Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection

David Pérez-Pascual; Sol Vendrell-Fernández; Bianca Audrain; Joaquín Bernal-Bayard; Rafael Patiño-Navarrete; Vincent Petit; Dimitri Rigaudeau; Jean-Marc Ghigo

doi:10.1371/journal.ppat.1009302

. 2021 Jan 29;17(1):e1009302. doi: 10.1371/journal.ppat.1009302

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection

David Pérez-Pascual ^1,^*, Sol Vendrell-Fernández ¹, Bianca Audrain ¹, Joaquín Bernal-Bayard ^1,^¤, Rafael Patiño-Navarrete ², Vincent Petit ³, Dimitri Rigaudeau ⁴, Jean-Marc Ghigo ^1,^*

Editor: Karen Guillemin⁵

PMCID: PMC7875404 PMID: 33513205

Abstract

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.

Author summary

The protection provided by host commensal microbiota against pathogens is a long-known phenomenon fostering the notion that introducing beneficial bacteria could reduce or prevent infections. However, the identification of such protective microorganisms is hampered by the poor reproducibility and relevance of current in vivo models. We developed procedures to raise germ-free rainbow trout larvae to study the determinants of microbiota-associated resistance to infection. We showed that the fish pathogen Flavobacterium columnare rapidly kills infected germ-free but not conventional larvae. We then re-colonized germ-free larvae with bacteria originating from cultured fish microbiota and identified two bacterial species providing full protection against infection. Our approach constitutes a rational and potentially high throughput in vivo strategy to study host-pathogen interactions and resistance to infection in fish. The identification of probiotic bacteria protecting rainbow trout and potentially other fish against F. columnare could also contribute to improve aquaculture sustainability and health.

Introduction

As wild fish stock harvests have reached biologically unsustainable limits, aquaculture has grown to provide over half of all fish consumed worldwide [1]. However, intensive aquaculture facilities are prone to disease outbreaks and the high mortality rate in immunologically immature juveniles, in which vaccination is unpractical, constitutes a primary bottleneck for fish production [2–4]. These recurrent complications prompt the prophylactic or therapeutic use of antibiotics and chemical disinfectants to prevent fish diseases [5,6] but may lead to final consumer safety risks, environmental pollution and spread of antibiotic resistance [7]. In this context, the use of bacterial probiotics to improve fish health and protect disease-susceptible juveniles is an economic and ecological sensible alternative to antibiotic treatments [8,9].

Probiotics are live microorganisms conferring health benefits on the host via promotion of growth, immuno-stimulation or direct inhibition of pathogenic microorganisms [10,11]. The native host microbiota plays a protective role against pathogenic microorganisms by a process known as colonization resistance [12,13]. In fish, the endogenous microbial community, whether residing in gastrointestinal tract or in the fish mucus, was early considered as a source of protective bacteria [14–18]. However, selection of probiotic bacteria is often empirical or hampered by the poor reproducibility of in vivo challenges, frequently performed in relatively uncontrolled conditions with high inter-individual microbial compositions [15,19].

To improve evidence-based identification of fish probiotics and their efficacy in disease prevention, the use of germ-free (GF) or fully controlled gnotobiotic hosts is a promising strategy [20,21]. In addition to laboratory fish models such as zebrafish (Danio rerio) [22–24], several fish species have been successfully reared in bacteria-free conditions to test probiotic-based protection against pathogenic bacteria, including Atlantic cod (Gadus morhua) [25], Atlantic halibut (Hippoglossus hippoglossus) [26], and European sea bass (Dicentrarchus labrax) [19] (for a review, see [27]).

Salmonids, especially rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), are economically important species, whose production in intensive farming is associated with increased susceptibility to diseases caused by viruses, bacteria and parasites [28]. Here we studied the probiotic potential of endogenous members of the rainbow trout microbiota to protect against infection by Flavobacterium columnare, a fresh-water fish pathogen causing major losses in aquaculture of fish such as Channel catfish, Nile tilapia and salmonids [29]. We developed a new protocol to rear GF trout larvae and showed that GF larvae were extremely sensitive to infection by F. columnare. We then identified two bacterial species originating either from the trout microbiota (a commensal Flavobacterium sp.) or the zebrafish microbiota (Chryseobacterium massiliae) that fully restored protection against F. columnare infection. Our in vivo approach opens perspectives for the rational and high throughput identification of probiotic bacteria protecting rainbow trout and other fish against columnaris disease. It also provides a new model for the study of host-pathogen interactions and colonization resistance in a relevant teleostean fish model.

Results

Germ-free trout show normal development and growth compared to conventional larvae

To produce microbiologically controlled rainbow trout and investigate the potential protection conferred by endogenous or exogenous bacteria against incoming pathogens, we produced (GF) trout larvae by sterilizing the chorion of fertilized eggs with a cocktail of antibiotics and antifungals, 0.005% bleach and a iodophor disinfection solution. GF eggs were then kept at 16°C under bacterial-free conditions and both conventional (Conv) and treated eggs hatched spontaneously 5 to 7 days after reception, indicating that the sterilization protocol did not affect the timing of hatching. However, hatching percentage was 72 ± 5.54% for sterilized eggs versus 48.6 ± 6.2% for non-treated. Once hatched, all larvae were transferred into vented-cap cell culture flasks containing fresh sterile water without antibiotics renewed every 48 hours (h). GF and Conv fish relied on their vitellus reserves until 20 days post-hatching (dph) after which they were fed with sterilized fish food powder every 48 h (Fig 1). Sterility tests were performed at 24 h, 7 days and 21 days post-sterilization treatment and before each water change until the end of the experiment (35 dph) (S1 Fig). To test the physiological consequences of raising GF larvae, we compared the growth of Conv and GF larvae reared from the same batch of fertilized eggs and observed no significant difference in standard body length (2.51 ± 0.24 cm vs. 2.58 ± 0.21 cm) or weight (1.17 ± 0.20 g vs. 1.17 ± 0.10 g) at 35 dph for Conv and GF, respectively (S2 Fig). To compare Conv and GF trout anatomy, we developed an approach combining iDISCO solvent-based method to generate transparent fish tissue and lightsheet 3D imaging of the whole body. This analysis did not reveal any anatomical differences at 21 dph, even regarding organs in direct contact with fish microbiota such as gills (Fig 2D and 2I) and intestine (Figs 2C and 2H and S3). No difference was seen in other organs potentially influenced by gut-microbiota such as the brain (Fig 2A and 2F), spleen (Fig 2B and 2G) and head kidney (Fig 2E and 2J) [30]. These results suggested that the natural microbiota had no major macroscopic impact on fish growth, development or anatomy at this stage of rainbow trout development in our rearing conditions.

Fig 1 — Eyed eggs were sterilized 5 days before hatching and kept in sterile, autoclaved mineral water at 16°C in Petri dishes until hatching. Once hatched (0 day post hatching = dph), rainbow trout larvae were transferred into vented cap cell culture flasks for the duration of the experiment. Larvae were fed every 2 days with sterile powder food from 21 dph until the end of the experiment; water was renewed 30 minutes after feeding. To test the protective effect of potential probiotic strains, larvae were re-conventionalized by one or several commensal bacteria diluted in water at 22 dph. Pathogenic bacteria were added to the water at 24 dph for 24 h and then larvae were washed with fresh sterile water. Survival after infection was monitored twice per day.

Fig 2 — 3D deep imaging of whole trout body corresponding to autofluorescence signal acquired by lightsheet microscopy after novel fish clearing processing. Selected optical sections of 21 dph were presented for Conv (A, B, C D and E) and GF (F, G, H, I and J) rainbow trout larvae. Brain (A and F), spleen (black arrow in B and G), gut (C and H) (see also S3 Fig), gills (black arrows in D and I), and head kidney (E and J). Images representative of two different fish per condition.

Identification of susceptibility to fish pathogens in germ-free but not conventional trout larvae

To identify bacterial pathogens able to infect GF rainbow trout larvae by the natural infection route, we exposed the 24 dph larvae for 24 h to 10⁷ colony forming units (CFU)/ml of several trout bacterial pathogens, including Flavobacterium psychrophilum strain THCO2-90, F. columnare strain Fc7, Lactococcus garvieae JIP 28/99, Vibrio anguillarum strain 1669 and Yersinia ruckeri strain JIP 27/88 [31]. Larvae were then washed with sterile water, renewing 90% of the infection water three times and kept at 16°C under sterile conditions. Among all tested pathogens, only F. columnare strain Fc7 led to high and reproducible mortality of GF trout larvae within 48 h post-exposure (Fig 3). In contrast, Conv larvae reared from non-sterilized eggs survived F. columnare strain Fc7 infection under tested conditions (Figs 4A and S4). Histological analysis performed at 25 dph (24 h post infection) on GF and Conv larvae did not show any sign of intestinal damage (S5 Fig). However, we observed an increase in goblet cells number in infected vs non-infected GF larvae, whereas Conv infected larvae showed the opposite phenotype when compared to non-infected Conv larvae (S5 Fig).

Fig 3 — Kaplan-Meier graph of GF larvae survival after bath exposure to F. *psychrophilum* strain THCO2-90, F. *columnare* strain Fc7, L. *garvieae* strain JIP 28/99, V. *anguillarum* strain 1669 and Y. *ruckeri* strain JIP 27/88. Mean and SD plot representing average survival percentage of fish for 10 days after exposure to different pathogenic microorganisms. For each condition n = 10 larvae. All surviving fish were euthanized at day 10 post-infection. Asterisks indicate significant difference from non-infected population (**p<0.01; ***p<0.001; ****p<0.0001).

