Pseudomonas sivasensis sp. nov. isolated from farm fisheries in Turkey

https://doi.org/10.1016/j.syapm.2020.126103Get rights and content

Abstract

A study of 91 isolates from fish farms in Turkey showed that isolates P7T, P11, P24b, P29, P72, P73 and P158 belonged to the genus Pseudomonas according to 16S rRNA nucleotide sequence analysis. The analysis of the sequences of the RNA polymerase sigma factor gene (rpoD) located these strains in the Pseudomonas fluorescens lineage of species within the P. fluorescens subgroup, close to the cluster composed of the species Pseudomonas grimontii, Pseudomonas marginalis and Pseudomonas panacis. Based on similarities in the 16S rRNA and rpoD gene sequences of three previously isolated strains from other origins (CCUG 57209, CCUG 62357 and W5.2-93) linked them to the same cluster. A polyphasic taxonomic approach including phenotypic characterization, fatty acid composition, and multilocus sequence analysis, together with whole-cell MALDI-TOF data, corroborated this assumption. The genome G+C mol% contents were 59.48 and 59.71, respectively. The average nucleotide indices based on BLAST analysis and the genome-to-genome distance calculation for the P7T and CCUG 57209 strains with their closest relative, P. grimontii, were 88.16–88.29% and 38.10–38.20%, respectively. These data confirm that isolates P7T, P11, P24b, P29, P72, P73, P158, CCUG 57209, CCUG 62357 and W5.2-93 represent a new species for which the name Pseudomonas sivasensis is proposed, with P7T as a type strain (=CCUG 74260T= and CECT30107T).

Introduction

Rainbow trout is one of the top cultured fish species because of its resistance to disease, variable water quality and stress factors compared with other cultured species. Similar to other countries, some of the most important bacterial fish pathogens, such us to genera such as genera Aeromonas, Vibrio, Flavobacterium, Streptococcus and Pseudomonas, can cause mass death of rainbow trout in aquaculture farms in Turkey, which limits fish production [4], [25], [31]. To identify and implement appropriate and adequate prevention measures, reduce fish mortality and to achieve effective vaccine production, the correct identification of the bacterial species present in the aquatic habitat is necessary.

The genus Pseudomonas includes a large number of species, more than 200, and is divided into 3 lineages and several groups and subgroups [14]. The P. fluorescens lineage comprises the P. fluorescens group of species, involved in plant growth promotion, plant disease, antibiotic resistance, and biocontrol activities, and shows high host diversity. This group is characterized by a large average genome size [33]. It is composed of 72 species that are phylogenetically organized into eight subgroups. One of these subgroups is the P. fluorescens subgroup, which exhibits a saprophytic lifestyle and is characterized by a large number of species (32). Most species in the subgroup have been recovered from water and plant surfaces [22]. Approximately half of the type strains representing this subgroup have been isolated from aquatic environments, such as drinking water reservoirs [9], natural mineral water [1], [5] and spring water [2], [3], which seems to indicate that members of the P. fluorescens group are probably typical inhabitants of unpolluted waters.

An initial screening of bacterial isolates was performed in water and fish samples of aquaculture farms of Turkey in the period of 2013–2018 (Kayseri, Sivas, Elazig). Seven strains were considered representatives of a putative new species because the similarity of their rpoD gene sequences to currently known Pseudomonas species type strains was below the threshold established for members of the same species in the genus [21]; these strains were then deeply analyzed taxonomically. Three other strains from different origins, including one from the collection in our laboratory and two from the culture collection of the University of Gothenburg (CCUG) that show high similarity of their gene sequences, were included in this study to place this potential new species in context. The genomes of two strains selected as representatives of this group were sequenced and analyzed to obtain genomic insights into the novel proposed species. The main objective of this study was to characterize and clarify the taxonomy of this previously undescribed putative new Pseudomonas species found in water samples and rainbow trout from fisheries in Turkey.

