North Sea.

<p>Values may vary from “0” to “1”, where 1 indicates 100% identity of the mass-spectral patterns.</p

Comparison of <i>Pseudomonas stutzeri</i> and <i>Proteus vulgaris</i> MALDI-TOF mass spectra.

<p>Isolates S04, S24 and S38 were identified as <i>P. stutzeri</i> through 16S rRNA gene sequencing; however isolate S24 showed a different peak pattern and was identified as <i>Proteus vulgaris</i> by the Biotyper software. Examples of common peaks are indicated by vertical lines and unique peaks are indicated by the arrows.</p

Identification of marine bacteria from artificial ballast water.*.

<p><b>IC</b>: isolate code. <b>B</b>: Blyth; <b>W</b>: Whitley bay; <b>BS</b>: Biotyper software score.The score ranged from 0–3, scores >1.7 indicates a positive genus ID, scores <1.7 mean no reliable identification; <b>PL</b> Presence of the species in Biotyper mass spectra Library, (+): presence and (−): absence. % DNA indicates the % of rRNA gene nucleotides matching of the query and publically available sequences at NCBI by BLAST search. Accession numbers were from DDBJ.</p

Heat-map of <i>Pseudoalteromonas</i> (A) and <i>Pseudomonas</i> (B) isolates generated by the Biotyper software.

<p>The spectra were split into 8 or 11 groups according to the directory structure. Red colours indicate closely related groups with identical peaks, and blue colours mark non-related groups.</p

MALDI-TOF mass spectra from <i>Pseudoalteromonas (PAL)</i> isolates.

<p>The mass spectrum of <i>P. tetraodonis</i> was added to the database after identification of an isolate by 16S rRNA gene sequencing. Isolate S49, with some similarities to the mass spectral pattern of <i>PAL</i> is also included in the comparisons. Panel A shows spectra of all <i>PAL</i> isolates for the range of <i>m/z</i> 3000–10000. Panels B to E show similarities and differences between the <i>PAL</i> isolates at different <i>m/z</i>. The order of isolates in all panels is as labelled in panel A.</p

CCV analysis of MALDI-TOF data of four <i>Vibrio</i> isolates using the in-house method developed for this study.^*.

*<p>Numbers are correlation coefficients indicating similarity of the isolates through their entire mass-spectra. Values may vary from “0” to “1”, where 1 indicates 100% identity.</p

In-house CCV analysis of MTB fingerprints of <i>Pseudomonas</i> isolates from the North Sea.

<p>Values may vary from “0” to “1”, where 1 indicates 100% identity of the mass-spectral patterns.</p

Mass spectral peak list comparing three marine bacterial species isolated in this study with previously published data.^*.

*<p>MALDI-TOF mass spectra peak list for <i>Serratia liquifaciens</i>, <i>Vibrio alginolyticus</i> and <i>Proteus vulgaris</i> obtained in this study and from data previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038515#pone.0038515-Bhme2" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038515#pone.0038515-FernndezNo1" target="_blank">[37]</a>. Mass to charge ration (±<i>m/z</i>) column indicates mass unit differences between the two studies and (–) indicates no data were available.</p