Fig 4 — A: F. *columnare* strain Fc7 kills GF but not Conv rainbow trout. Mean and SD plot representing average day post-infection at which infected fish die. For each condition n = 10 larvae. All surviving fish were euthanized at day 10 post-infection. Asterisks indicate significant difference from non-infected population (****p<0.0001). B: GF trout larvae exposed to water used to raise Conv fish at 22 dph show similar survival rates to F. *columnare* infection than Conv trout larvae. C: The 11 strains identified from Conv fish microbiota were added to rainbow trout larvae at 22 dph, followed by F. *columnare* infection at 24 dph. This bacterial mixture is able to protect re-conventionalized larvae from infection. For each condition n = 10 larvae. All surviving fish were euthanized at day 10 after infection (****p<0.0001).

Conventional rainbow trout microbiota protects against F. columnare infection

Considering the high sensitivity of GF but not Conv trout larvae to infection by F. columnare Fc7, we hypothesized that resistance to infection could be provided by some components of the Conv larvae microbiota. To test this, we exposed GF rainbow trout larvae to water from Conv larvae flasks at 22 dph. Re-conventionalized (Re-Conv) rainbow trout larvae survived as well as Conv larvae to F. columnare Fc7 infection, whereas those maintained in sterile conditions died within the first 48h after infection (Fig 4B). These results suggested that microbiota associated with Conv rainbow trout provide protection against F. columnare Fc7 infection. To identify culturable species potentially involved in this protection, we plated bacteria recovered from 3 whole Conv rainbow trout larvae at 35 dph on various agar media. 16S rRNA-based analysis of each isolated morphotype led to the identification of 11 different bacterial strains corresponding to 9 different species that were isolated and stored individually (Table 1).

Table 1. The 11 strains isolated from Conv rainbow trout larvae.

Bacterial strains isolated from trout microbiota
Aeromonas rivipollensis 1 strain 4512
Pseudomonas helmanticensis strain 4513
Aeromonas rivipollensis 2 strain 4514
Pseudomonas baetica strain 4515
Aeromonas hydrophila strain 4516
Flavobacterium plurextorum 1 strain 4517
Acinetobacter sp. strain 4518
Flavobacterium plurextorum 2 strain 4519
Delftia acidovorans strain 4465
Flavobacterium sp. strain 4466
Pseudomonas sp. strain 4520

Open in a new tab

We then re-conventionalized GF rainbow trout larvae at 22 dph with an equiratio mix of all 11 identified bacterial strains (hereafter called Mix11), each at a concentration of 5.10⁵ CFU/ml. After exposure to F. columnare strain Fc7, these Re-Conv^Mix11 larvae survived as well as Conv fish (Fig 4C), demonstrating that the Mix11 isolated from the rainbow trout microbiota recapitulates full protection against F. columnare infection observed in Conv larvae.

Resistance to F. columnare infection is conferred by one member of the trout microbiota

To determine whether some individual members of the protective Mix11 could play key roles in infection resistance, we mono-re-conventionalized 22 dph GF trout by each of the 11 isolated bacterial strains at 5.10⁵ CFU/ml followed by challenge with F. columnare Fc7. We found that only Flavobacterium sp. strain 4466 restored Conv-level protection, whereas the other 10 strains displayed no protection, whether added individually (Fig 5A) or as a mix (Mix10 in Fig 5B). To evaluate the colonization of gastrointestinal tract by Flavobacterium sp. strain 4466 and/or F. columnare Fc7, we plated intestines from mono-reconventionalized fish 24 hours after infection in TYES agar after dissection in sterile conditions. Interestingly, whereas both Flavobacterium sp. strain 4466 and F. columnare Fc7 were able to successfully colonize the gut of mono-exposed rainbow trout (Fig 5C), we only detected Flavobacterium sp. after trout’s infection by F. columnare Fc7 (Fig 5C), suggesting a potential competition between both bacterial species. Consistently, although cell-free spent supernatant of Flavobacterium sp. strain 4466 showed no inhibitory activity against F. columnare Fc7 in an overlay assay (Fig 6A), Flavobacterium sp. strain 4466 colony growth inhibited the growth of F. columnare Fc7 (Fig 6B) and of all tested F. columnare strains (Fig 6C), suggesting a potential contact dependent inhibition. We identified a cluster of 12 genes potentially associated to this phenotype in the Flavobacterium sp. strain 4466 genome (tssB, tssC, tssD, tssE, tssF, tssG, tssH, tssI, tssK, tssN, tssP and tssQ) characteristic of type 6 secretion system (T6SS), T6SSⁱⁱⁱ, a contact-dependent antagonistic system only present in phylum Bacteroidetes [32]. To improve the taxonomic identification of the protective Flavobacterium isolated from the trout larvae microbiota, we performed whole genome sequencing followed by Average Nucleotide Identity (ANI) analysis. We determined that despite similarity with Flavobacterium spartansii (94.65%) and Flavobacterium tructae (94.62%), these values are lower than the 95% ANI needed to identify two organisms as the same species [33]. Furthermore, full-length 16S rRNA and recA genes comparisons also showed high similarity with F. spartansii and F. tructae, however, the obtained values were also below the 99% similarity threshold required to consider that two organisms belong to the same species (S1 Table). Similarly, a maximum likelihood based phylogenetic tree (S6 Fig) generated from sequences of 15 bacterial strains from the Flavobacterium genus revealed that the sequence of Flavobacterium sp. strain 4466 clustered with sequences of F. spartansii and F. tructae, but did not allow the identification of Flavobacterium sp. strain 4466 at species level.

Fig 5 — A: The 11 species isolated from Conv fish microbiota (Table 1) were added individually to rainbow trout larvae at 22 dph, followed by F. *columnare* Fc7 infection at 24 dph. From the 11 different strains, only *Flavobacterium* sp. strain 4466 protected re-conventionalized larvae from infection. B: Mix11, Mix10 (mix of all identified strain with the exception of *Flavobacterium* sp. strain 4466), were added to rainbow trout larvae at 22 dph, followed by F. *columnare* infection at 24 dph. Mix11 protected re-conventionalized larvae from infection, whereas Mix10 did not. For each condition n = 10 larvae. All surviving fish were euthanized at day 10 after infection. C: CFU/mL recovered from dissected intestines from GF fish exposed to F. *columnare* Fc7, *Flavobacterium* sp. strain 4466 or both, 24 hours post-infection. (****p<0.0001).

Fig 6 — A: lack of F. *columnare* Fc7 growth-inhibition after adding 5 μl of *Flavobacterium sp*. culture supernatant. B: Halo of F. *columnare* FC7 growth inhibition surrounding *Flavobacterium* sp. colony on a F. *columnare* strain Fc7 overlay. C: Halo of growth inhibition of F. *columnare* ALG-00-530, IA-S-4, and Ms-Fc-4. The agar overlay technique was performed by spreading F. *columnare* bacterial suspension on soft-agar solution over TYES agar, and then spotting 5 μl of an overnight culture of *Flavobacterium* sp. strain 4466. Incubation was performed at 28°C for 24 h. This experiment was performed in triplicate.

Endogenous Flavobacterium sp. strain 4466 protects germ-free rainbow trout against infection by different strains of F. columnare

To test whether the protective Flavobacterium sp. isolated from the Conv rainbow trout microbiome could protect rainbow trout we re-conventionalized GF fish larvae with Flavobacterium sp. 48 hours before exposure to four virulent F. columnare strains (Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4) belonging to genomovars I and II, and isolated from different geographical origins and host fish species. Flavobacterium sp. strain 4466 conferred protection to rainbow trout larvae against all F. columnare strains (Fig 7). Therefore, the Flavobacterium sp. strain identified from trout Mix11 is a putative probiotic potentially protecting trout from columnaris disease.

Fig 7 — *Flavobacterium* sp. Survival of GF trout larvae exposed to *Flavobacterium* sp. strain 4466 48 h before infection with F. *columnare* strains Fc7, IA-S-4, Ms-Fc-4 and ALG-00-530. All F. *columnare* strains rapidly killed GF fish, whereas trout larvae that were re-conventionalized with *Flavobacterium* sp strain 4466 survived to all strains of F. columnare. Mean and SD plot representing average day post-infection at which infected fish died. For each condition n = 10 larvae. All surviving fish were euthanized at day 10. Asterisks indicate significant difference from non-infected population (****p<0.0001).

Use of germ-free trout model to validate exogenous probiotics protecting against F. columnare infection

To determine whether our GF trout model could be used as a controlled gnotobiotic approach to screen for trout probiotics, we pre-exposed 22 dph GF rainbow trout larvae to Chryseobacterium massiliae, a bacterium that does not belong to trout microbiota but was previously shown to protect larval stage and adult zebrafish from infection by F. columnare [34]. After 48 h of bath in a C. massiliae suspension at 10⁵ CFU/ml, we infected trout larvae with F. columnare strains Fc7, ALG-00-530, IA-S-4 and Ms-Fc-4 and observed that C. massiliae protected against all tested F. columnare pathogens (Fig 8). These results showed that the GF rainbow trout model enables the evaluation of bacterial species, endogenous to trout or not, with probiotic potential against highly virulent F. columnare strains.

Fig 8 — GF trout larvae survival exposed to C. *massiliae* 48 h before infection with F. *columnare* strains Fc7, IA-S-4, Ms-Fc-4 and ALG-00-530. Mean and SD plot representing average day post-infection at which infected fish die. For each condition n = 10 larvae. All surviving fish were euthanized at day 10. Asterisks indicate significant difference from non-infected population (****p<0.0001; **p<0.01).

Discussion

Although the use of probiotics is a promising approach to improve fish growth and reduce disease outbreaks while limiting chemical and antibiotic treatments [17,35,36], rational and evidence-based procedures for the identification of protective bacteria are limited. Here, we established a controlled and robust model to study trout resistance to infection by bacterial pathogens and to identify trout probiotics in microbiologically controlled conditions using GF and gnotobiotic rainbow trout.

