Identification of bacterial pathogens in cultured fish using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Background Majority of infectious diseases of cultured fish is caused by bacteria. Rapid identification of bacterial pathogen is necessary for immediate management. The present study developed Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a fast identification of fish bacterial pathogens. Streptococcus agalactiae, S. iniae, Aeromonas hydrophila, A. veronii, and Edwardsiella tarda obtained from diseased fish were used as representative bacterial pathogens in this study. Bacterial peptides were extracted to create the Main Spectra Profile (MSP), and the MSP of each bacterial species was added into the MALDI Biotyper database. Ten additional field isolates of each bacterial species were tested to validate the technique.Results The MSP of all field isolates were clearly distinguishable, and the MSPs of the same species were clustered together. However, the species identification when matching with the public MALDI Biotyper library (Bruker MALDI Biotyper) showed unreliable result at the species level. The accurate identification only obtained when the custom-made database was used, giving a 100% matching result.Conclusion This study demonstrates a newly developed technique for the effective identification of fish bacterial pathogens. Further applications require a broad, well-established database to accommodate the prudent identification of many fish bacterial pathogens by MALDI-TOF MS.

Background Bacterial pathogens are a major etiology of infectious diseases ofcultured fish [1]. Among those bacteria, Streptococcus spp., Aeromonas spp., and Edwardsiella spp.,, are commonly found in several important aquaculture species, such as the Asian catfish Clarias batrachus [2], barramundi Lates calcarifer [3], and Nile tilapia Oreochromis niloticus 4]. In many cases of bacterial infection, clinical signs and lesions are not specific and may mislead the diagnosis. Therefore, identification of bacterial species causing disease is necessary in order to carry out proper disease management.
Conventional microbiology, including morphological, physiological and biochemical tests, and molecular techniques based on 16S rDNA sequencing, are the gold standard for bacterial species identification [5]. However, these techniques require a substantial amount of time and expensive reagents [6]. The recent development of mass spectrometry (MS), the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been implemented in human and veterinary medicine as an alternative diagnostic tool with increasing popularity due to its quickness, simplicity, cost-effectiveness, and strong discriminating power [7,8]. The MALDI-TOF MS detects the mass signals from bacterial proteins or peptides and determines their unique mass spectra or peptide mass fingerprints (PMFs). The obtained PMFs are then compared with reference bacterial strains in the public proteomics/genomics databases or in a dedicated mass spectra library (library based approach) [9], which is able to differentiate the bacteria at their genus, species or sub-group levels subject to sufficient reference strains existing in the database [10].
The MALDI-TOF MS approach has been adopted as a routine diagnostic tool for human medicine [11] and also has been widely evaluated for the ability to differentiate bacterial species of veterinary and public health importance. For example, S. equi at subspecies level [8], Streptococcus spp. isolated from diseased pigs [12], pathogenic Gram-negative bacteria in seafood [13] and Aeromonad species found in a drinking water system [14] have been assessed. In fish, MALDI-TOF was evaluated for the fast identification of Grampositive bacterial pathogens, including S. agalactiae, Lactococcus garvieae, S. iniae, and S. dysgalactiae isolated from Nile tilapia [15] and S. iniae isolated from the olive flounder Paralichthys olivaceus [16]. These studies found that the public database, Bruker MALDI

MALDI-TOF MS for bacterial species differentiation
The high reliability of the MALDI-TOF MS was indicated by the obtained 100% recognition capabilities and by the cross-validation values of 87.8%, 97.1%, 100%, 100%, and 100% for S. agalactiae, S. iniae. A. hydrophila, A. veronii, and E. tarda, respectively. The five bacterial species showed distinguishing spectral peaks ranging between 2,000-15,000 Da ( Fig. 1). The 3D-PCA scatterplot and the MSP dendrogram presented clearly distinguishable clusters for each bacterial species. For the MSP dendrogram, bacterial isolates within the same species were grouped in the same clade and the same genus were placed closer to each other ( Fig. 2A and 2B).

MALDI Biotyper database
The additional field isolates, when blasted against the reference strains available in the Bruker database, gave no-reliable identification for 12 isolates (log score 1.454-1.667), probable genus-level identification for 31 isolates (log score 1.707-1.997), and secure genus-level identification for seven isolates (log score 2.018-2.254). No species-level identification was obtained, especially for S. iniae since it is not available in the Bruker database.
A custom database of the five bacterial fish pathogens was then established. Matching results of the field strain were improved, showing nine isolates with a secure genus-level identification to a probable species-level identification (log score 2.115-2.264) and 41 isolates with a highly probable species-level identification (log score 2.301-2.601) ( Table   2). Specificity of the method was indicated by the 100% species identification compared with the 16S rDNA sequencing, and no mis-identification at species level was observed.

Discussion
An accurate and repeatable method for the identification of bacterial pathogens of aquaculture species was developed in this study. It can be performed in a relatively short time with a lower cost compared to conventional biochemical methods. However, the correct identification of bacterial species was only obtained when a custom database was constructed because the reference database does not accommodate the tested pathogens.
The reference database may only contain bacterial species that are significant to humans but not species of veterinary importance, particularly from aquatic species [21]. The failure in species identification from the Bruker database may also result from inconsistent protein profiles due to the use of different sample preparation protocols. The extraction method usually involves the use of organic acid to extract small-sized protein molecules, such as ribosomal proteins, cold shock proteins, and nucleic-acid binding proteins [22]. The different percentage of acid used in other studies [50% ACN and 2.5% TFA] [16, 23, 24] may alter the pattern of those extracted proteins. Nevertheless, ability to tailor a database expands the application of MALDI Biotyper to be used as an identification tool for bacterial species specific to a host or location, and at below specieslevel, such as sub-species, strain, or serotype [25,26].
Besides the identification purpose of MALDI-TOF, the 3D-PCA scatterplot generated from the protein profile can be used for grouping and differentiation of the protein profile of the organism. The protein profile analyzed with MALDI-TOF MS in the present study clearly showed separate clusters of each bacterial species on the 3D-PCA scatterplot and MSP dendrogram, indicating the discriminating power of the method. The MALDI-TOF MS approach has also been used to distinguish antimicrobial resistant Enterobacteriaceae [27], differentiate Burkholderia pseudomallei mutants [22], and even in screening for early or late stage cancer [28]. Therefore, MALDI-TOF MS can be a useful tool for investigating aquatic pathogens in many aspects.
In the present study, we provide an example of a MSP dendrogram created by the Biotyper software, by grouping the bacteria based on their phenotypic traits instead of their genetic traits. Interestingly, the ATCC strains from the reference database showed a high distance level to the field strains, and this may explain the failure of species identification described previously. Genotyping is usually based on phylogenetic analysis of a highly

Bacterial samples
All bacterial isolates were obtained from clinical cases that were submitted to the Faculty of Veterinary Science, Chulalongkorn University for disease diagnosis. Bacterial isolation was performed on kidney tissues using Columbia blood agar supplemented with 5% sheep blood (Oxiod, Basingstoke, UK) and incubated at 28 °C for 24 h. Bacterial species were identified by conventional microbiology methods, including Gram staining, catalase and oxidase production tests, and API identification (BioMérieus ® , France). Bacterial species were confirmed using PCR amplification and sequencing of the 16S rDNA [17]. Pure bacterial isolates were stored in nutrient broth (NB; Oxiod) containing 10% fetal calf serum and 20% glycerol at -80 o C for further analysis.

Sample preparation for MALDI-TOF MS
Each bacterial isolate was revived from the stock onto Columbia blood agar and incubated at 28 °C for 18 h. Extraction of bacterial proteins was performed as previously described by [18]. Briefly, a loopfull of bacterial colonies was suspended in 70% ethanol and the suspension was centrifuged at 11,000 g for 2 min. The supernatant was removed, and the bacterial pellet was resuspended and mixed thoroughly with 100% acetonitrile (ACN) containing 5% (w/v) trifluoroacetic acid (TFA). The suspension was centrifuged and the supernatant was collected for peptide measurement using Lowry's assay at 690 nm absorbance [19]. The concentration of peptide was adjusted to 0.1 µg µL -1 for the MALDI-TOF MS analysis.

MALDI-TOF MS for database generation
Five bacterial isolates of each bacterial species were used as a representative for the MSP database preparation (Table 1)

Method validation and bacterial species identification
The same bacterial isolate was processed twice using a similar protocol and analyzed by MAILDI-TOF MS to evaluate the repeatability of the method. To assess the specificity of the method, additional field isolates of the five pathogens (10 additional isolates/pathogen) were used. All isolates were identified using 16S rDNA PCR and sequencing, as previously described    Figure 1 Mass peptide fingerprints of five bacterial pathogens (S. agalactiae, S. iniae, A. hydrophila, A. veronii and E. tarda) isolated from cultured fish.