Five P. salmonis isolates from Atlantic salmon in Chile provided by Prof. Sergio Marshall (Laboratory of Genetics and Molecular Immunology, Pontifical Catholic University of Valparaíso, Chile) were used in this study: one from the LF-89 genogroup (Psal-013) and four from the EM-90 genogroup (Psal-051, Psal-068, Psal-163, and Psal-182) ( Table 1 ). The bacteria were stored at −80°C with glycerol and 20% fetal bovine serum (FBS) stocks and routinely cultured on cysteine heart agar plates (CHAB) supplemented with bovine blood (Norwegian Veterinary Institute, Oslo, Norway) and in FN2 broth medium (Bravo and Martinez, 2016) with shaking at 100 rpm and incubated at 18°C.

To measure growth, the FN2 broth medium was used. Freshly grown bacterial cultures were adjusted to an initial OD of 0.02 at 600 nm and transferred into a 250 mL sterile flask with broth medium (25 mL). The flasks were maintained in agitation (100 rpm) at 18°C for 6 days for mono- and co-culture growth. The start point of the genogroup proportion for the co-cultures was a 1:1 ratio.

Each culture assay was performed in four independent replicates. Growth data were measured using a PV4 Visible Spectrophotometer (VWR) every 24 h. In addition, 1 mL samples were taken at the same time to extract total DNA, and at 48 h, 96 h, and 120 h a 500 μL sample was taken for further RNA extraction to monitor changes in P. salmonis gene expression during mono- and co-culture growth.

To check for contamination and viability, 100 μL of each liquid culture were replated on CHAB agar plates and incubated at 18°C for 10 days to evaluate colonies and further DNA extraction to perform an RFLP-PCR analysis specific for P. salmonis (Aravena et al., 2020).

All the samples taken from bacterial growth kinetics were first centrifuged at 10,000×g for 10 min. The pellets were then resuspended in lysis buffer (1 M Tris–HCl, 0.2 M EDTA, 10% Triton X-100) with lysozyme and incubated for 30 min at 37°C. Protein kinase (Qiagen, Hilden, Germany) was added and incubated at 56°C for 30 min. DNA was extracted using QIAcube and QIAGEN DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions and stored at −20°C until use. A NanoDrop™ ND‐1000 spectrophotometer (Thermo Scientific) was used to determine the concentration and quality of DNA.

To determine the growth rate of P. salmonis genogroups, DNA samples from the co-cultures were used in triplicate for qPCR assays using an AriaMx Real-Time PCR System (Agilent Technologies). Specific primers targeting each genogroup’s unique gene were designed ( Table 2 ). The PCR reaction mixture contained 10 μL of Maxima SYBR Green Master Mix (Thermo Scientific), 300 nM of each specific forward and reverse primer and 15 ng of DNA template in a total volume of 20 μL. The thermal profile started with an initial denaturation at 95°C for 3 min followed by 40 cycles at 95°C for 15 s and 60°C for 60 s, and a final melting curve between 60°C and 95°C. For all primers, efficiencies were established using the equation E = 10^(−1/slope). Relative quantification was estimated using threshold cycle (ct) values, normalizing the Ct of the target gene to the housekeeping gene for the same sample (at the same time), and then calculating the ratio between genogroups in fold changes of EM-90 compared to LF-89.

To evaluate the gene expression of target biomarkers in P. salmonis at 48 h, 96 h, and 120 h, 500 µL of each sample was centrifuged at 10,000×g for 10 min at 4°C to recover the pellet, which was used for RNA extraction using the Qiagen RNeasy® Mini Kit (Qiagen) according to the manufacturer’s protocol. A NanoDrop™ ND‐1000 spectrophotometer (Thermo Scientific) was used to determine the quality and quantify each RNA sample. Then cDNA was synthesized using the QuantiTect® Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions.

cDNA samples were used in three technical replicates for each of the four biological replicates. Briefly, for RT-qPCR MIX, 15 ng of cDNA was used for each sample along with 10 μL of Maxima SYBR Green Master Mix (Thermo Scientific) and 300 nM of forward and reverse primers in a total volume of 20 μL. The thermal profile started with an initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s, and a final melting curve between 60°C and 95°C.

Specific primers were used to establish key gene expression changes in the co-cultures compared to the monoculture using glyA as the normalizer ( Table 2 ). For all primers, efficiencies were established using the equation E = 10^(−1/slope). Relative gene expression was estimated using threshold cycle (Ct) values and the Livak 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Frozen stocks at −80°C from the five isolates were streaked onto cysteine heart agar plates (CHAB) supplemented with bovine blood (Norwegian Veterinary Institute, Oslo, Norway) and incubated at 18°C until growth. Single colonies of the isolates were used to inoculate 3 mL of FN2 broth, which was then incubated at 18°C. After the cultures reached the exponential phase, the optical density (OD) of each culture at 600 nm was adjusted to 0.02 to be used as an inoculum for the 96 well microplates to perform a biofilm formation assay.

Three independent experiments were performed by inoculating eight wells per isolate and co-culturing with 150 µL of the bacterial suspension in FN2 broth. FN2 medium without bacteria was used as the negative control. The microplates were incubated at 18°C with shaking at 100 rpm. After 120 h, all the medium and planktonic bacteria were carefully removed from the inoculated and control wells. Bacteria that adhered to the wells of the microplates were stained with 180 µl of crystal violet solution (0.1%) at room temperature for 10 min. The crystal violet (CV) solution was removed, and the walls were rinsed thoroughly with sterile distilled water. The microplates were turned upside down and dried for 1 h. For CV solubilization, absolute ethanol (200 µL/well) was added and left at room temperature for 10 min. The resulting solutions were homogenized by repeated pipetting before reading the microplates at 620 nm using a PV4 Visible Spectrophotometer (VWR).

One isolate from each genogroup was selected for in vivo cultivation, Psal-013 (LF-89) and Psal-182 (EM-90). Each isolate was cultivated in FN2 broth medium at 18°C with shaking (100 rpm) until obtaining an OD_600nm of 0.2 at the beginning of the exponential phase. Thereafter, nine semi-permeable implants were made using 20 cm length dialysis tubes with a 12 kDa–14 kDa molecular weight cut-off (Spectrum Laboratories Inc., LA, CA, USA) as described by Colquhoun and Sørum (1998). One end was sealed and autoclaved in PBS 1X. Then, each tube was inoculated with a total volume of 5 mL for every bacterial culture (0.25 mL + 0.25 mL for the co-cultures) and sealed with a triple knot. Three main groups of bacterial cultures were used: Psal-013 alone, Psal-182 alone, and Psal-013 (0.25 mL) with Psal-182 (0.25 mL) mixed.

For in vivo culturing of P. salmonis isolates, nine Atlantic salmon (S. salar) post-smolts with an average weight of 1 kg were implanted with one dialysis tube (three fish per group). Fish were kept at the Norwegian Institute for Water Research (NIVA, Solbergstrand, Norway) throughout the experiment. The implants were carefully inserted into the abdominal cavity of the fish through a small 4 cm incision along with a pit-tag for marking and later identification. The cut was then sewn along with three to four sutures through all layers of the tissue. The fish were transferred to a common tank containing seawater at an average temperature of 12.5°C. After 6 days, and before any symptoms could be detected, the fish were euthanized with an overdose of benzocaine (Benzoak 200 mg/mL) for implant removal. The implants were washed with PBS 1×, and the bacterial culture was removed and stored on ice until further use. Norwegian Food Safety Authorities (FOTS ID 26316) approved the animal trial in accordance with the laws and regulations for experiments on live animals in the EU (Directive 2010/637EU) and Norway (FOR-2015-06-18-761).