Our gnotobiotic protocol is based on the survival of rainbow trout eggs to chemical sterilization eliminating the microbial community associated to the egg surface. The lower hatching efficiency observed for Conv eggs compared to germ-free eggs could be due to different factors such as physico-chemical parameters modification induced by endogenous microbiota (i.e. dissolved O₂ concentration or water pH), or higher susceptibility to opportunistic infections. Similarly to gnotobiotic protocols used for zebrafish [24,37], cod [25] and stickleback (Gasterosteus aculeatus) [38], our approach produced larvae that were GF from hatching to 35 dph at 16°C without continued exposure to antibiotics, therefore avoiding possible effects of prolonged antibiotic exposure on fish development [39]. Similarly to GF stickleback larvae at 14 dph [38], we observed no development or growth differences between GF and Conv trout larvae at 21 dph. In contrast, GF sea bass (D. labrax L.) larvae grew faster and had a more developed gut compared to conventionally raised larvae [40]. These discrepancies could come from the fact that, in our study and in the GF stickleback study, anatomical analyses were performed before first-feeding, whereas the GF sea bass were already fed when examined [40]. Indeed, trout larvae initially acquire nutrients by absorbing their endogenous yolk until the intestinal track is open from the mouth to the vent. We therefore cannot rule out that at later stages of development, when fish begin to rely on external feeding, differences between GF and Conv fish may occur, especially in the structure and size of organs or in body weight. However, the hurdles associated with long-term fish husbandry while keeping effective sterility control, de facto limits our approach to relatively short-term experiments on larvae with limited feeding time and low complexity microbiota.

While GF conditions cannot be compared to those prevailing in the wild or used in fish farming [25], our results showed that GF rainbow trout larvae are highly susceptible to F. columnare, the causative agent of columnaris disease affecting many aquaculture fish species [29,41].

Although our histology analysis comparing GF and Conv larvae infected or not by F. columnare Fc7 did not show any major sign of inflammation of damage, we observed that the number of Goblet cells per crypt increases in infected GF larvae and decresed in Conv larvae. A healthy intestine is determined by biological markers such as Goblet cells count, which secrete mucus with bactericidal properties [42]. Interestingly, a significative decrease in the number of Goblet cells was also observed of non-infected GF larvae compared to those of Conv larvae, as previously reported in zebrafish [37]. The absence of stimulating microorganisms in GF larvae could lead to a dysregulated acute immune response after F. columnare infection. These results suggest that the microbiota influence cell differentiation (or maturation) in trout gut epithelium [43], potentially affecting for some aspect of the protection against F. columnare infection.

Interestingly, our GF rainbow trout larvae model also revealed the protective activity of C. massiliae, a potential probiotic bacterium isolated from Conv zebrafish [34], against various F. columnare strains from different fish host and geographical origins. These results demonstrate that GF rainbow trout is a robust animal model for the study of F. columnare pathogenicity and support C. massiliae as a potential probiotic to prevent columnaris diseases in teleost fish other than its original host.

Furthermore, we demonstrated that the relatively simple culturable bacteria isolated from microbiota harbored by Conv trout larvae effectively protect against F. columnare. Interestingly, different studies have demonstrated that highly diverse gut communities are more likely to protect the host from pathogens [44,45]. This constitutes the base for the paradoxical negative health effect associated with the massive utilization of antibiotics in aquaculture: the reduction in microbial diversity facilitates colonization by opportunistic pathogens [46]. While this advocates for practices leading to enrichment of fish microbial communities to minimize pathogenic invasions in aquaculture [16], our results demonstrate that resistance to a bacterial pathogen can also be achieved by a single bacterial strain in a low complexity microbiota. Moreover, previous studies of resistance to infection provided by controlled bacterial consortia in gnotobiotic hosts often relied on community composition, rather than individual members of the microbiota [47–50]. We showed that the observed protection in larvae is mainly due to the presence of Flavobacterium sp. strain 4466. We cannot exclude, however, that at later developmental stages, the presence of other bacterial species may be needed for more efficient implantation or stability of protective members in the trout microbiota.

The high genetic variability of F. columnare and its broad host range constitute an important limitation for the identification of effective probiotics against this widespread pathogen. Several probiotic candidates isolated from the host provided partial protection against F. columnare infection in other conventional fish species such as walleye (Sander vitreous) and brook char (Salvelinus fontinalis) [51,52]. However, high variability in protection provided by probiotic strains against F. columnare was observed depending on the fish batch used, indicating a resistance directly dependent on the fish host genetics [51] or immunological status. Here we reduced this variability using GF and gnotobiotic trout larvae and demonstrated the ability of Flavobacterium sp. strain 4466 isolated from Conv trout larvae microbiota to protect against F. columnare infection. Furthermore, this bacterium, but not its supernatant, inhibits F. columnare growth in vitro, which suggests a direct interaction between Flavobacterium sp. strain 4466 and F. columnare. Intriguingly, Flavobacterium sp. strain 4466 encodes a complete subtype T6SSⁱⁱⁱ, a molecular mechanism that delivers antimicrobial effector proteins upon contact with target cells and is unique to the phylum Bacteroidetes [53]. The members of Flavobacterium genus are ubiquitous inhabitants of freshwater and marine fish microbiota and both commensal and pathogenic Flavobacterium often share the same ecological niche [54–56]. Whether the Flavobacterium sp. strain 4466 T6SSⁱⁱⁱ contact-dependent killing system contributes to colonization resistance by inhibiting F. columnare Fc7 growth is currently under investigation. We cannot, however, exclude other mechanisms such as competition for nutrients or pathogen exclusion upon direct competition for adhesion to host tissues. This process has been suggested for infected zebrafish with efficient colonization of highly adhesive probiotic strains and enhanced life expectancy [24,57,58].

For the past 30 years, the fish farming industry has dedicated considerable efforts to identify probiotic microorganisms for rainbow trout, including Gram-positive and Gram-negative bacteria and yeast [59]. However, the high interindividual and seasonal variability of trout microbiota [60,61] and the random or time-limited colonization ability of exogenous microorganism rarely enables consistent probiotic efficacy. Despite some studies of rainbow trout proposing different endogenous bacterial strains as probiotic candidates, few have demonstrated protective properties against pathogenic bacteria in vivo [62–65]. Short-residing probiotics may limit unintended consequences to the microbial community and host system, but the use of endogenous residents may stably modulate the community and protect the fish against reoccurring disease outbreaks over longer timescales [66,67]. The probiotic efficacy of Flavobacterium sp. strain 4466 against different strains of F. columnare from different fish hosts and geographical origins, suggests that it could be used as a broad probiotic to prevent infections.

In conclusion, we showed that germ-free and gnotobiotic trout larvae enable effective experimental study of microbiota-determined sensitivity to major salmonid freshwater pathogens, leading to the identification of endogenous and exogenous potential probiotic strains. This approach will be instrumental in studying the molecular basis of probiosis against fish pathogens as well as host-pathogen mechanisms, ultimately contributing to the mitigation of rainbow trout diseases in aquaculture.

Materials and methods

Ethics statement

All animal experiments described in the present study were conducted at the Institut Pasteur according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and were approved by the Institut Pasteur institutional Animal Health and Care Committees under permit # dap200024.

Handling of rainbow trout larvae

Rainbow trout (AQUALANDE breeding line) “eyed” eggs of 210 to 230 degree-days (21–23 days after fertilization at 10°C) (dd) were obtained from Aqualande Group trout facility in Pissos, France. Upon arrival, the eggs were progressively acclimatized to 16°C before manipulation. All procedures were performed under a laminar microbiological cabinet and with single-use disposable plastic ware. Eggs were kept in 145 x 20 mm Petri dishes with 75 mL autoclaved dechlorinated tap water at 16°C until hatching. After hatching, fish were transferred and kept in 250 mL vented cap culture flasks in 100 mL sterile tap water at 16°C. Fish were fed starting 21 days post-hatching with gamma-ray sterilized fish food powder every 48 h, 30 minutes before water renewal of half the volume of sterile tap water to avoid waste (NH₄⁺, NO₂^-, NO₃^-) accumulation and oxygen limitation.

Sterilization and raising of germ-free rainbow trout

The eyed rainbow trout eggs received at 210–230 dd were transferred to sterile Petri dishes (140 mm diameter, 150 eggs/dish) and washed twice with a sterile methylene blue solution (0.05 mg/ml) in autoclaved dechlorinated tap water, previously filtered with 0.22 μm system. The eggs, kept in 75 ml of methylene blue solution, were then exposed to a previously described antibiotic cocktail [24] (750 μl penicillin G (10,000 U/ml), streptomycin (10 mg/ml); 300 μl of filtered kanamycin sulfate (100 mg/ml) and 75 μl of the antifungal drug amphotericin B solution (250 μg/ml)) for 24 hours by gentle agitation at 16°C. Eggs were then washed 3 times with fresh sterile water and treated with bleach (0.005%) for 15 minutes. Following 3 washes with sterile water, eggs were treated for 10 minutes with 10 ppm Romeiod (COFA, France), a iodophor disinfection solution. Finally, eggs were washed 3 times and kept in a class II hood at 16°C in 75 ml of sterile water supplemented with the previously mentioned antibiotic cocktail until hatching spontaneously 5 to 7 days following the disinfection process. Once hatched, fish were immediately transferred to 75 cm³ vented cap culture flasks containing 100 ml of fresh sterile water without antibiotics (12 larvae/flask). The hatching percentage was determined by comparing the number of hatched and alive larvae relative to the total number of eggs in the Petri dish. Conventionally raised eggs followed the same procedure from their acclimatization until hatching without exposing them to the sterilization protocol mentioned above.

Sterility: Sterility was monitored by culture-based and 16S rRNA PCR-based tests at 24 h, 7- and 21-day post-treatment. After feeding started, 50 μl of GF fish flask water was sampled before each water change as well as one larva every week to perform culture-based and 16S rRNA-based PCR sterility tests. 50 μl of rearing water from each flask was plated on LB, YPD and TYES agar plates, all incubated at 16°C under aerobic conditions. Fish larvae were also checked for bacterial contamination every week using the following methods. Randomly chosen fish were sacrificed by an overdose of filtered tricaine methane sulfonate solution (tricaine, Sigma, 300 mg/L). Whole fish were mechanically disrupted in Lysing Matrix tubes containing 1 ml of sterile water and 425–600 μm glass beads (Sigma). Samples were homogenized at 6.0 m s^-1 for 45 s on a FastPrep Cell Disrupter (BIO101/FP120 QBioGene) and serial dilutions of the homogenized solution were plated on LB, YPD and TYES agars. When water samples or fish homogenates showed any bacterial CFU on the different culture media used, the corresponding animals (or flasks) were removed from the experiment. The absence of any contamination in the fish larvae was further confirmed by PCR as follows. Total bacterial DNA was extracted from fish homogenate sample using QIAmp DNA Microbiome Kit (Qiagen) following manufacturer instructions. All reagents used were molecular grade and supplied by Sigma-Aldrich (UK). To detect the presence of microbial DNA, universal specific primers for the chromosomal 16S rRNA (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-GGTTACCTTGTTACGACTT-3') following the protocol described in [68], the presence of a band of ~1400 bp on an agarose gel indicated contamination and the flask was removed from the experiment.

Bacterial strains and growth conditions

F. columnare strains Fc7 [69], Ms-Fc-4 [70] and IA-S-4 [71] (genomovar I), ALG-00-530 [72] (genomovar II), and Chryseobacterium massiliae [34] were grown in tryptone yeast extract salts (TYES) broth [0.4% (w/v) tryptone, 0.04% yeast extract, 0.05% (w/v) MgSO₄ 7H₂O, 0.02% (w/v), CaCl₂ 2H₂O, 0.05% (w/v) D-glucose, pH 7.2] at 150 rpm and 18°C. F. psychrophilum strain THCO2-90 was grown in TYES broth at 150 rpm and 18°C. Yersinia ruckeri strain JIP 27/88 was grown in Luria-Bertani (LB) medium at 150 rpm and 28°C. V. anguillarum strain 1669 was grown in tryptic soy broth (TSB) at 150 rpm and 28°C. L. garvieae JIP 28/99 was grown in brain heart infusion (BHI) broth at 150 rpm and 28°C. When required, 15 g/L of agar was added to the broth media to obtain the corresponding solid media. Stock cultures were preserved at -80°C in the respective broth media supplemented with 15% (vol/vol) glycerol.

Fish infection challenge

Pathogenic bacteria were grown in suitable media at different temperatures until advanced stationary phase. Then, each culture was pelleted (10,000 rpm for 5 min) and washed once in sterile water. Bacteria were resuspended in sterile water and added to culture flasks at a final concentration of 10⁷ CFU/ml. After 24 hours of incubation with pathogenic bacteria at 16°C, fish were washed three times by water renewal. Bacterial concentrations were confirmed at the beginning and at the end of the immersion challenge by plating serial dilutions of water samples on specific medium for each pathogen. Ten to twelve larvae were used per condition and experiment and each experiment was repeated at least twice. Virulence was evaluated by daily monitoring of fish mortality up to 10 days post-infection.

Characterization of culturable conventional rainbow trout microbiota

To identify the species constituting the cultivable microbiota of Conv trout larvae, 3 individuals were sacrificed with an overdose of tricaine at 35 dph, homogenized following the protocol described above and serial dilutions of the homogenates were plated on TYES, LB, R2A and TS agars. The plates were incubated a 16°C for 48 to 72 hours. All morphologically distinct colonies (based on form, size, color, texture, elevation and margin) were then isolated and conserved at -80°C in respective broth medium supplemented with 15% (vol/vol) glycerol.

In order to identify individual morphotypes, individual colonies were picked for each morphotype from each agar plates, vortexed in 200 μl DNA-free water and boiled for 20 min at 90°C. Five μl of this bacterial suspension was used as template for colony PCR to amplify the 16S rRNA gene with the universal primer pair 27f and 1492R. 16S rRNA gene PCR products were verified on 1% agarose gels, purified with the QIAquick PCR purification kit and two PCR products for each morphotype were sent for sequencing (Eurofins, Ebersberg, Germany). Individual 16S rRNA- gene sequences were compared with those available in the EzBioCloud database [73]. A whole genome-based bacterial species identification was performed for Flavobacterium sp. strain 4466 with the TrueBac ID system (v1.92, DB:20190603) (https://www.truebacid.com/) [74]. Species-level identification was performed based on the algorithmic cut-off set at 95% ANI or when the 16S rRNA gene sequence similarity was >99%.

Whole genome sequencing

Chromosomal DNA of Flavobacterium sp. strain 4466 isolated from rainbow trout larvae microbiota was extracted using the DNeasy Blood & Tissue kit (QIAGEN) including RNase treatment. DNA quality and quantity was assessed on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA sequencing libraries were made using the Nextera DNA Library Preparation Kit (Illumina Inc.) and library quality was checked using the High Sensitivity DNA LabChip Kit on the Bioanalyzer 2100 (Agilent Technologies). Sequencing clusters were generated using the MiSeq reagents kit v2 500 cycles (Illumina Inc.) according to manufacturer’s instructions. DNA was sequenced at the Mutualized Platform for Microbiology at Institut Pasteur by bidirectional sequencing, producing 2 x 150 bp paired-end (PE) reads. Reads were quality filtered, trimmed and adapters removed with fastq-mcf [75] and genomes assembled using SPAdes 3.9.0 [76].

Phylogenomic analysis

The proteomes for the 15 closest Flavobacterium strains identified by the ANI analysis were retrieved from the NCBI RefSeq database (S2 Table).

These sequences together with the Flavobacterium sp. strain 4466 proteome were analyzed with Phylophlan (version 0.43, march 2020) [77]. This method uses the 400 most conserved proteins across the proteins and builds a Maximum likelihood phylogenetic tree using RAxML (version 8.2.8) [78]. Maximum likelihood tree was boostrapped with 1000 replicates.

Germ free rainbow trout microbial re-conventionalization

Each isolated bacterial species was grown for 24 hours in suitable medium at 150 rpm and 20°C. Bacteria were then pelleted, washed twice in sterile water and diluted to a final concentration of 5.10⁷ CFU/ml. At 22 dph, GF rainbow trout were mono-re-conventionalized by adding 1 ml of each bacterial suspension per flask (5.10⁵ CFU/ml, final concentration). In the case of fish re-conventionalization with bacterial consortia, individual bacterial strains were washed, then mixed in the same aqueous suspension, each at a concentration of 5.10⁷ CFU/ml. The mixed bacterial suspension was then added to the flask containing GF rainbow trout as previously described. In all cases, fish re-conventionalization was performed for 48 h and the infection challenge with F. columnare was carried out immediately after water renewal.

Histological analyzes

Histological sections were used to compare microscopical lesions between GF and Conv fish following infection with F. columnare. Sacrificed animals were fixed for 1 week in 4% methanol-free paraformaldehyde. Whole fixed animals were then dehydrated in a graded series of ethanol solutions (4 hours in 80% ethanol solution; 4 hours in 95% ethanol solution, and 4 hours in 100% ethanol). Final dehydration was performed by 100% xylene solution 2 × 4 hours. Then, samples were embedded in paraffin wash solution (3 x 4 hours) and embedded in paraffin wax (3 × 4 hours) for polymerization.

Semi-thin sections (thickness 5 μm) were cut with a “Leica Ultracut UCT” ultramicrotome (Leica Microsysteme GmbH, Wien, Austria), and mounted on adhesive slides (Klinipath- KP-PRINTER ADHESIVES). Paraffin-embedded sections were deparaffinized and stained with Alcian Blue (AB) and Periodic-Acid Schiff (PAS) to observe both neutral and acidic mucins and Goblet cells quantification. All slides were scanned with the Pannoramic Scan 150 (3D Histech) and analyzed with the CaseCenter 2.9 viewer (3D Histech). Goblet cells quantification was estimated by manual counting of total AB positive cells in blue per villi of the posterior gut.

Whole fish clearing and 3D imaging

For a 3D imaging of cleared whole fish, fish were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) overnight at 4°C. Fixed samples were rinsed with PBS. To render tissue transparent, fish were first depigmented by pretreatment in SSC 0.5X twice during 1 hour at room temperature followed by an incubation in saline sodium citrate (SSC) 0.5X + KOH 0.5% + H₂O₂ 3% during 2 hours at room temperature. Depigmentation was stopped by incubation in PBS twice for 15 minutes. Fish were then post-fixed with 2% formaldehyde in PBS for 2 hours at room temperature and then rinsed twice with PBS for 30 min. Depigmented fish were cleared with the iDISCO+ protocol [79]. Briefly, samples were progressively dehydrated in ascending methanol series (20, 40, 60 and 80% in H₂O, then twice in 100% methanol) during 1 hour for each step. The dehydrated samples were bleached by incubation in methanol + 5% H₂O₂ at 4°C overnight, followed by incubation in methanol 100% twice for 1 hour. They were then successively incubated in 67% dichloromethane + 33% methanol for 3 hours, in dichloromethane 100% for 1 hour and finally in dibenzylether until fish became completely transparent. Whole sample acquisition was performed on a light-sheet ultramicroscope (LaVision Biotec, Bielefeld, Germany) with a 2X objective using a 0.63X zoom factor. Autofluorescence was acquired by illuminating both sides of the sample with a 488 nm laser. Z-stacks were acquired with a 2 μm z-step.

