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Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

Emami K, Askari V, Ullrich M, Mohinudeen K, Anil AC, Khandeparker L, Burgess JG, Mesbahi E - PLoS ONE (2012)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom. kaveh.emami@ncl.ac.uk

ABSTRACT
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.

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MALDI-TOF mass spectra from Pseudoalteromonas (PAL) isolates.The mass spectrum of P. tetraodonis 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 PAL is also included in the comparisons. Panel A shows spectra of all PAL isolates for the range of m/z 3000–10000. Panels B to E show similarities and differences between the PAL isolates at different m/z. The order of isolates in all panels is as labelled in panel A.
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pone-0038515-g006: MALDI-TOF mass spectra from Pseudoalteromonas (PAL) isolates.The mass spectrum of P. tetraodonis 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 PAL is also included in the comparisons. Panel A shows spectra of all PAL isolates for the range of m/z 3000–10000. Panels B to E show similarities and differences between the PAL isolates at different m/z. The order of isolates in all panels is as labelled in panel A.

Mentions: Members of the genus Pseudoalteromonas were initially described as belonging to the genus Pseudomonas in 1963 [33]. In 1995, Gauthier et al. [34] by describing 13 species which previously were identified as Alteromonas, proposed a new genus Pseudoalteromonas. Pseudomonads as well as some Pseudomonas-like species are widespread throughout nature and some such as P. aeruginosa are opportunistic pathogens of humans and plants [35]. Pseudomonas-like species that were isolated in this study are listed in Table 1. MALDI mass-spectra from pseudomonads are presented in Fig. 5 while Pseudoalteromonas isolates are shown in Fig 6. Dieckmann et al. [36] identified Pseudomonas-like bacteria present in marine sponges by MS but found ambiguities both at the inter- and intra-species level when comparing 16S rRNA gene sequences. They observed that for species of Pseudoalteromonas and Alteromonas inter-species diversity had been as high as intra-species diversity. In the former study, some Pseudoalteromonas isolates differed by only 3 or 4 nucleotide positions over the ∼1500 bp of their 16S rRNA gene sequence but were easily discriminated by MTB. Dieckmann et al. [36] used different instruments and chemicals in their study. For example, they obtained data by running the samples on a VOYAGER mass spectrometer (Applied Biosciences). Moreover, the bacteria were grown on different media at different temperatures. Finally, the sample preparation method and matrix (2, 5-Dihydroxybenzoic acid; DHB) used were not the same. Nevertheless, there were significant similarities between their characteristic Pseudoalteromonas mass spectra and the spectra we obtained for the corresponding isolates. For example, we observed peaks at m/z 4233±2 while they observed one at 4238. In four isolates of the ballast water a peak at m/z 5093±1 was detected, and the former authors measured one at m/z 5094 in two out of three of their Pseudoalteromonas isolates. For five of our Pseudoalteromonas isolates, we identified a peak at m/z 6072±3 and they also found a peak at m/z 6074. Isolates S06 and S49 showed a peak at m/z 7152 that was close to the peak of m/z 7151 observed in the Dieckmann et al. study. These results indicate the robustness of the MTB method across different laboratories and conditions.


Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

Emami K, Askari V, Ullrich M, Mohinudeen K, Anil AC, Khandeparker L, Burgess JG, Mesbahi E - PLoS ONE (2012)

MALDI-TOF mass spectra from Pseudoalteromonas (PAL) isolates.The mass spectrum of P. tetraodonis 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 PAL is also included in the comparisons. Panel A shows spectra of all PAL isolates for the range of m/z 3000–10000. Panels B to E show similarities and differences between the PAL isolates at different m/z. The order of isolates in all panels is as labelled in panel A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369924&req=5

pone-0038515-g006: MALDI-TOF mass spectra from Pseudoalteromonas (PAL) isolates.The mass spectrum of P. tetraodonis 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 PAL is also included in the comparisons. Panel A shows spectra of all PAL isolates for the range of m/z 3000–10000. Panels B to E show similarities and differences between the PAL isolates at different m/z. The order of isolates in all panels is as labelled in panel A.
Mentions: Members of the genus Pseudoalteromonas were initially described as belonging to the genus Pseudomonas in 1963 [33]. In 1995, Gauthier et al. [34] by describing 13 species which previously were identified as Alteromonas, proposed a new genus Pseudoalteromonas. Pseudomonads as well as some Pseudomonas-like species are widespread throughout nature and some such as P. aeruginosa are opportunistic pathogens of humans and plants [35]. Pseudomonas-like species that were isolated in this study are listed in Table 1. MALDI mass-spectra from pseudomonads are presented in Fig. 5 while Pseudoalteromonas isolates are shown in Fig 6. Dieckmann et al. [36] identified Pseudomonas-like bacteria present in marine sponges by MS but found ambiguities both at the inter- and intra-species level when comparing 16S rRNA gene sequences. They observed that for species of Pseudoalteromonas and Alteromonas inter-species diversity had been as high as intra-species diversity. In the former study, some Pseudoalteromonas isolates differed by only 3 or 4 nucleotide positions over the ∼1500 bp of their 16S rRNA gene sequence but were easily discriminated by MTB. Dieckmann et al. [36] used different instruments and chemicals in their study. For example, they obtained data by running the samples on a VOYAGER mass spectrometer (Applied Biosciences). Moreover, the bacteria were grown on different media at different temperatures. Finally, the sample preparation method and matrix (2, 5-Dihydroxybenzoic acid; DHB) used were not the same. Nevertheless, there were significant similarities between their characteristic Pseudoalteromonas mass spectra and the spectra we obtained for the corresponding isolates. For example, we observed peaks at m/z 4233±2 while they observed one at 4238. In four isolates of the ballast water a peak at m/z 5093±1 was detected, and the former authors measured one at m/z 5094 in two out of three of their Pseudoalteromonas isolates. For five of our Pseudoalteromonas isolates, we identified a peak at m/z 6072±3 and they also found a peak at m/z 6074. Isolates S06 and S49 showed a peak at m/z 7152 that was close to the peak of m/z 7151 observed in the Dieckmann et al. study. These results indicate the robustness of the MTB method across different laboratories and conditions.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom. kaveh.emami@ncl.ac.uk

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus