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Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for the identification of Pseudomonas species isolated from fish
Microbial Pathogenesis 132 (2019) 313–318
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for the identification of Pseudomonas species isolated from fish
T
Miroslava Kačániováa,b,∗, Alīna Klūgac, Attila Kántord, Juraj Medoa, Jana Žiarovskáe, Czeslaw Puchalskib, Margarita Terentjevac a
Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia Department of Bioenergy and Food Technology, Faculty of Biology and Agriculture, University of Rzeszow, Zelwerowicza St. 4, PL, 35601, Rzeszow, Poland c Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Life Sciences and Technologies, K. Helmaņa iela 8, LV, 3004, Jelgava, Latvia d Department of Storing and Processing Plant Products, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia e Department of Plant Genetics and Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Freshwater fish Mass spectrometry DNA Sequencing Pseudomonas
Effective and reliable methods of identification of Pseudomonas species are important for the characterization of microorganisms. Freshwater ecosystems are an important source of Pseudomonas species, including those pathogenic to fish and humans. The aim of the present study was to compare the identification conducted with MALDI-TOF MS Biotyper and 16S rDNA sequencing of fish-borne Pseudomonas spp. Altogether, 13 different Pseudomonas spp. were isolated from freshwater fish. Phylogenetic analysis showed a clear taxonomic placement only for 13 out of 15 Pseudomonas isolates. Accordance of identification method was found only in 6 out of 15 isolates. The human pathogenic Pseudomonas spp. were not found in our study, indicating that the fish could be considered as safe for consumption. The present study revealed a high discriminatory power of the mass spectra investigation and 16S rDNA gene sequencing technology for the identification of Pseudomonas spp. associated with freshwater fish.
1. Introduction The genus Pseudomonas of the family Pseudomonadaceae within the class Gammaproteobacteria was described by Migula [1]. Since that time, the genus has undergone various taxonomic revisions with many new species being introduced [2–5]. Nowadays, the genus Pseudomonas consists of more than 200 species and 11 subspecies with validly published names [6]. Pseudomonas spp. are widely distributed in the environment and were isolated from water, soils, plants, insects, animals and other natural habitats [7]. Aquaculture is an expanding industry and fish diseases caused by bacteria belong to the most significant causes of economic loss for entrepreneurs [8,9]. Fish microbiota consist of diverse groups of microorganisms, which are important for fish and environmental health. Pseudomonas spp. comprise a significant part of fish microbiota with a significant proportion possibly serving as fish pathogens or opportunistic pathogens, which may cause diseases in stressed fish [10,11].
P. fluorescens is the most important spp. relevant to fish pathology and is very often associated with skin and fish disease [12]. Infections caused by P. fluorescens can lead to a 100% mortality in rainbow trout farms. Other Pseudomonas (P. putida or P. luteola) were isolated from internal organs of fish without significant clinical impact. Pseudomonas spp. can cause the strawberry disease in rainbow trout and tench. Systemic infections with septicaemia symptoms were identified in crucian carp and silver carp [12]. Among the Pseudomonas spp., P. fluorescens, P. anguilliseptica, P. putida, P. putrefaciens and P. aeruginosa are affecting fish health more frequently [13]. Pseudomonas spp. has been isolated from foods because of its ubiquitous occurrence in raw materials and processing environment of the food enterprises. Bacteria can cause a spoilage of fish and seafood [14,15] and be present in ready-to-eat products [16]. Pseudomonas spp. are not considered as a foodborne pathogen in Europe and other regions. Microbiological testing of imported fish and shellfish may include various bacterial pathogens - Escherichia coli, Clostridium
∗ Corresponding author. Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia. E-mail address: [email protected] (M. Kačániová).
https://doi.org/10.1016/j.micpath.2019.04.024 Received 7 March 2019; Received in revised form 11 April 2019; Accepted 12 April 2019 Available online 15 April 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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The number of isolated microorganisms
botulinum, Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii and Salmonella sp., but not Pseudomonas spp. (Canadian Food Inspection Agency [17], European Comission [18], American Food Safety and Inspection Service (FSIS) [19]. Human infections caused by bacterial pathogens originated from fish are quite common, and the severity of disease depends on the season, contact of fish or environment, preparation of fish and the immune condition of individual [20]. Transmission of Pseudomonas spp. and the risk of infection in healthy population have been considered as low in developed countries [21]. However, the presence of enterotoxigenic Pseudomonas spp. in food and drinking water has been reported in previous studies [22,23]. Contamination of fish, food and drinking water with enterotoxigenic Pseudomonas spp. may result in diarrhoea and skin infections in immunodeficient individuals [13]. Reliable and easy applicable methods for the identification of microbiota are necessary to characterize the fish microbiota in order to prevent a spread of fish disease and to ensure the safety of fish meat for consumption. Hence, we analyzed the bacterial microbiota of most common freshwater fish in Latvia with the emphasis on Pseudomonas spp., which may serve as a fish and human pathogen. Therefore, changes occurring in freshwater fish seem to be fundamental in the development of any disease, including emerging ones [12]. The aim of the present study was to compare the accuracy of identification obtained with the automated systems Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDITOF MS) (Bruker Daltonics, Bremen, Germany) and 16S rDNA sequencing in discrimination of Pseudomonas spp.
Yersinia ruckeri Yersinia intermedia Stenotrophonomas sp. Serratia liquefaciens Serratia grimesii Serratia fonticola Rahnella aquatilis Pseudomonas veronii Pseudomonas thivervalensis Pseudomonas taetrolens Pseudomonas synxantha Pseudomonas sp. Pseudomonas rhodesiae Pseudomonas putida Pseudomonas proteolytica Pseudomonas orientalis Pseudomonas marginalis Pseudomonas mandelii Pseudomonas lundensis Pseudomonas libanensis Pseudomonas koreensis Pseudomonas kilonensis Pseudomonas jessenii Pseudomonas graminis Pseudomonas gessardii Pseudomonas fulva Pseudomonas… Pseudomonas fragi Pseudomonas fluorescens Pseudomonas… Pseudomonas corrugata Pseudomonas brenneri Pseudomonas brassicacearum Pseudomonas antarctica Providencia rustigianii Providencia heimbachae Pantoea sp. Pantoea agglomerans Moellerella wisconsensis Janthinobacterium lividum Enterobacter cloacae Debaryomyces hansenii Candida pelliculosa Buttiauxella gaviniae Buttiauxella ferragutiae Aeromonas veronii Aeromonas ichtiosmia Aeromonas bestiarium
2. Material and methods 2.1. Sampling Altogether, 22 samples of freshwater fish were collected in Latvia throughout 2015. Among the samples, 17 were wild freshwater fish caught in inland waters and five samples were aquacultured fish bought at the retail market. The sampled wild fish included roach (Rutulis rutulis, n = 7), chub (Leuciscus cephalus, n = 1), crucian carp (Carassius carassius, n = 5), bream (Abramis brama, n = 4). Roach (Rutulis rutulis, n = 3) and bream (Abramis brama, n = 2) were purchased from the retail market outlets. The fish were transported to the laboratory in sterile bags on ice, and the testing was initiated within 2 h. The skin, gill and gut were used for microbiological studies and at least 1 g of material was used for the preparation of the primary dilution. One gram of skin, gill and gut were added to 45 ml of physiological saline solution for a decimal dilution (10−2). Tissues of interest were aseptically removed and investigated separately. The samples were transferred into a corresponding amount of physiological saline solution 0,89% (1:10) to obtain initial and decimal dilutions according ISO [24]. An amount of 0.1 ml of sample suspension was plated out on Pseudomonas agar (Pseudomonas agar base, Sigma Aldrich, UK; 5 ml of glycerol; CFC Supplement, FD036). Inoculated agars were incubated at 25 °C for 48 h aerobically. Then the agars were evaluated for bacterial growth and 3–5 colonies or more (depending on the morphological characteristics of colonies) per plate were selected for further confirmation with MALDI-TOF MS Biotyper (Bruker Daltonics, Germany).
0
1 1 2 1 1 6 7 3 1 5 1 2 2 1 9 1 1 2 2 3 13 1 1 1 1 3 7 10 6 8 3 4 3 6 1 5 3 6 1 1 1 2 11 1 6 2 1 1 5
10
15
Fig. 1. The microbial species and number of isolates originated from fish.
discarded and the same spin was repeated on the pellet. Remains of ethanol were removed, and the pellet was allowed to dry. Ten μl of 70% formic acid were added to the pellet by pipetting and vortexing. Then, 10 μl of acetonitrile were added. The suspension was centrifuged at 14,000 rpm for 2 min and 1 μl of the supernatant was transferred to the MALDI target. Once dry, every spot was overlaid with 1 μl of HCCA (2Cyano-3-(4-hydroxyphenyl) acrylic acid) (Bruker Daltonics, Germany) matrix and left to dry at room temperature before analysis. The spectra were generated by MALDI-TOF and analyzed with Microflex LT (Bruker Daltonics, Germany) instrument using Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC specific software. Criteria for a successful identification were within a confidence score of ≥2.0 for the species level and ≥1.7 for the genus level according to the manufacturer [25].
2.2. Identification of isolates with MALDI -TOF MS Biotyper 2.3. DNA analysis Altogether, 161 bacterial colonies were selected for further testing after storage. The isolates were transferred onto Tryptone soya agar at 37 °C for 24 h and a loopful of bacterial colonies was transferred into 300 μl of distilled water. After the addition of 900 μl of ethanol, the samples were centrifuged for 2 min at 14,000 rpm. The supernatant was
DNA was extracted using a Sigma kit (GenElute™ Bacterial Genomic DNA Kit, Sigma Aldrich, UK) from 24-h old bacterial culture following the manufacturer's instructions. Amplification reactions were carried out in 25 μl volumes containing 200 mM dNTPs, 1x DreamTaq buffer, 314
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Table 1 Comparison of identification of Pseudomonas spp. with MALDI-TOF MS Biotyper and 16S rDNA sequencing. Strain
Species
Concordance (%)
Sequence identity
MALDI
Score
AB1_7 AB1_8 AB1_9 AB1_11 AB1_12 AB2_1 AB2_2 AB2_3 AB2_4 AB2_5 AB2_6 AB2_7 AB2_8 AB2_9 AB2_10
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
99% 99% 99% 99% 99% 99% 99% 100% 100% 99% 99% 99% 99% 99% 99%
1472/1475 1472/1476 1447/1452 1473/1475 1473/1475 1459/1463 1475/1478 1478/1478 1478/1478 1473/1476 1471/1476 1466/1468 1472/1478 1472/1476 1467/1468
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
2.063 2.157 2.103 2.099 1.964 2.086 2.158 2.168 2.038 2.418 2.346 2.193 2.174/2.221 2.18 2.183
extremaustralis orientalis deceptionensis extremaustralis weihenstephanensis brenneri rhodesiae veronii silesiensis proteolytica kilonensis helmanticensis libanensis/gessardii orientalis helmanticensis
extremorientalis fluorescens taetrolens extremorientalis fragi frederiksbergensis Antarctica veronii mandelii proteolytica brassicacearum koreensis libanensi/gessardii synxantha proteolytica
Fig. 2. Dendrogram of selected Pseudomonas species created with MALDI-TOF MS Biotyper.