Agar overlay assay for growth inhibition detection

The growth inhibitory effect of Flavobacterium sp. 4466 has been evaluated using an agar spot test. Briefly, 125 μl from an overnight culture of different strains of F. columnare adjusted to OD 1 were mixed to 5 ml of top agar (0.7% agar) and overlaid on plates of TYES agar. Five μL of overnight culture of Flavobacterium sp. 4466 were then spotted on the overlay of targeted bacteria. The plates were incubated at 28°C for 24 hours. Growth inhibition of F. columnare was recorded by observation of a clear halo surrounding Flavobacterium sp. colony. Sterile TYES broth was used as a mock and the experiment were performed in triplicate.

Statistical methods

Statistical analyses were performed using unpaired, non-parametric Mann-Whitney test or ANOVA using Kruskal-Wallis multiple comparison tests for average survival analysis and the log rank (Mantel-Cox) test for Kaplan–Meier survival curves. Analyses were performed using Prism v8.2 (GraphPad Software). A cut-off of p-value of 5% was used for all tests. * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001.

Supporting information

S1 Table. Flavobacterium sp. strain 4466 taxonomic identification based on genomic similarities.

(PDF)

Click here for additional data file.^{(110.5KB, pdf)}

S2 Table. Flavobacterium species genomes retrieved from public databases.

(PDF)

Click here for additional data file.^{(125.5KB, pdf)}

S1 Fig. Sterility test of rainbow trout larvae raised under GF and Conv conditions.

(PDF)

Click here for additional data file.^{(1.4MB, pdf)}

S2 Fig. Growth performance of rainbow trout larvae raised under GF and Conv conditions.

(PDF)

Click here for additional data file.^{(233.4KB, pdf)}

S3 Fig. Anatomical comparison of the gut of Conv and GF rainbow trout larvae.

(PDF)

Click here for additional data file.^{(2.3MB, pdf)}

S4 Fig. Survival of re-conventionalized trout larvae against F. columnare Fc7 infection.

(PDF)

Click here for additional data file.^{(246.1KB, pdf)}

S5 Fig. Histological comparison of the gut of infected and non-infected Conv and GF rainbow trout larvae.

(PDF)

Click here for additional data file.^{(2.4MB, pdf)}

S6 Fig. Phylogenetic tree illustrating the relationship between Flavobacterium sp. strain 4466 and the closest 15 Flavobacterium species based on ANI analysis.

(PDF)

Click here for additional data file.^{(625.9KB, pdf)}

Acknowledgments

We thank Rebecca Stevick, Jean-Pierre Levraud, Pierre Boudinot, Eric Duchaud, Mark McBride and Christophe Beloin for critical reading of the manuscript. We thank Laurent Debruyne and Jérémie Vieville from the Pissos Aqualande Trout breeding station. We are grateful to Jean-François Bernardet and Mark McBride for kindly providing us some of pathogenic microorganisms used in this study. We thank Rustem Uzbekov for his help in histology analyses performed in the context of a service provided by the IBiSA Microscopy facility, Tours University, France. iDISCO imaging was established and performed by Christelle Langevin and Maxence Fretaud (INRAE EMERG’IN IERP phenotyping platform) and light-sheet images were acquired at the Institut de la Vision. This work has benefited from the facilities and expertise of @BRIDGe (Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350 Jouy-en-Josas, France).

Data Availability

The genome of Flavobacterium sp. 4466 was deposited at European Nucleotide Archive (ENA) databank, accession number: ERS4574862, https://www.ebi.ac.uk/ena/browser/submit.