From every fish, the entire content of the dialysis tube was aseptically collected and centrifuged at 5,000×g for 10 min at 4°C for pellet collection. Total RNA was extracted from all samples using the Qiagen RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. QIAseq FastSelect−5S/16S/23S (Qiagen) was used according to the manufacturer’s instructions for rRNA depletion. The concentration and quality were measured using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific). Library preparation and RNA-seq were performed at the Norwegian Sequencing Center (UiO, Norway) using TruSeq Stranded mRNA library prep and Illumina NovaSeq SP System (150 bp paired-end RNA sequencing).

To process the raw files, the Orion High Performance Computing Cluster at the Norwegian University of Life Sciences (NMBU) was used, according to Morales-Lange et al. (2021). Raw reads from the RNA sequencing were trimmed using BBDuk (v34.56). Then, the clean reads were aligned to P. salmonis genome Psal-013 (ASM970875v1—RefSeq assembly accession: GCF_009708755.1) or Psal-182 genome (ASM970941v1—RefSeq assembly accession: GCF_009709415.1) using HISAT (v2.1.0). The fragments mapped were counted with featureCounts (v1.4.6-1) and differentially expressed genes (DEGs) between groups were estimated using SARTools (v1.7.3) package in Rstudio (v4.2.0). Significant DEGs were identified when the adjusted p-value (padj) was <0.05.

Data from growth rate, gene expression of target biomarkers and biofilm production were analysed and visualized using GraphPad Prism (v8.0.1). One-way ANOVA and Tukey’s multiple comparison test were used to determine significant differences (p <0.05). The growth rate data were correlated using the Corrplot package (v0.84) (Wei and Simko, 2017) in Rstudio (v4.2.0). Correlations were considered significant when the p-value was <0.05.

Growth data showed that in monocultures, the LF-89-like isolate (Psal-013) exhibited the lowest growth of all P. salmonis isolates, reaching an OD_{600 nm} of 3.30 after 120 h ( Figures 1A–D ). In comparison, the EM-90 Psal-051 isolate reached an OD_{600 nm} of 5.46 after 120 h ( Figure 1A ). Psal-051 exhibited the shortest lag phase of 24 h among the remaining EM-90 isolates. Interestingly, the co-culture of Psal-013 and Psal-051 showed a decrease in growth after 120 h, reaching an OD_{600 nm} of 4.42.

The monoculture of EM-90 isolate Psal-068 reached the logarithmic phase after 72 h of growth ( Figure 1B ). Psal-068, thereby, had the longest lag phase of all P. salmonis isolates, with a peak growth of 5.01 measured at OD_{600 nm} after 120 h. Similar growth was observed when Psal-068 was co-cultured with Psal-013, following the same curve as Psal-068 alone ( Figure 1B ).

Moreover, the monoculture growth of the EM-90 isolate Psal-163 ( Figure 1C ) reached the logarithmic phase after 72 h, and the peak of growth was measured as 6.18 at OD_{600 nm}. In the co-culture with Psal-013, no differences were observed in the growth curve ( Figure 1C ).

The EM-90 isolate Psal-182 exhibited the fastest logarithmic growth, reaching the stationary phase after 72 h, and a peak growth of 6.06 measured at OD_{600 nm} after 120 h ( Figure 1D ). In the co-culture experiment with Psal-013, the lag phase was shorter and after 96 h reached a growth of 5.20 at OD_{600 nm}, a decline phase started with an OD_{600 nm} of 3.29. For the re-plating results, all cultures were viable, and no contamination was detected at any time (data not shown).

After determining the effect of co-culturing the LF-89 isolate (Psal-013) with different isolates from EM-90, the growth ratio of the two genotypes in the co-culture was investigated. The results displayed in Figure 2A shows that all EM-90 isolates exhibited improved growth at the expense of LF-89. The Psal-013/051 co-culture showed the lowest difference in fold change. Co-cultures of Psal-013/068 and Psal-013/163 exhibited a similar trend, with a peak in growth after 96 h, with significant differences in time. However, in the co-culture of Psal-013 and Psal-182, the peak was reached at 72 h, after which the ratio between the genogroups began to decline.