2.4. Data analysis
0.5 U DreamTaq DNA polymerase (Life technologies, USA, NY), 0.5 mM of the corresponding 16S rDNA sequencing primer [26], and 0.5 μL of non-diluted DNA. Conditions of PCR reactions were as follows: initial denaturation at 95 °C for 3 min and subsequent 35 cycles; denaturation at 95 °C for 30 s, annealing at the corresponding temperature for each primer set for 45 s, elongation at 72 °C for 90 s, and the final elongation at 72 °C for 10 min. PCR and sequencing reactions were carried out in the Biorad MJ mini thermal cycler (BioRad Corp., USA, CA). Primers 27F and 1492R [26] were used for PCR. PCR products were enzymatically purified by ExoI/FastAP (Life technologies, USA, NY). Sequencing was performed bi-directionally using primers 785F and 907R [25] using the Macrogen Inc. South Korea on Applied biosystems 3730XL and Big Dye 3.1 chemistry.
The acquired sequences were assembled and processed using the Seaview software [27,28]. Similarity of sequences to type the species was checked by BLASTn. The alignment was made using Clustal Omega [29]. Reference sequences of Pseudomonas type spp. were acquired from GenBank. A phylogenetic tree was constructed by maximum likelihood algorithm using PhyML [28] with following settings: GTR model, BioNJ starting tree, best of NNI and SPR tree searching, optimized across site rate variation, 1000 bootstraps.
3. Results and discussion Pseudomonas spp. were the most distributed microbial species isolated from fish in the present study. Meanwhile, other species included Aeromonas, Buttiauxella, Candida, Debaromyces, Enterobacter, 315
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Fig. 3. Maximum likelihood phylogenetic analysis of 15 Pseudomonas strains isolated from fish. Numbers above branches indicate bootstrap values (only ≥50 are shown). Green lines indicate identification by 16S rDNA and purple lines by MALDI TOF.
frequently isolated. The isolated microbial species are shown in Fig. 1. MALDI-TOF MS analysis is a method that has been used extensively for the identification of microbial species as it provides an opportunity to receive reliable results rapidly. The speed of identification is crucial
Jantinobacterium, Moellerella, Pantoea, Providencia, Rahnella, Serratia, Stenotrophonomonas and Yersinia intermedia. Pseudomonas koreensis (13 isolates), Candida pelliculosa (11 isolates), Pseudomonas fragi (10 isolates) and Pseudomonas proteolytica (9 isolates) were the most 316
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polyphasic attitude including morphological and physiological traits [49]. The incorporation of MALDI-TOF MS Biotyper to a polyphasic system could increase the speed and resolution of Pseudomonas spp. identification.
for diagnostic, especially in clinical settings [30–35]. For the identification, the Biotyper software compares the sample spectrum to its spectra database, generated by the characterized isolates [25]. MALDITOF MS analysis has not been applied for fish microbiota identification previously, however, this method provided an acceptable score for all isolates selected for identification. As a preliminary test of the applicability of MALDI-TOF mass spectrometry for the identification of Pseudomonas spp., a set of 27 strains were analyzed. The results of the identification with a log score are listed in Table 1. With the exception of isolate AB1_12, other Pseudomonas spp. were identified with a score above 2.00, which means high confidence identification to the spp. level. Altogether, 14 Pseudomonas spp. were identified with MALDI-TOF. The analyzed strains formed 3 clusters and the Pseudomonas spp. similarity is reflected in the MSP dendrogram (Fig. 2). A total of 15 Pseudomonas isolates previously identified by MALDITOF MS were analyzed by 16S rDNA amplicon sequencing. 16S rDNA sequencing may be applicable for bacterial species identification in various fields because of its highly discriminative power. Additionally, the method has been used for routine clinical diagnostics as well [36–38]. BLASTn analysis of 16S rDNA sequences (Table 1) showed 99 or 100% similarity for all species. Only 2 isolates revealed sequences identical to type species while others shared up to 6 mismatches. The general concept of species differentiation considers 95 or 97% similarity as a criterion for species delimitation, however between some types of Pseudomonas species only 5 or even less bases could make a difference (e.g. P. gessardi and P. libanensis differ by 3 bases). Pseudomonas is one of the most complex and diverse bacterial genera comprising more than 200 species described to date [12]. Several groups and sub-groups have been identified within this genus [39]. The phylogenetic analysis showed a clear taxonomic placement only for 13 out of 15 of Pseudomonas isolates in the present study. The dendrogram in Fig. 3 contains a comparison of all type species with at least 99% similarity to any isolate. There are at least 10 well defined clades. Identified clusters respect the general grouping but they are not in full congruence to previously published sub-groups of Pseudomonas spp. [40], however, clustering depends on all sequences in the collection. Fish isolates are apparently members of P. fluorescens, P. gessardii, P. fragi, P. koreensis and P. mandelii subgroups but no none from the P. aeruginosa group was identified. P. fluorescens was reported to be the a fish pathogen, while other isolated Pseudomonas spp. were expected to be non-harmful to fish [41–43]. Since human pathogenic Pseudomonas spp. were not isolated in the present study, our findings indicate that the fish collected are safe for consumption. P. aeruginosa was found in fish previously and is known to pose a threat to the human health [17,44–48]. As such, our report shows that an accurate identification of Pseudomonas spp. is important to ensure fish protection from bacterial infections and to evaluate the bacterial quality of fish and its safety for consumption. The isolate AB2_1 did not cluster with P. brenneri which was the most similar in sequence. MALDI-TOF MS Biotyper identified this isolate as P. frederiksbergensis which was clustered in a different branch than P. brenneri. AB2_8 felt to cluster among P. azotoformans strains despite the most similar species identified both by MALDI and 16S was P. libanensis. MALDI probably did not correctly identify the isolates AB1_8 and AB2_9 as P. fluorescens and P. synxantha, respectively, as the genetic analysis placed them to the cluster with P. orientalis. AB2_6 was the most similar to the type strain of P. kilonensis (5 bp diff, 0 gaps) but it was clustered with P. brassicacearum (7bp diff, 2 gaps) and congruently identified by MALDI. The accuracy of MALDI-TOF MS identification was found only in 6 out of 15 isolates. Isolates AB2_1 and AB2_8 should be processed by a deep phylogenetic analysis to confirm their status. Phylogeny of Pseudomonas spp. is very difficult, generally based on multilocus sequencing analysis of DNA (MLSA), but the correct identification of species requires a