Funding Statement

This work was supported by the Institut Pasteur, the French Government’s Investissement d’Avenir program: Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant n°. ANR-10-LABX-62-IBEID to J.M.G.), the Fondation pour la Recherche Médicale (grant n°. DEQ20180339185 to J.M.G.). In addition, D.P.-P. was the recipient of an Institut Carnot Pasteur MS post-doctoral fellowship. S.V.-F. was supported by an ERASMUS scholarship and J.B.-B. was the recipient of a long-term post-doctoral fellowship from the Federation of European Biochemical Societies (FEBS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.FAO. The state of World Fisheries and Aquaculture. Rome: Food and Agriculture Organization of the United Nations, 2016 [Google Scholar]
2.Vadstein O, Bergh Ø, Gatesoupe F-J, Galindo-Villegas J, Mulero V, Picchietti S, et al. Microbiology and immunology of fish larvae. Reviews in Aquaculture. 2013;5(s1):S1–S25. 10.1111/j.1753-5131.2012.01082.x [DOI] [Google Scholar]
3.Embregts CW, Forlenza M. Oral vaccination of fish: Lessons from humans and veterinary species. Dev Comp Immunol. 2016;64:118–37. Epub 2016/03/29. 10.1016/j.dci.2016.03.024 . [DOI] [PubMed] [Google Scholar]
4.Perez-Sanchez T, Mora-Sanchez B, Balcazar JL. Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges. Trends Microbiol. 2018;26(11):896–903. Epub 2018/05/29. 10.1016/j.tim.2018.05.002 . [DOI] [PubMed] [Google Scholar]
5.Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A, et al. Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health. Environmental Microbiology. 2013;15(7):1917–42. 10.1111/1462-2920.12134 [DOI] [PubMed] [Google Scholar]
6.Santos L, Ramos F. Antimicrobial resistance in aquaculture: Current knowledge and alternatives to tackle the problem. International Journal of Antimicrobial Agents. 2018;52(2):135–43. 10.1016/j.ijantimicag.2018.03.010 [DOI] [PubMed] [Google Scholar]
7.Brandt KK, Amezquita A, Backhaus T, Boxall A, Coors A, Heberer T, et al. Ecotoxicological assessment of antibiotics: A call for improved consideration of microorganisms. Environ Int. 2015;85:189–205. 10.1016/j.envint.2015.09.013 . [DOI] [PubMed] [Google Scholar]
8.Defoirdt T, Sorgeloos P, Bossier P. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr Opin Microbiol. 2011;14(3):251–8. 10.1016/j.mib.2011.03.004 . [DOI] [PubMed] [Google Scholar]
9.Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiology and Molecular Biology Reviews. 2000;64(4):655–71. 10.1128/mmbr.64.4.655-671.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Tinh NT, Dierckens K, Sorgeloos P, Bossier P. A review of the functionality of probiotics in the larviculture food chain. Mar Biotechnol (NY). 2008;10(1):1–12. 10.1007/s10126-007-9054-9 . [DOI] [PubMed] [Google Scholar]
11.Martínez Cruz P, Ibáñez AL, Monroy Hermosillo OA, Ramírez Saad HC. Use of probiotics in aquaculture. ISRN microbiology. 2012;2012:916845–. 10.5402/2012/916845 . [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Buffie CG, Pamer EG. Microbiota-mediated colonization resistance against intestinal pathogens. Nature Reviews Immunology. 2013;13:790 10.1038/nri3535 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Stecher B, Hardt W-D. Mechanisms controlling pathogen colonization of the gut. Current Opinion in Microbiology. 2011;14(1):82–91. 10.1016/j.mib.2010.10.003 [DOI] [PubMed] [Google Scholar]
14.de Bruijn I, Liu Y, Raaijmakers JM, Wiegertjes GF. Exploring fish microbial communities to mitigate emerging diseases in aquaculture. FEMS Microbiology Ecology. 2017;94(1). 10.1093/femsec/fix161 [DOI] [PubMed] [Google Scholar]
15.Gomez-Gil B, Roque A, Turnbull JF. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture. 2000;191(1):259–70. 10.1016/S0044-8486(00)00431-2. [DOI] [Google Scholar]
16.Xiong JB, Nie L, Chen J. Current understanding on the roles of gut microbiota in fish disease and immunity. Zool Res. 2019;40(2):70–6. 10.24272/j.issn.2095-8137.2018.069 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Hai NV. The use of probiotics in aquaculture. Journal of Applied Microbiology. 2015;119(4):917–35. 10.1111/jam.12886 [DOI] [PubMed] [Google Scholar]
18.Lazado CC, Caipang CMA. Mucosal immunity and probiotics in fish. Fish & Shellfish Immunology. 2014;39(1):78–89. 10.1016/j.fsi.2014.04.015 [DOI] [PubMed] [Google Scholar]
19.Dierckens K, Rekecki A, Laureau S, Sorgeloos P, Boon N, Van den Broeck W, et al. Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae. Environmental Microbiology. 2009;11(2):526–33. 10.1111/j.1462-2920.2008.01794.x [DOI] [PubMed] [Google Scholar]
20.Fiebiger U, Bereswill S, Heimesaat MM. Dissecting the Interplay Between Intestinal Microbiota and Host Immunity in Health and Disease: Lessons Learned from Germfree and Gnotobiotic Animal Models. Eur J Microbiol Immunol (Bp). 2016;6(4):253–71. 10.1556/1886.2016.00036 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Yi P, Li L. The germfree murine animal: An important animal model for research on the relationship between gut microbiota and the host. Veterinary Microbiology. 2012;157(1):1–7. 10.1016/j.vetmic.2011.10.024 [DOI] [PubMed] [Google Scholar]
22.Caruffo M, Navarrete NC, Salgado OA, Faundez NB, Gajardo MC, Feijoo CG, et al. Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae. Front Cell Infect Microbiol. 2016;6:127 10.3389/fcimb.2016.00127 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Qin C, Zhang Z, Wang Y, Li S, Ran C, Hu J, et al. EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish. Frontiers in Microbiology. 2017;8(2406). 10.3389/fmicb.2017.02406 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Rendueles O, Ferrieres L, Fretaud M, Begaud E, Herbomel P, Levraud JP, et al. A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria. PLoS Pathog. 2012;8(7):e1002815 10.1371/journal.ppat.1002815 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Forberg T, Arukwe A, Vadstein O. A protocol and cultivation system for gnotobiotic Atlantic cod larvae (Gadus morhua L.) as a tool to study host microbe interactions. Aquaculture. 2011;315(3):222–7. 10.1016/j.aquaculture.2011.02.047. [DOI] [Google Scholar]
26.Verner-Jeffreys DW, Shields RJ, Birkbeck TH. Bacterial influences on Atlantic halibut Hippoglossus hippoglossus yolk-sac larval survival and start-feed response. Dis Aquat Organ. 2003;56(2):105–13. 10.3354/dao056105 . [DOI] [PubMed] [Google Scholar]
27.Vestrum R.I., Luef B., Forberg T., Bakke I., Emerging O. V. Issues in Fish Larvae Research. In: M. Y, editor.: Springer; 2018. [Google Scholar]
28.Quesada SP, Paschoal JAR, Reyes FGR. Considerations on the Aquaculture Development and on the Use of Veterinary Drugs: Special Issue for Fluoroquinolones—A Review. Journal of Food Science. 2013;78(9):R1321–R33. 10.1111/1750-3841.12222 [DOI] [PubMed] [Google Scholar]
29.Declercq AM, Haesebrouck F, Van den Broeck W, Bossier P, Decostere A. Columnaris disease in fish: a review with emphasis on bacterium-host interactions. Veterinary Research. 2013;44(1):27 10.1186/1297-9716-44-27 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Butt RL, Volkoff H. Gut Microbiota and Energy Homeostasis in Fish. Frontiers in endocrinology. 2019;10:9–. 10.3389/fendo.2019.00009 . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Austin B, Austin DA. Bacterial fish pathogens: disease of farmed and wild fish. 4th ed Dordrecht Chichester: Springer; Published in association with Praxis Pub.; 2007. xxvi, 552 p. p. [Google Scholar]
32.Russell Alistair B, Wexler Aaron G, Harding Brittany N, Whitney John C, Bohn Alan J, Goo Young A, et al. A Type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism. Cell Host & Microbe. 2014;16(2):227–36. 10.1016/j.chom.2014.07.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Konstantinidis KT, Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(7):2567–72. 10.1073/pnas.0409727102 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Stressmann FA, Bernal-Bayard J, Perez-Pascual D, Audrain B, Rendueles O, Briolat V, et al. Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection. The ISME Journal 2020. Epub 2020/10/21. 10.1038/s41396-020-00807-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Banerjee G, Ray AK. The advancement of probiotics research and its application in fish farming industries. Research in Veterinary Science. 2017;115:66–77. 10.1016/j.rvsc.2017.01.016 [DOI] [PubMed] [Google Scholar]
36.Hoseinifar SH, Sun Y-Z, Wang A, Zhou Z. Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives. Frontiers in Microbiology. 2018;9(2429). 10.3389/fmicb.2018.02429 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Rawls JF, Samuel BS, Gordon JI. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(13):4596–601. 10.1073/pnas.0400706101 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Milligan-Myhre K, Small CM, Mittge EK, Agarwal M, Currey M, Cresko WA, et al. Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations. Disease Models & Mechanisms. 2016;9(2):187–98. 10.1242/dmm.021881 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Forberg T, Milligan-Myhre K. Chapter 6—Gnotobiotic Fish as Models to Study Host–Microbe Interactions. In: Schoeb TR, Eaton KA, editors. Gnotobiotics: Academic Press; 2017. p. 369–83. [Google Scholar]
40.Rekecki A, Dierckens K, Laureau S, Boon N, Bossier P, Van den Broeck W. Effect of germ-free rearing environment on gut development of larval sea bass (Dicentrarchus labrax L.). Aquaculture. 2009;293(1):8–15. 10.1016/j.aquaculture.2009.04.001. [DOI] [Google Scholar]
41.Olivares-Fuster O, Baker JL, Terhune JS, Shoemaker CA, Klesius PH, Arias CR. Host-specific association between Flavobacterium columnare genomovars and fish species. Systematic and Applied Microbiology. 2007;30(8):624–33. 10.1016/j.syapm.2007.07.003 [DOI] [PubMed] [Google Scholar]
42.Elsabagh M, Mohamed R, Moustafa EM, Hamza A, Farrag F, Decamp O, et al. Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquaculture Nutrition. 2018;24(6):1613–22. 10.1111/anu.12797. [DOI] [Google Scholar]
43.Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE, Guillemin K. Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation. Developmental Biology. 2006;297(2):374–86. 10.1016/j.ydbio.2006.05.006 [DOI] [PubMed] [Google Scholar]
44.De Schryver P, Vadstein O. Ecological theory as a foundation to control pathogenic invasion in aquaculture. The Isme Journal. 2014;8:2360 10.1038/ismej.2014.84 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Zhu J, Dai W, Qiu Q, Dong C, Zhang J, Xiong J. Contrasting Ecological Processes and Functional Compositions Between Intestinal Bacterial Community in Healthy and Diseased Shrimp. Microbial Ecology. 2016;72(4):975–85. 10.1007/s00248-016-0831-8 [DOI] [PubMed] [Google Scholar]
46.He S, Wang Q, Li S, Ran C, Guo X, Zhang Z, et al. Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis. Science China Life Sciences. 2017;60(11):1260–70. 10.1007/s11427-016-9072-6 [DOI] [PubMed] [Google Scholar]
47.Brugiroux S, Beutler M, Pfann C, Garzetti D, Ruscheweyh HJ, Ring D, et al. Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium. Nat Microbiol. 2016;2:16215 Epub 2016/11/22. 10.1038/nmicrobiol.2016.215 . [DOI] [PubMed] [Google Scholar]
48.Kim YG, Sakamoto K, Seo SU, Pickard JM, Gillilland MG 3rd, Pudlo NA, et al. Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens. Science. 2017;356(6335):315–9. Epub 2017/04/22. 10.1126/science.aag2029 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Little AEF, Robinson CJ, Peterson SB, Raffa KF, Handelsman J. Rules of Engagement: Interspecies Interactions that Regulate Microbial Communities. Annual Review of Microbiology. 2008;62(1):375–401. 10.1146/annurev.micro.030608.101423 . [DOI] [PubMed] [Google Scholar]
50.Stecher B, Chaffron S, Kappeli R, Hapfelmeier S, Freedrich S, Weber TC, et al. Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog. 2010;6(1):e1000711 Epub 2010/01/12. 10.1371/journal.ppat.1000711 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Boutin S, Bernatchez L, Audet C, Derôme N. Antagonistic effect of indigenous skin bacteria of brook charr (Salvelinus fontinalis) against Flavobacterium columnare and F. psychrophilum. Veterinary Microbiology. 2012;155(2):355–61. 10.1016/j.vetmic.2011.09.002 [DOI] [PubMed] [Google Scholar]
52.Seghouani H, Garcia-Rangel C-E, Füller J, Gauthier J, Derome N. Walleye Autochthonous Bacteria as Promising Probiotic Candidates against Flavobacterium columnare. Frontiers in Microbiology. 2017;8(1349). 10.3389/fmicb.2017.01349 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Chatzidaki-Livanis M, Geva-Zatorsky N, Comstock LE. Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species. Proceedings of the National Academy of Sciences. 2016;113(13):3627 10.1073/pnas.1522510113 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Bernardet J-F, Bowman JP. The Genus Flavobacterium. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass New York, NY: Springer New York; 2006. p. 481–531. [Google Scholar]
55.Loch TP, Faisal M. Emerging flavobacterial infections in fish: A review. Journal of Advanced Research. 2015;6(3):283–300. 10.1016/j.jare.2014.10.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Lowrey L, Woodhams DC, Tacchi L, Salinas I. Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin. Appl Environ Microbiol. 2015;81(19):6915–25. Epub 2015/07/26. 10.1128/AEM.01826-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Boirivant M, Strober W. The mechanism of action of probiotics. Curr Opin Gastroenterol. 2007;23(6):679–92. 10.1097/MOG.0b013e3282f0cffc . [DOI] [PubMed] [Google Scholar]
58.Wang C, Chuprom J, Wang Y, Fu L. Beneficial bacteria for aquaculture: nutrition, bacteriostasis and immunoregulation. J Appl Microbiol. 2019. Epub 2019/07/16. 10.1111/jam.14383 . [DOI] [PubMed] [Google Scholar]
59.Pérez-Sánchez T, Ruiz-Zarzuela I, de Blas I, Balcázar JL. Probiotics in aquaculture: a current assessment. Reviews in Aquaculture. 2014;6(3):133–46. 10.1111/raq.12033 [DOI] [Google Scholar]
60.Huber I, Spanggaard B, Appel KF, Rossen L, Nielsen T, Gram L. Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum). Journal of Applied Microbiology. 2004;96(1):117–32. 10.1046/j.1365-2672.2003.02109.x [DOI] [PubMed] [Google Scholar]
61.Huyben D, Sun L, Moccia R, Kiessling A, Dicksved J, Lundh T. Dietary live yeast and increased water temperature influence the gut microbiota of rainbow trout. Journal of Applied Microbiology. 2018;124(6):1377–92. 10.1111/jam.13738 [DOI] [PubMed] [Google Scholar]
62.Araújo C, Muñoz-Atienza E, Nahuelquín Y, Poeta P, Igrejas G, Hernández PE, et al. Inhibition of fish pathogens by the microbiota from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment. Anaerobe. 2015;32:7–14. 10.1016/j.anaerobe.2014.11.001 [DOI] [PubMed] [Google Scholar]
63.Brunt J, Austin B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases. 2005;28(12):693–701. 10.1111/j.1365-2761.2005.00672.x [DOI] [PubMed] [Google Scholar]
64.Mohammadian T, Nasirpour M, Tabandeh MR, Heidary AA, Ghanei-Motlagh R, Hosseini SS. Administrations of autochthonous probiotics altered juvenile rainbow trout Oncorhynchus mykiss health status, growth performance and resistance to Lactococcus garvieae, an experimental infection. Fish & Shellfish Immunology. 2019;86:269–79. 10.1016/j.fsi.2018.11.052 [DOI] [PubMed] [Google Scholar]
65.Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I, et al. Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae. Journal of Fish Diseases. 2011;34(7):499–507. 10.1111/j.1365-2761.2011.01260.x [DOI] [PubMed] [Google Scholar]
66.Lemon KP, Armitage GC, Relman DA, Fischbach MA. Microbiota-targeted therapies: an ecological perspective. Sci Transl Med. 2012;4(137):137rv5 10.1126/scitranslmed.3004183 [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Newaj-Fyzul A, Al-Harbi AH, Austin B. Review: Developments in the use of probiotics for disease control in aquaculture. Aquaculture. 2014;431:1–11. 10.1016/j.aquaculture.2013.08.026. [DOI] [Google Scholar]
68.Vergin KL, Urbach E, Stein JL, DeLong EF, Lanoil BD, Giovannoni SJ. Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl Environ Microbiol. 1998;64(8):3075–8. Epub 1998/08/04. 10.1128/AEM.64.8.3075-3078.1998 [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Michel C, Messiaen S, Bernardet J-F. Muscle infections in imported neon tetra, Paracheirodon innesi Myers: limited occurrence of microsporidia and predominance of severe forms of columnaris disease caused by an Asian genomovar of Flavobacterium columnare. Journal of Fish Diseases. 2002;25(5):253–63. 10.1046/j.1365-2761.2002.00364.x [DOI] [Google Scholar]
70.Evenhuis JP, Mohammed H, LaPatra SE, Welch TJ, Arias CR. Virulence and molecular variation of Flavobacterium columnare affecting rainbow trout in Idaho, USA. Aquaculture. 2016;464:106–10. 10.1016/j.aquaculture.2016.06.017. [DOI] [Google Scholar]
71.LaFrentz BR, García JC, Waldbieser GC, Evenhuis JP, Loch TP, Liles MR, et al. Identification of Four Distinct Phylogenetic Groups in Flavobacterium columnare With Fish Host Associations. Frontiers in microbiology. 2018;9:452–. 10.3389/fmicb.2018.00452 . [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Olivares-Fuster O, Bullard SA, McElwain A, Llosa MJ, Arias CR. Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge. Diseases of aquatic organisms. 2011;96(3):221–7. 10.3354/dao02371 . [DOI] [PubMed] [Google Scholar]
73.Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology. 2017;67(5):1613–7. 10.1099/ijsem.0.001755 [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Sung-Min H, Chang Ki K, Juhye R, Jung-Hyun B, Seung-Jo Y, Seon-Bin C, et al. Application of the Whole Genome-Based Bacterial Identification System, TrueBac ID, Using Clinical Isolates That Were Not Identified With Three Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Systems. Annals of Laboratory Medicine. 2019;39(6):530–6. Epub 2019/11/01. 10.3343/alm.2019.39.6.530 [DOI] [PMC free article] [PubMed] [Google Scholar]
75.Aronesty E. Command-line tools for processing biological sequencing data. ea-utils [Internet]. 2011. Available from: https://github.com/ExpressionAnalysis/ea-utils.
76.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology: a journal of computational molecular cell biology. 2012;19(5):455–77. Epub 2012/04/18. 10.1089/cmb.2012.0021 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Segata N, Bornigen D, Morgan XC, Huttenhower C. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nat Commun. 2013;4:2304 Epub 2013/08/15. 10.1038/ncomms3304 [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. Epub 2014/01/24. 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Renier N, Wu Z, Simon David J, Yang J, Ariel P, Tessier-Lavigne M. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell. 2014;159(4):896–910. 10.1016/j.cell.2014.10.010 [DOI] [PubMed] [Google Scholar]

