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. 2023 May 17;11(5):1320.
doi: 10.3390/microorganisms11051320.

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri

Nadia Stoppani  1 , Silvia Colussi  1 , Paolo Pastorino  1 , Marino Prearo  1 , Simona Sciuto  1 , Ilhan Altinok  2 , Rafet Çağrı Öztürk  2 , Mustafa Ture  3 , Ana Isabel Vela  4 , Maria Del Mar Blanco  4 , Charalampos Kotzamanidis  5 , Konstantina Bitchava  6 , Andigoni Malousi  7 , Lucio Fariano  8 , Donatella Volpatti  9 , Pier Luigi Acutis  1 , Jose Francisco Fernández-Garayzábal  4
Affiliations

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri

Nadia Stoppani et al. Microorganisms. .

Abstract

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

Keywords: 16S-23S internal transcribed spacer region; Lactococcus garvieae; Lactococcus petauri; diagnostic technique; genome; lactococcosis.

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Conflict of interest statement

The authors declare no conflict of interest or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Multiple 16S-23S ITS sequences alignment of the L. garvieae and L. petauri control strains used in this study (accession number in parentheses). Polymorphism positions refer to the strain L. garvieae LMG9472 (accession number HM241914.1).
Figure 2
Figure 2
Evolutionary relationship of taxa based on an ITS analysis. The neighbor-joining method was used to infer the evolutionary history. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 22 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 535 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. Lactococcus lactis strain KCTC 3768 was used as the outgroup. Strains reported in the tree as L. petauri Turkey, L. petauri Greece, and L. petauri Spain are each one representative of 20 different clinical strains per country. The L. garvieae Italy strain reported in the tree is representative of 22 different clinical strains.
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