4. Conclusion In this study, a total 161 colonies of microorganisms have been isolated from freshwater fish and identified with MALDI -TOF MS analysis. Thirteen Pseudomonas isolates were further confirmed by 16S rDNA gene sequencing and the isolates were identified as P. antarctica, P. brassicacearum, P. extremorientalis, P. frederiksbergensis, P. fluorescens, P. fragi, P. libanensis, P. koreensis, P. mandelii, P. proteolytica, P. synxantha, P. taetrolens and P. veronii. Overall, our study revealed a high discriminatory power of the mass spectra investigation and 16S rDNA gene sequencing technology for the identification of Pseudomonas spp. associated with freshwater fish. Conflicts of interest The authors declare that there is no conflict of interest. Acknowledgments The study was supported by the European Community project No 26220220180: Building Research Centre „AgroBioTech" and by the grants APVV-15-0544. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.04.024. References [1] W. Migula, Ubereinneues system der Bakterien, Arb, Bakteriol. Inst. Karlsruhe 1 (1984) 235–238. [2] P.H.A. Sneath, M. Stevens, M.J. Sackin, Numerical taxonomy of Pseudomonas based on published records of substrateutilization, Antonie Leeuwenhoek 47 (5) (1981) 423–448 https://doi.org/10.1007/bf00426004. [3] H. Oyaizu, K. Komagata, Grouping of Pseudomonas species on the basis of cellular fatty acid composition and the quinone system with special reference to the existence of 3-hydroxy fatty acids, J. Gen. Appl. Microbiol. 29 (1) (1983) 17–40 https://doi.org/10.2323/jgam.29.17. [4] M. Vancanneyt, S. Witt, W.R. Abraham, K. Kersters, H.L. Fredrickson, Fatty acid content in whole-cell hydrolysates and phosphor lipid fractions of pseudomonads: a taxonomic evaluation, Syst. Appl. Microbiol. 19 (4) (1996) 528–540 https://doi. org/10.1016/s0723-2020(96)80025-7. [5] Y. Anzai, H. Kim, J.Y. Park, H. Wakabayashi, H. Oyaizu, Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence, Int. J. Syst. Evol. Microbiol. 50 (4) (2000) 1563–1589 https://doi.org/10.1099/00207713-50-4-1563. [6] A.C. Parte, List of prokaryotic names with standing in nomenclature, Nucleic Acids Res. 42 (D1) (2014) D613–D616 https://doi.org/10.1093/nar/gkt1111. [7] N.J. Palleroni, Introduction to the family Pseudomonadaceae, in: A. Balows, H. Trüper, G.G.M. Dworkin, W. Harder, K.H. Schleifer (Eds.), The Prokaryotes, second ed., New York & Springer, USA, 1992, pp. 3071–3085. [8] A.E. Toranzo, B. Magariños, J.L. Romalde, A review of the main bacterial fish diseases in mariculture systems, Aquaculture 246 (1–4) (2005) 37–61 https://doi. org/10.1016/j.aquaculture.2005.01.002. [9] J.R. López, J.I. Navas, N. Thanantong, R. de la Herran, O.A.F. Sparagano, Simultaneous identification of five marine fish pathogens belonging to the genera Tenacibaculum, Vibrio, Photobacterium and Pseudomonas by reverse line blot hybridization, Aquaculture 324–325 (2012) 33–38 https://doi.org/10.1016/j. aquaculture.2011.10.043. [10] FAO/WHO, food and agriculture organization of the united nations/world health organization. Microbiological risk assessment series, Risk Characterization of Microbiological Hazards in Hood. Guidelines, vol. 17, WHO, Geneva, Switzerland, 2009, p. 116. [11] T. Sakata, Microflora of healthy animals, in: B. Austin, D.A. Austin (Eds.), Methods for the Microbiological Examination of Fish and Shellfish Chichester, Ellis Horwood Ltd., England, UK, 1989, pp. 141–163. [12] A. Pękala-Safińska, Contemporary threats of bacterial infections in freshwater fish, J. Vet. Res. 62 (3) (2018) 261–267. [13] A. Ashraf, E.I. Tawab, A. Ahmed, A.A. Maarouf, Nesma, M.G. Ahmed, Detection of Virulence factors of Pseudomonas species isolated from fresh water fish by PCR,
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Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for the identification of Pseudomonas species isolated from fish
T
Miroslava Kačániováa,b,∗, Alīna Klūgac, Attila Kántord, Juraj Medoa, Jana Žiarovskáe, Czeslaw Puchalskib, Margarita Terentjevac a
Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia Department of Bioenergy and Food Technology, Faculty of Biology and Agriculture, University of Rzeszow, Zelwerowicza St. 4, PL, 35601, Rzeszow, Poland c Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Life Sciences and Technologies, K. Helmaņa iela 8, LV, 3004, Jelgava, Latvia d Department of Storing and Processing Plant Products, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia e Department of Plant Genetics and Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Freshwater fish Mass spectrometry DNA Sequencing Pseudomonas
Effective and reliable methods of identification of Pseudomonas species are important for the characterization of microorganisms. Freshwater ecosystems are an important source of Pseudomonas species, including those pathogenic to fish and humans. The aim of the present study was to compare the identification conducted with MALDI-TOF MS Biotyper and 16S rDNA sequencing of fish-borne Pseudomonas spp. Altogether, 13 different Pseudomonas spp. were isolated from freshwater fish. Phylogenetic analysis showed a clear taxonomic placement only for 13 out of 15 Pseudomonas isolates. Accordance of identification method was found only in 6 out of 15 isolates. The human pathogenic Pseudomonas spp. were not found in our study, indicating that the fish could be considered as safe for consumption. The present study revealed a high discriminatory power of the mass spectra investigation and 16S rDNA gene sequencing technology for the identification of Pseudomonas spp. associated with freshwater fish.