When investigating the correlation of all co-cultures, a positive correlation between Psal-013/068 and Psal-013/163 was found ( Figure 2B ). This finding suggests a similar growth ratio for EM-90 and LF-89 in these two co-cultures. Furthermore, the correlation of co-cultures by time showed a positive correlation between 96 h and 120 h ( Figure 2C ), which means that the ratio at 96 h and 120 h remained similar.

As the establishment of biofilms may influence bacterial pathogenesis, it was of interest to explore differences in biofilm formation between the P. salmonis LF-89 and EM-90 strains, and when the two genotypes were co-cultivated.

The results ( Figure 3 ) showed that the monoculture of Psal-013 (LF-89-like) had the lowest biofilm production (OD_{620 nm} = 0.123 ± 0.08). While Psal-182 reached a higher biofilm production in co-cultures (OD_{620 nm} = 0.70 ± 0.22) than in monoculture (OD_{620 nm} = 0.60 ± 0.08) ( Figure 3D ). Psal-051 in monocultures, reached a higher production (OD_{620 nm} = 0.507 ± 0.12) than in co-cultures (OD_{620 nm} = 0.45 ± 0.19), but without significant differences ( Figure 3A ). Psal-068 ( Figure 3B ) significantly increased biofilm production from monoculture (OD_{620 nm} = 0.17 ± 0.07) to co-culture (OD_{620 nm} = 0.27 ± 0.08). Although, Psal-163 showed the same trend ( Figure 3C ), a lower biofilm production in monoculture (OD_{620 nm} = 0.41 ± 0.10) than in co-cultures (OD_{620 nm} = 0.48 ± 0.15), there was no significant differences.

Co-cultivation of P. salmonis LF-89 and EM-90 strains affected bacterial growth phenotypes. Therefore, the next step was to investigate the effects of co-cultivation on gene expression. First we compared the expression of four marker genes (flaA, relA, luxR and cheA genes) after 120 h of growth. flaA encodes the major subunit of flagellin in flagella, while relA activates (p)ppGpp, which is involved in regulating growth and different stress responses in bacteria, luxR is related to communication through N-acylhomoserine lactone (AHL) quorum-sensing (QS) signaling and cheA to chemotaxis.

For flagellin (flaA) ( Figure 4A ), Psal-182 alone showed the highest expression at 120 h compared with all other cultures, with a significant difference from the co-culture with Psal-013. In contrast, Psal-068 and Psal-013 co-culture showed the lowest expression among all cultures, with a significant difference compared to Psal-068 alone. Psal-051 and Psal-163 in the co-culture presented a similar level of expression to that of Psal-013 grown alone.

RelA gene expression ( Figure 4B ) was not significantly downregulated in the Psal-051 monoculture. For luxR ( Figure 4C ), a significant difference was observed in the high expression during the co-culture of Psal-051 and Psal-013 compared to Psal-051 monoculture. However, no expression was observed for the LF-89 isolate Psal-013 alone. Moreover, for Psal-068 and Psal-163, there were no significant differences related to their co-cultures.

Psal-013/182 co-culture showed the highest expression of cheA but was not significantly higher than that in the Psal-182 monoculture ( Figure 4D ). In addition, Psal-163 in mono- and co-cultures showed no differences in expression related to Psal-013 or between them. In contrast, Psal-051 co-cultured with Psal-013 showed significantly higher expression than Psal-051 monoculture. Finally, the co-culture of Psal-013/068 showed the lowest expression among all the cultures, with a significant difference from the Psal-168 monoculture.

RNA-seq analysis was performed from the co-cultivation of P. salmonis genotypes in vivo (within the dialysis bag in the fish). The DEG distribution from each pairwise comparison is shown in Table 3 (co-culture vs. Psal-182, co-culture vs. Psal-013, and Psal-182 vs. Psal-013). The metadata (normalized gene expression, description, and statistical analysis) are listed in Table S1 .