Section snippets

Bacterial isolation

Fish were sampled from rainbow trout farms with high production capacities established using spring water, stream water, and dammed lake water in four different regions of Turkey (Aegean, Central Anatolia, Black Sea, and Eastern Anatolia). Ninety-one samples of fish and pond water were randomly collected from fishes classified as asymptomatic (seemingly healthy), subclinical, clinical, or moribund each month during each sampling period from 2013 to 2017, in addition to two cases in 2018. The

Isolation

As shown in Table S1, the 10 studied strains were isolated from several different origins and dates of isolation. Three strains were isolated from pond water samples (strains P11, P72 and P73) and four strains from the yolk sac and surface of Oncorhynchus mykiss (rainbow trout) alevins (0.4 g average weight) with no apparent lesions according to an external and internal examination. No fish mortality was detected in the pond where sampling was performed (strains P7, P24b, P29 and P158). Three

Conclusion

The study confirms that ten strains from different countries, locations and dates of isolation conform to a homogeneous cluster of a new Pseudomonas species. The 16S rRNA nucleotide sequence permitted the positioning of the strains in the genus. WC-MALDI-TOF analysis revealed their corresponding group in the genus. The results of multilocus sequence analyses indicated that they did not belong to any already known species. The phenotypic and chemotaxonomic data showed differences from the most

Nucleotide sequence accession numbers

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this study are as follows: LR777868 to LR777875 (strains P. sivasensis P7T, P11, P24b, P29, P72, P73, P158, W5.2-93) for the 16S rRNA; LR777883 to LR777890 (strains P. sivasensis P7T, P11, P24b, P29, P72, P73, P158, W5.2-93) for the gyrB gene; LR777891 to LR777900 (strains P. sivasensis P7T, P11, P24b, P29, P72, P73, P158, CCUG 57209, CCUG 62357, W5.2-93) for the rpoB gene and LR777876 to LR777882 (strains P.

Acknowledgements

This research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [No: 118O420]. We are thankful to Bursa Uludag University for additional support. This research was approved by the Local Ethics Commission (report 2012-14-04).

The authors acknowledge the CCUG (Culture Collection of the University of Gothenburg) for providing the strains CCUG 57209 and CCUG 62357 and Dr. Sandra Mattijs of the Institut de Recherches Microbiologiques-Wiame, Campus du CERIA,

References (33)

  • J. Felsenstein

    Evolutionary trees from DNA sequences: a maximum likelihood approach

    J. Mol. Evol.

    (1981)
  • D. Garrido-Sanz et al.

    genomic and genetic diversity within the Pseudomonas fluorescens complex

    PLOS ONE

    (2016)
  • E.P. Ivanova et al.

    Pseudomonas extremorientalis sp. nov., isolated from a drinking water reservoir

    Int. J. Syst. Evol. Microbiol.

    (2002)
  • N.H. Khan et al.

    Isolation of Pseudomonas aeruginosa from open ocean and comparison with freshwater, clinical, and animal isolates

    Microb. Ecol.

    (2007)
  • N. Kimata et al.

    Pseudomonas aeruginosa isolated from marine environments in Tokyo Bay

    Microb. Ecol.

    (2004)
  • J. Lalucat et al.

    Genomics in bacterial taxonomy: impact on the genus Pseudomonas

    Genes

    (2020)
  • Cited by (11)

    • Pseudomonas sp. as the causative agent of anomalous blue discoloration in Brazilian fresh soft cheese (Minas Frescal)

      2021, International Dairy Journal
      Citation Excerpt :

      To reveal the genetic relationships and to identify a possible connection of the isolates identified here with strains characterised in previous studies as capable of producing blue discoloration, an in silico multi-locus sequence typing (MLST) was performed at pubMLST (https://pubmlst.org/organisms/pseudomonas-fluorescens), and a circular phylogenetic tree based Neighbor-Joining was generated using iTol v.4.2 (https://itol.embl.de/; Letunic & Bork, 2016). Furthermore, the genomes of the isolates A006 and B157 were related to the genomes of the all species type strains in the P. fluorescens phylogenetic subgroup available in database platforms (Duman et al., 2020; Gomila, Peña, Mulet, Lalucat, & García-Valdés, 2015). Relatedness was estimated, using several approaches: average nucleotide identity (ANI) based on BLASTN algorithm (ANIb); ANI based on the MUMMER ultra-rapid aligning tool (ANIm); tetranucleotide frequency correlation coefficients (TETRA); and, digital DNA:DNA hybridisation (DDH) by the genome-to-genome distance (GGDC) method.

    • Pseudomonas anatoliensis sp. nov and Pseudomonas iridis sp. nov. isolated from fish

      2021, Systematic and Applied Microbiology
      Citation Excerpt :

      Growth in the presence of NaCl (0–10% w/v) was tested in NB medium (Nutrient Broth, Difco). The fluorescence test was performed in King B medium as previously described [6,16]. Pyocyanin production was tested in King A medium (Himedia, M1543).

    • Employed Bacterial Species and Bacterial Cellulose (BC) Applications: The State of Play

      2022, Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology
    View all citing articles on Scopus
    View full text