1. Introduction The genus Pseudomonas of the family Pseudomonadaceae within the class Gammaproteobacteria was described by Migula [1]. Since that time, the genus has undergone various taxonomic revisions with many new species being introduced [2–5]. Nowadays, the genus Pseudomonas consists of more than 200 species and 11 subspecies with validly published names [6]. Pseudomonas spp. are widely distributed in the environment and were isolated from water, soils, plants, insects, animals and other natural habitats [7]. Aquaculture is an expanding industry and fish diseases caused by bacteria belong to the most significant causes of economic loss for entrepreneurs [8,9]. Fish microbiota consist of diverse groups of microorganisms, which are important for fish and environmental health. Pseudomonas spp. comprise a significant part of fish microbiota with a significant proportion possibly serving as fish pathogens or opportunistic pathogens, which may cause diseases in stressed fish [10,11].
P. fluorescens is the most important spp. relevant to fish pathology and is very often associated with skin and fish disease [12]. Infections caused by P. fluorescens can lead to a 100% mortality in rainbow trout farms. Other Pseudomonas (P. putida or P. luteola) were isolated from internal organs of fish without significant clinical impact. Pseudomonas spp. can cause the strawberry disease in rainbow trout and tench. Systemic infections with septicaemia symptoms were identified in crucian carp and silver carp [12]. Among the Pseudomonas spp., P. fluorescens, P. anguilliseptica, P. putida, P. putrefaciens and P. aeruginosa are affecting fish health more frequently [13]. Pseudomonas spp. has been isolated from foods because of its ubiquitous occurrence in raw materials and processing environment of the food enterprises. Bacteria can cause a spoilage of fish and seafood [14,15] and be present in ready-to-eat products [16]. Pseudomonas spp. are not considered as a foodborne pathogen in Europe and other regions. Microbiological testing of imported fish and shellfish may include various bacterial pathogens - Escherichia coli, Clostridium
∗ Corresponding author. Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, SK, 94976, Nitra, Slovakia. E-mail address: [email protected] (M. Kačániová).
https://doi.org/10.1016/j.micpath.2019.04.024 Received 7 March 2019; Received in revised form 11 April 2019; Accepted 12 April 2019 Available online 15 April 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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The number of isolated microorganisms
botulinum, Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii and Salmonella sp., but not Pseudomonas spp. (Canadian Food Inspection Agency [17], European Comission [18], American Food Safety and Inspection Service (FSIS) [19]. Human infections caused by bacterial pathogens originated from fish are quite common, and the severity of disease depends on the season, contact of fish or environment, preparation of fish and the immune condition of individual [20]. Transmission of Pseudomonas spp. and the risk of infection in healthy population have been considered as low in developed countries [21]. However, the presence of enterotoxigenic Pseudomonas spp. in food and drinking water has been reported in previous studies [22,23]. Contamination of fish, food and drinking water with enterotoxigenic Pseudomonas spp. may result in diarrhoea and skin infections in immunodeficient individuals [13]. Reliable and easy applicable methods for the identification of microbiota are necessary to characterize the fish microbiota in order to prevent a spread of fish disease and to ensure the safety of fish meat for consumption. Hence, we analyzed the bacterial microbiota of most common freshwater fish in Latvia with the emphasis on Pseudomonas spp., which may serve as a fish and human pathogen. Therefore, changes occurring in freshwater fish seem to be fundamental in the development of any disease, including emerging ones [12]. The aim of the present study was to compare the accuracy of identification obtained with the automated systems Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDITOF MS) (Bruker Daltonics, Bremen, Germany) and 16S rDNA sequencing in discrimination of Pseudomonas spp.