Of the 12 upregulated DEGs in the co-culture compared to Psal-182, 11 belonged to the chromosome and one to plasmid 4. Moreover, of the four downregulated DEGs, three corresponded to the chromosome and one to plasmid 4. In the co-culture, compared to Psal-013, 10 upregulated DEGs corresponded to chromosome and seven to plasmid 1. The unique downregulated DEG belongs to chromosome ( Table S1 ). Psal-182 monoculture compared to Psal-013 showed seven upregulated DEGs belonging to the chromosome, two to plasmid 4, and one to plasmid 2. In the same pairwise comparison, six of the 14 downregulated DEGs belonged to the chromosome, three to plasmid 4, four to plasmid 2 and one to plasmid 1 ( Table S1 ).

Regarding Hypothetical Protein (HP), among the four downregulated DEGs detected in co-cultures compared to Psal-182, two were identified as HP. Similarly, in co-cultures compared with Psal-013, seven upregulated DEGs were identified as HP. In addition, in the Psal-182 monoculture compared to Psal-013, two of the 10 upregulated DEGs were identified as HP and eight out of 14 downregulated DEGs were identified as HP.

During co-culture, compared to Psal-182, the most upregulated gene was IS30 family transposase (Psal182_RS17815) located in plasmid 4, with 3.641 of fold change, followed by IS4 family transposase (helix-turn-helix domain-containing protein) (Psal182_RS12950) 3.478-fold change, IS3 family transposase (Psal182_RS02820) 3.358-fold change, DNA transposase (Psal182_RS01495) 3.085-fold change, IS30 family transposase (Psal182_RS12275) 2.966-fold change, flagellar protein export ATPase FliI (Psal182_RS02060) 2.547-fold change, and flagellar hook-associated protein FlgK (Psal182_RS10205) 2.114-fold change. The downregulated DEGs were P-II family nitrogen regulator (Psal182_RS15510) with a −2.205 of fold change and ATP-dependent Clp protease proteolytic subunit (Psal182_RS08200) with a −1.521-fold change ( Figure 5A ).

Compared to Psal-013, during co-culture, the most upregulated gene was the type II toxin–antitoxin system RelE/ParE family (Psal013_RS16670) with 3.558-fold change, followed by HigA family addiction module antitoxin (Psal013_RS16660) with 2.581-fold change, flagellar hook-associated protein FlgK (Psal013_RS06465) with 2.522-fold change, NAD(P)-dependent alcohol dehydrogenase (Psal013_RS16675) with 2.354-fold change, thiol reductant ABC exporter subunit CydD (Psal013_RS11795) with 2.268-fold change, flagellar protein export ATPase FliI (Psal013_RS14360) with 2.22-fold change, and cadherin-like domain-containing protein (Psal013_RS10175) with 2.205-fold change. In contrast, the only downregulated DEG was the ATP-dependent Clp protease proteolytic subunit (Psal013_RS08085) with a fold change of −1.767 ( Figure 5B ).

In Psal-182 in vivo monocultures compared to Psal-013, the upregulated genes were IS4 family transposase (Psal182_RS12950) with 4.991 of fold change, followed by IS30 family transposase (Psal182_RS17815) (from plasmid 4) 4.86-fold change, flagellar protein export ATPase FliI (Psal182_RS02060) 3.28-fold change, IS30 family transposase (Psal182_RS12275) 2.809-fold change, Transposase DDE domain (Psal182_RS01495) 2.403-fold change, FAD-dependent 5-carboxymethylaminomethyl-2-thiouridine(34) oxidoreductase MnmC (Psal182_RS09240) 1.85-fold change, ABC transporter permease (Psal182_RS11665) 1.609-fold change, and IS30 family transposase (Psal182_RS17360) (located in plasmid 2) 1.552-fold change. The downregulated DEGs were AAA family ATPase (Psal182_RS17775) with a −7.26 fold change, followed by class I SAM-dependent methyltransferase (Psal182_RS01935) with a −2.903-fold change, Smc ATPase (Psal182_RS02460) −2.405-fold change, Protein kinase (Psal182_RS16425) with a−2.225-fold change, and 50S ribosomal protein L35 (Psal182_RS02405) with a −2.087-fold change ( Figure 5C ).