PLoS Pathog. doi: 10.1371/journal.ppat.1009302.r001

Decision Letter 0

Karen Guillemin, Nina R Salama

14 Aug 2020

Dear Prof. GHIGO,

Thank you very much for submitting your manuscript "Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Karen Guillemin

Guest Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Strengths: directly studies trout, which are an economically important but under utilised experimental system. Logical experiments establish a system using gnotobiotic larvae to model antibiotic-associated opportunistic infections, demonstrate protection by cocktail and then individual species of normal microbiome.

Well written and presented. Missing a few connections (ie CFU recovery) that would strengthen the manuscript by providing additional readouts. Missing mechanistic insight into the mode of protection at this stage but I believe the authors could use the system to rapidly establish a mechanism of protection.

Reviewer #2: This manuscript describes the generation of a gnotobiotic protocol for rainbow trout, and the ability of the trout microbiota to protect against pathogenic bacteria. The paper also describes use of a mock community of trout microbiota that conferred resistance to the infection, and that monoassociations and mock community exposures indicate a specific member of the microbiota produces anti-bacterial activity against the pathogen. This is a significant finding both for trout aquaculture and for gnotobiotic studies in general. As far as I can tell, similar studies have not been done in trout. In general, these experiments were executed well, although I do have some concerns, most of which may be clarified with some editing.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Figure 2 and SFig 4

Need to have at least some basic microscopic analysis of histology. Eg Alcian blue staining for intestinal goblet cell enumeration. Additionally, the SFig4 uninfected samples do not appear well matched.

SFig 4B: there appear to be significant signs of IEC vacuolation and, potentially, mucus staining abnormalities in the CONV-infected (there's something in the lumen). This concern needs to be addressed by higher resolution imaging of more appropriately-stained tissue (Alcian blue, H&E etc)

Figure 4 onwards "mean death day" plots need to be complemented by CFU recovery or bacterial 16s DNA/RNA quantification.

3. Mechanistic evidence

The in vitro data in SFig5 is central to the mechanism of the manuscript and needs to be included as a full figure. The in vitro data should be complemented by in vivo CFU recovery showing Flavobacterium colonisation prevents/reduces colonisation of the GI tract by F. columnare.

Lines 299-303 suggest mechanism that could be investigated. This paper would be significantly strengthened by the demonstration of at least one of these mechanisms in vivo.

Reviewer #2: Supplement figure 5: Was this done in triplicate and these are representative? If so, indicate. If not, these should be repeated.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 120: Is hatching efficiency directly related to embryo survival or could the increased hatching of GF embryos be due to weakened chorions?

Line 145: 10^7 CFU/ml is a lot of bacteria. Was the mortality rate dose responsive? Surprising that V. anguillarium did not kill larvae.

Figure 4 "mean death day" plots: Please show survival curves too for completeness. Ok as supplementary Fig.

Line 174: No crucial, but do the Mix11 species colonise at equal proportions? Is there a hint (that could be tied into later observations) that Flavobacteria outperform the other species?

Sentence in lines 281-283 does not seem related to the rest of the paragraph.

Line 292: highlight protection against "multiple" strains of F. columnare in zebrafish embryos.

Related note: the switch to zebrafish embryos for demonstrating protection against multiple pathogen strains was not well justified. Paper would be strengthened by showing protection of trout larvae against multiple pathogenic strains. Is this omission patent related?

T tests are not appropriate for the multiple comparisons performed in Fig 4 and 5. Reanalyse with ANOVA

Reviewer #2: Minor points:

Introduction:

90: “sterile” indicates the fish in those studies had no living organisms. Reference 27 uses antibiotics to kill microbes and only examined growth in aerobic, high nutrient conditions, not by 16S PCR. Check other references for sterility confirmation, or change “sterile conditions” to “reduced microbial load” or “reduced bacterial load”

Methods:

Line 335: Were these all from the same clutch of fish, or several different clutches? Given the reference to host genetic background on the ability of the microbiota to protect against pathogens, this could be significant.

Line 340: kept at 16C while in petri dishes? Add that detail, or in line 338 that fish were maintained at 16C until the end of the experiment (or whenever their temp was changed).

Also line 340: Is the water tap water, ocean water, what salinity? More details needed.

Lines 341, 348 and elsewhere: how was the water and methylene blue solution sterilized? What type of water was used (instant ocean, tap water, RO water? Is it the autoclaved dechlorinated water from line 340?)

Line 343: specify sterile water was added.

Line 347: this is at odds with line 335, which says fish were collected at 210-230 degree days. Were only the 210 dd fish derived germ free?

379: add “Following the protocol in [68]. Presence of a band of X bp on an agarose gel indicated contamination and the flask was removed from the experiment.”

401: Is that 10-12 fish per flask, or is that 5-6 fish in each flask and each flask is in triplicate? This relates to the issues with replication.

Need to include a method for the microbe-microbe interaction studies.

Results:

In some places the manuscript states fish were reconventionalized at 21 dph, others it’s 22 dph. Be consistent.

Figure 1: Label the timeline with “dph”

Table 1: add the identification of the strains so that if people contact you to get the strains, they can give you the correct strain identification. For example, if I contacted you and asked for Aeromonas rivipollensis 1, how would you ensure that I received the strain from this paper? Related, indicate whether you deposited the strains to a repository.

Supplement figure 1 and elsewhere: Is figure S1 a representation of the PCR gels used, or is that all the flasks that were used in these experiments? It appears that there were 16 germ free flasks; if the pathogen exposures were done in triplicate flasks of 10 fish each (not sure how you would get the error bars in figures 3 & 4 without that) that would be 15 of the 16 flasks, not enough flasks left for the Re-conv, mix 11, etc experiments. State that the PCR gel is representative (if it is) of the PCR results and clarify how many flasks were used for each experiment.

Supplement figure 4: Can’t read the scale bars to determine whether the images are at the same scale.

Supplement figure 5: Scale bars?

Line 188: “Consistently” – how many times was the sequencing performed and/or reanalyzed? This just seems like an odd word here.

Discussion:

Line 242: Your fish were not GF up to 35 dph. They were GF from -5dph to 35 dph. As far as I can tell, your fish were conventional for 210-230 degree days before they were treated. Fish undergo major development while in their corions.