Yersinia ruckeri Yersinia intermedia Stenotrophonomas sp. Serratia liquefaciens Serratia grimesii Serratia fonticola Rahnella aquatilis Pseudomonas veronii Pseudomonas thivervalensis Pseudomonas taetrolens Pseudomonas synxantha Pseudomonas sp. Pseudomonas rhodesiae Pseudomonas putida Pseudomonas proteolytica Pseudomonas orientalis Pseudomonas marginalis Pseudomonas mandelii Pseudomonas lundensis Pseudomonas libanensis Pseudomonas koreensis Pseudomonas kilonensis Pseudomonas jessenii Pseudomonas graminis Pseudomonas gessardii Pseudomonas fulva Pseudomonas… Pseudomonas fragi Pseudomonas fluorescens Pseudomonas… Pseudomonas corrugata Pseudomonas brenneri Pseudomonas brassicacearum Pseudomonas antarctica Providencia rustigianii Providencia heimbachae Pantoea sp. Pantoea agglomerans Moellerella wisconsensis Janthinobacterium lividum Enterobacter cloacae Debaryomyces hansenii Candida pelliculosa Buttiauxella gaviniae Buttiauxella ferragutiae Aeromonas veronii Aeromonas ichtiosmia Aeromonas bestiarium
2. Material and methods 2.1. Sampling Altogether, 22 samples of freshwater fish were collected in Latvia throughout 2015. Among the samples, 17 were wild freshwater fish caught in inland waters and five samples were aquacultured fish bought at the retail market. The sampled wild fish included roach (Rutulis rutulis, n = 7), chub (Leuciscus cephalus, n = 1), crucian carp (Carassius carassius, n = 5), bream (Abramis brama, n = 4). Roach (Rutulis rutulis, n = 3) and bream (Abramis brama, n = 2) were purchased from the retail market outlets. The fish were transported to the laboratory in sterile bags on ice, and the testing was initiated within 2 h. The skin, gill and gut were used for microbiological studies and at least 1 g of material was used for the preparation of the primary dilution. One gram of skin, gill and gut were added to 45 ml of physiological saline solution for a decimal dilution (10−2). Tissues of interest were aseptically removed and investigated separately. The samples were transferred into a corresponding amount of physiological saline solution 0,89% (1:10) to obtain initial and decimal dilutions according ISO [24]. An amount of 0.1 ml of sample suspension was plated out on Pseudomonas agar (Pseudomonas agar base, Sigma Aldrich, UK; 5 ml of glycerol; CFC Supplement, FD036). Inoculated agars were incubated at 25 °C for 48 h aerobically. Then the agars were evaluated for bacterial growth and 3–5 colonies or more (depending on the morphological characteristics of colonies) per plate were selected for further confirmation with MALDI-TOF MS Biotyper (Bruker Daltonics, Germany).
0
1 1 2 1 1 6 7 3 1 5 1 2 2 1 9 1 1 2 2 3 13 1 1 1 1 3 7 10 6 8 3 4 3 6 1 5 3 6 1 1 1 2 11 1 6 2 1 1 5
10
15
Fig. 1. The microbial species and number of isolates originated from fish.
discarded and the same spin was repeated on the pellet. Remains of ethanol were removed, and the pellet was allowed to dry. Ten μl of 70% formic acid were added to the pellet by pipetting and vortexing. Then, 10 μl of acetonitrile were added. The suspension was centrifuged at 14,000 rpm for 2 min and 1 μl of the supernatant was transferred to the MALDI target. Once dry, every spot was overlaid with 1 μl of HCCA (2Cyano-3-(4-hydroxyphenyl) acrylic acid) (Bruker Daltonics, Germany) matrix and left to dry at room temperature before analysis. The spectra were generated by MALDI-TOF and analyzed with Microflex LT (Bruker Daltonics, Germany) instrument using Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC specific software. Criteria for a successful identification were within a confidence score of ≥2.0 for the species level and ≥1.7 for the genus level according to the manufacturer [25].
2.2. Identification of isolates with MALDI -TOF MS Biotyper 2.3. DNA analysis Altogether, 161 bacterial colonies were selected for further testing after storage. The isolates were transferred onto Tryptone soya agar at 37 °C for 24 h and a loopful of bacterial colonies was transferred into 300 μl of distilled water. After the addition of 900 μl of ethanol, the samples were centrifuged for 2 min at 14,000 rpm. The supernatant was
DNA was extracted using a Sigma kit (GenElute™ Bacterial Genomic DNA Kit, Sigma Aldrich, UK) from 24-h old bacterial culture following the manufacturer's instructions. Amplification reactions were carried out in 25 μl volumes containing 200 mM dNTPs, 1x DreamTaq buffer, 314
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Table 1 Comparison of identification of Pseudomonas spp. with MALDI-TOF MS Biotyper and 16S rDNA sequencing. Strain
Species
Concordance (%)
Sequence identity
MALDI
Score
AB1_7 AB1_8 AB1_9 AB1_11 AB1_12 AB2_1 AB2_2 AB2_3 AB2_4 AB2_5 AB2_6 AB2_7 AB2_8 AB2_9 AB2_10
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
99% 99% 99% 99% 99% 99% 99% 100% 100% 99% 99% 99% 99% 99% 99%
1472/1475 1472/1476 1447/1452 1473/1475 1473/1475 1459/1463 1475/1478 1478/1478 1478/1478 1473/1476 1471/1476 1466/1468 1472/1478 1472/1476 1467/1468
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
2.063 2.157 2.103 2.099 1.964 2.086 2.158 2.168 2.038 2.418 2.346 2.193 2.174/2.221 2.18 2.183
extremaustralis orientalis deceptionensis extremaustralis weihenstephanensis brenneri rhodesiae veronii silesiensis proteolytica kilonensis helmanticensis libanensis/gessardii orientalis helmanticensis
extremorientalis fluorescens taetrolens extremorientalis fragi frederiksbergensis Antarctica veronii mandelii proteolytica brassicacearum koreensis libanensi/gessardii synxantha proteolytica
Fig. 2. Dendrogram of selected Pseudomonas species created with MALDI-TOF MS Biotyper.
2.4. Data analysis
0.5 U DreamTaq DNA polymerase (Life technologies, USA, NY), 0.5 mM of the corresponding 16S rDNA sequencing primer [26], and 0.5 μL of non-diluted DNA. Conditions of PCR reactions were as follows: initial denaturation at 95 °C for 3 min and subsequent 35 cycles; denaturation at 95 °C for 30 s, annealing at the corresponding temperature for each primer set for 45 s, elongation at 72 °C for 90 s, and the final elongation at 72 °C for 10 min. PCR and sequencing reactions were carried out in the Biorad MJ mini thermal cycler (BioRad Corp., USA, CA). Primers 27F and 1492R [26] were used for PCR. PCR products were enzymatically purified by ExoI/FastAP (Life technologies, USA, NY). Sequencing was performed bi-directionally using primers 785F and 907R [25] using the Macrogen Inc. South Korea on Applied biosystems 3730XL and Big Dye 3.1 chemistry.
The acquired sequences were assembled and processed using the Seaview software [27,28]. Similarity of sequences to type the species was checked by BLASTn. The alignment was made using Clustal Omega [29]. Reference sequences of Pseudomonas type spp. were acquired from GenBank. A phylogenetic tree was constructed by maximum likelihood algorithm using PhyML [28] with following settings: GTR model, BioNJ starting tree, best of NNI and SPR tree searching, optimized across site rate variation, 1000 bootstraps.
3. Results and discussion Pseudomonas spp. were the most distributed microbial species isolated from fish in the present study. Meanwhile, other species included Aeromonas, Buttiauxella, Candida, Debaromyces, Enterobacter, 315
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Fig. 3. Maximum likelihood phylogenetic analysis of 15 Pseudomonas strains isolated from fish. Numbers above branches indicate bootstrap values (only ≥50 are shown). Green lines indicate identification by 16S rDNA and purple lines by MALDI TOF.
frequently isolated. The isolated microbial species are shown in Fig. 1. MALDI-TOF MS analysis is a method that has been used extensively for the identification of microbial species as it provides an opportunity to receive reliable results rapidly. The speed of identification is crucial
Jantinobacterium, Moellerella, Pantoea, Providencia, Rahnella, Serratia, Stenotrophonomonas and Yersinia intermedia. Pseudomonas koreensis (13 isolates), Candida pelliculosa (11 isolates), Pseudomonas fragi (10 isolates) and Pseudomonas proteolytica (9 isolates) were the most 316
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polyphasic attitude including morphological and physiological traits [49]. The incorporation of MALDI-TOF MS Biotyper to a polyphasic system could increase the speed and resolution of Pseudomonas spp. identification.
for diagnostic, especially in clinical settings [30–35]. For the identification, the Biotyper software compares the sample spectrum to its spectra database, generated by the characterized isolates [25]. MALDITOF MS analysis has not been applied for fish microbiota identification previously, however, this method provided an acceptable score for all isolates selected for identification. As a preliminary test of the applicability of MALDI-TOF mass spectrometry for the identification of Pseudomonas spp., a set of 27 strains were analyzed. The results of the identification with a log score are listed in Table 1. With the exception of isolate AB1_12, other Pseudomonas spp. were identified with a score above 2.00, which means high confidence identification to the spp. level. Altogether, 14 Pseudomonas spp. were identified with MALDI-TOF. The analyzed strains formed 3 clusters and the Pseudomonas spp. similarity is reflected in the MSP dendrogram (Fig. 2). A total of 15 Pseudomonas isolates previously identified by MALDITOF MS were analyzed by 16S rDNA amplicon sequencing. 16S rDNA sequencing may be applicable for bacterial species identification in various fields because of its highly discriminative power. Additionally, the method has been used for routine clinical diagnostics as well [36–38]. BLASTn analysis of 16S rDNA sequences (Table 1) showed 99 or 100% similarity for all species. Only 2 isolates revealed sequences identical to type species while others shared up to 6 mismatches. The general concept of species differentiation considers 95 or 97% similarity as a criterion for species delimitation, however between some types of Pseudomonas species only 5 or even less bases could make a difference (e.g. P. gessardi and P. libanensis differ by 3 bases). Pseudomonas is one of the most complex and diverse bacterial genera comprising more than 200 species described to date [12]. Several groups and sub-groups have been identified within this genus [39]. The phylogenetic analysis showed a clear taxonomic placement only for 13 out of 15 of Pseudomonas isolates in the present study. The dendrogram in Fig. 3 contains a comparison of all type species with at least 99% similarity to any isolate. There are at least 10 well defined clades. Identified clusters respect the general grouping but they are not in full congruence to previously published sub-groups of Pseudomonas spp. [40], however, clustering depends on all sequences in the collection. Fish isolates are apparently members of P. fluorescens, P. gessardii, P. fragi, P. koreensis and P. mandelii subgroups but no none from the P. aeruginosa group was identified. P. fluorescens was reported to be the a fish pathogen, while other isolated Pseudomonas spp. were expected to be non-harmful to fish [41–43]. Since human pathogenic Pseudomonas spp. were not isolated in the present study, our findings indicate that the fish collected are safe for consumption. P. aeruginosa was found in fish previously and is known to pose a threat to the human health [17,44–48]. As such, our report shows that an accurate identification of Pseudomonas spp. is important to ensure fish protection from bacterial infections and to evaluate the bacterial quality of fish and its safety for consumption. The isolate AB2_1 did not cluster with P. brenneri which was the most similar in sequence. MALDI-TOF MS Biotyper identified this isolate as P. frederiksbergensis which was clustered in a different branch than P. brenneri. AB2_8 felt to cluster among P. azotoformans strains despite the most similar species identified both by MALDI and 16S was P. libanensis. MALDI probably did not correctly identify the isolates AB1_8 and AB2_9 as P. fluorescens and P. synxantha, respectively, as the genetic analysis placed them to the cluster with P. orientalis. AB2_6 was the most similar to the type strain of P. kilonensis (5 bp diff, 0 gaps) but it was clustered with P. brassicacearum (7bp diff, 2 gaps) and congruently identified by MALDI. The accuracy of MALDI-TOF MS identification was found only in 6 out of 15 isolates. Isolates AB2_1 and AB2_8 should be processed by a deep phylogenetic analysis to confirm their status. Phylogeny of Pseudomonas spp. is very difficult, generally based on multilocus sequencing analysis of DNA (MLSA), but the correct identification of species requires a
4. Conclusion In this study, a total 161 colonies of microorganisms have been isolated from freshwater fish and identified with MALDI -TOF MS analysis. Thirteen Pseudomonas isolates were further confirmed by 16S rDNA gene sequencing and the isolates were identified as P. antarctica, P. brassicacearum, P. extremorientalis, P. frederiksbergensis, P. fluorescens, P. fragi, P. libanensis, P. koreensis, P. mandelii, P. proteolytica, P. synxantha, P. taetrolens and P. veronii. Overall, our study revealed a high discriminatory power of the mass spectra investigation and 16S rDNA gene sequencing technology for the identification of Pseudomonas spp. associated with freshwater fish. Conflicts of interest The authors declare that there is no conflict of interest. Acknowledgments The study was supported by the European Community project No 26220220180: Building Research Centre „AgroBioTech" and by the grants APVV-15-0544. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.04.024. References [1] W. Migula, Ubereinneues system der Bakterien, Arb, Bakteriol. Inst. Karlsruhe 1 (1984) 235–238. [2] P.H.A. Sneath, M. Stevens, M.J. Sackin, Numerical taxonomy of Pseudomonas based on published records of substrateutilization, Antonie Leeuwenhoek 47 (5) (1981) 423–448 https://doi.org/10.1007/bf00426004. [3] H. Oyaizu, K. Komagata, Grouping of Pseudomonas species on the basis of cellular fatty acid composition and the quinone system with special reference to the existence of 3-hydroxy fatty acids, J. Gen. Appl. Microbiol. 29 (1) (1983) 17–40 https://doi.org/10.2323/jgam.29.17. [4] M. Vancanneyt, S. Witt, W.R. Abraham, K. Kersters, H.L. Fredrickson, Fatty acid content in whole-cell hydrolysates and phosphor lipid fractions of pseudomonads: a taxonomic evaluation, Syst. Appl. Microbiol. 19 (4) (1996) 528–540 https://doi. org/10.1016/s0723-2020(96)80025-7. [5] Y. Anzai, H. Kim, J.Y. Park, H. Wakabayashi, H. Oyaizu, Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence, Int. J. Syst. Evol. Microbiol. 50 (4) (2000) 1563–1589 https://doi.org/10.1099/00207713-50-4-1563. [6] A.C. Parte, List of prokaryotic names with standing in nomenclature, Nucleic Acids Res. 42 (D1) (2014) D613–D616 https://doi.org/10.1093/nar/gkt1111. [7] N.J. Palleroni, Introduction to the family Pseudomonadaceae, in: A. Balows, H. Trüper, G.G.M. Dworkin, W. Harder, K.H. Schleifer (Eds.), The Prokaryotes, second ed., New York & Springer, USA, 1992, pp. 3071–3085. [8] A.E. Toranzo, B. Magariños, J.L. Romalde, A review of the main bacterial fish diseases in mariculture systems, Aquaculture 246 (1–4) (2005) 37–61 https://doi. org/10.1016/j.aquaculture.2005.01.002. [9] J.R. López, J.I. Navas, N. Thanantong, R. de la Herran, O.A.F. Sparagano, Simultaneous identification of five marine fish pathogens belonging to the genera Tenacibaculum, Vibrio, Photobacterium and Pseudomonas by reverse line blot hybridization, Aquaculture 324–325 (2012) 33–38 https://doi.org/10.1016/j. aquaculture.2011.10.043. [10] FAO/WHO, food and agriculture organization of the united nations/world health organization. Microbiological risk assessment series, Risk Characterization of Microbiological Hazards in Hood. Guidelines, vol. 17, WHO, Geneva, Switzerland, 2009, p. 116. [11] T. Sakata, Microflora of healthy animals, in: B. Austin, D.A. Austin (Eds.), Methods for the Microbiological Examination of Fish and Shellfish Chichester, Ellis Horwood Ltd., England, UK, 1989, pp. 141–163. [12] A. Pękala-Safińska, Contemporary threats of bacterial infections in freshwater fish, J. Vet. Res. 62 (3) (2018) 261–267. [13] A. Ashraf, E.I. Tawab, A. Ahmed, A.A. Maarouf, Nesma, M.G. Ahmed, Detection of Virulence factors of Pseudomonas species isolated from fresh water fish by PCR,
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