Major concerns:

It is not clear what the biological replication of the experiments are. For example, in the pathogen exposures: how many flasks of fish were exposed to the pathogens? For examples of this, see line 916, 144-148, 159-161, 171-175. Line 402 indicates that each experiment was repeated at least twice; does that mean from derivation of germ free fish to termination of the experiment, or does that mean the germ free fish were divided and at least two flasks were used in each experiment?

Lines 120-122 and elsewhere: Were conventional eggs made germ free and then microbes were added, or were they untreated (that is, didn’t go through the germ free derivation protocol)? If the latter, the process of deriving eggs germ free may have affected the fish in lines 120-122 and elsewhere.

Line 121-122: “Possibly due to higher susceptibility of Conv eggs to….” This is speculative. A number of things can effect hatching efficiency.

Regarding the iDISCO, 3D imaging, and histology (described in results in 131-138 and histological results in lines 153-154): What measurements were taken? What were the results from the measurements? Was the person doing the analysis blind to the treatment? “No damage” doesn’t speak to what aspects of inflammation/lesions/delayed growth/color, etc, was measured and used to determine there was no damage or delay in development.

Lines 339-340: Were the petri dishes with treated fish sealed to ensure that microbes weren’t able to get into the petri dishes? Fungi in particular are able to get into unsealed petri dishes. These would not be detected by plating at 16C for 2 days or by 16S sequencing.

The methods for the germ free derivation and the fish handling are confusing. I would recommend combining the two into one section called “Sterilization and husbandry of fish”.

Line 916 and 144-148: Was the water tested after the exposure and at the end of the experiment to ensure these were monocultures? Line 401 indicates specific media was used for each pathogen; if these are selective media, other microbes that contaminate the water/fish would not be detected. Yersinia ruckeri, V. anguillarum, and L. garvieae were all grown at 28C. F. columnae, Chryseobacterium massiliae and F. psychromphilum were grown at 18C. It could be assumed that most of the general microbiota and other microbes that may contaminate the flasks grow at 16C. These temperature differences may inhibit the ability to detect contaminants in the cultures with the pathogens or other microbe exposures.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Stefan Oehlers

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

PLoS Pathog. 2021 Jan 29;17(1):e1009302. doi: 10.1371/journal.ppat.1009302.r002

Author response to Decision Letter 0

25 Nov 2020

Attachment

Submitted filename: Perez-Pascual.PPATHOGENS-D-20-01341R.Resp to reviewers.VF.docx

Click here for additional data file.^{(12MB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1009302.r003

Decision Letter 1

Karen Guillemin, Nina R Salama

24 Dec 2020

Dear Prof. GHIGO,

We are pleased to inform you that your manuscript 'Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection' has been provisionally accepted for publication in PLOS Pathogens, pending some textual changes requested by the reviewers.

Before your manuscript can be formally accepted you will need to make some textual changes to address issues raised by the reviewers, as detailed in the comments below.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Karen Guillemin

Guest Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have addressed all of my comments with new data or in their direct response.

Reviewer #2: This is a resubmission of a manuscript I previously reviewed. This revised version is significantly improved as the authors have address most of the previous reviewer's concerns. As I said previously, this paper describes the generation of a gnotobiotic protocol for rainbow trout, and the ability of the trout microbiota to protect against pathogenic bacteria. The paper also describes use of a mock community of trout microbiota that conferred resistance to the infection, and that monoassociations and mock community exposures indicate a specific member of the microbiota produces anti-bacterial activity against the pathogen. This is a significant finding both for trout aquaculture and for gnotobiotic studies in general. As far as I can tell, similar studies have not been done in trout. In general, these experiments were executed well, although I do have some concerns that may be clarified with some editing.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: No further experiments are requested.

Reviewer #2: “Sterile” is incorrectly used in this manuscript. “Sterile” and “sterilized” indicates the fish in those studies had no living organisms. The fish and water were not tested for viral material or for fungi. Fungi will not necessarily grow under the conditions described for sterility testing (16C for several days on three types of media) and absence of fungi was not confirmed by PCR/sequencing. Please use “bacteria-free”, as is standard in fish gnotobiotic studies in which the above have not been performed. Germ free is also fine, as this generally indicates bacteria free.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Extensive proof reading is required.

Reviewer #2: Line 90: I checked several of the references. Most do not claim to derive fish as sterile (no microorganisms) but instead refer to their systems as “bacteria-free”. In the references I checked, and in most papers I have read on this topic, sterile is used to describe the water the fish are in (as in the autoclaved or filtered water) or the tools used, but the fish themselves, especially if they are treated with antibiotics but not soaked in bleach or PVPI, are considered “bacteria-free” or “reduced microbial load”. This is especially true for studies which don’t test specifically for virus or fungal species, which includes most fish studies. Change the word “sterile” to “bacteria-free” or something similar. Also, the cod reference (Forberg et al 2011) was fine and did not need to be removed.

Line 116-119: Hatching timing does not indicate viability. These are two separate measurements. Revise line 119 to indicate that you measuring timing to hatch.

Line 120-123 belong in discussion, not results.

Figure 3: axis should be labeled Time (days post exposure)

Figure S5: Panel A, left side images of GF and CV are in different orientations. Panel B, GF and Conv are in different parts of the plane of the fish, making comparisons difficult. In the right figure of panel B, the bottom image is either a different part of the gut or something else is going on because the folds are missing from the top of the image (on a closer examination, that may be the lumen of the gut. Indicate in the left images where the zoomed in images are from, if they are from the images on the left of the same panel). The folds in the infected CV fish also look smaller and more spaced out than the folds in the GF fish in the right side images of panel B. This may be due to the location in the gut that the image was taken, but that also appears to the be the case in the left images.

Figure S5: Scale bars are needed.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Stefan Oehlers

Reviewer #2: No

PLoS Pathog. doi: 10.1371/journal.ppat.1009302.r004

Acceptance letter

Karen Guillemin, Nina R Salama

24 Jan 2021

Dear Prof. GHIGO,

We are delighted to inform you that your manuscript, "Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Flavobacterium sp. strain 4466 taxonomic identification based on genomic similarities.

(PDF)

Click here for additional data file.^{(110.5KB, pdf)}

S2 Table. Flavobacterium species genomes retrieved from public databases.

(PDF)

Click here for additional data file.^{(125.5KB, pdf)}

S1 Fig. Sterility test of rainbow trout larvae raised under GF and Conv conditions.

(PDF)

Click here for additional data file.^{(1.4MB, pdf)}

S2 Fig. Growth performance of rainbow trout larvae raised under GF and Conv conditions.

(PDF)

Click here for additional data file.^{(233.4KB, pdf)}

S3 Fig. Anatomical comparison of the gut of Conv and GF rainbow trout larvae.

(PDF)

Click here for additional data file.^{(2.3MB, pdf)}

S4 Fig. Survival of re-conventionalized trout larvae against F. columnare Fc7 infection.

(PDF)

Click here for additional data file.^{(246.1KB, pdf)}

S5 Fig. Histological comparison of the gut of infected and non-infected Conv and GF rainbow trout larvae.

(PDF)

Click here for additional data file.^{(2.4MB, pdf)}

S6 Fig. Phylogenetic tree illustrating the relationship between Flavobacterium sp. strain 4466 and the closest 15 Flavobacterium species based on ANI analysis.

(PDF)

Click here for additional data file.^{(625.9KB, pdf)}

Attachment

Submitted filename: Perez-Pascual.PPATHOGENS-D-20-01341R.Resp to reviewers.VF.docx

Click here for additional data file.^{(12MB, docx)}

Data Availability Statement

The genome of Flavobacterium sp. 4466 was deposited at European Nucleotide Archive (ENA) databank, accession number: ERS4574862, https://www.ebi.ac.uk/ena/browser/submit.

[ppat.1009302.ref001] 1.FAO. The state of World Fisheries and Aquaculture. Rome: Food and Agriculture Organization of the United Nations, 2016 [Google Scholar]

[ppat.1009302.ref002] 2.Vadstein O, Bergh Ø, Gatesoupe F-J, Galindo-Villegas J, Mulero V, Picchietti S, et al. Microbiology and immunology of fish larvae. Reviews in Aquaculture. 2013;5(s1):S1–S25. 10.1111/j.1753-5131.2012.01082.x [DOI] [Google Scholar]

[ppat.1009302.ref003] 3.Embregts CW, Forlenza M. Oral vaccination of fish: Lessons from humans and veterinary species. Dev Comp Immunol. 2016;64:118–37. Epub 2016/03/29. 10.1016/j.dci.2016.03.024 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref004] 4.Perez-Sanchez T, Mora-Sanchez B, Balcazar JL. Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges. Trends Microbiol. 2018;26(11):896–903. Epub 2018/05/29. 10.1016/j.tim.2018.05.002 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref005] 5.Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A, et al. Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health. Environmental Microbiology. 2013;15(7):1917–42. 10.1111/1462-2920.12134 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref006] 6.Santos L, Ramos F. Antimicrobial resistance in aquaculture: Current knowledge and alternatives to tackle the problem. International Journal of Antimicrobial Agents. 2018;52(2):135–43. 10.1016/j.ijantimicag.2018.03.010 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref007] 7.Brandt KK, Amezquita A, Backhaus T, Boxall A, Coors A, Heberer T, et al. Ecotoxicological assessment of antibiotics: A call for improved consideration of microorganisms. Environ Int. 2015;85:189–205. 10.1016/j.envint.2015.09.013 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref008] 8.Defoirdt T, Sorgeloos P, Bossier P. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr Opin Microbiol. 2011;14(3):251–8. 10.1016/j.mib.2011.03.004 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref009] 9.Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiology and Molecular Biology Reviews. 2000;64(4):655–71. 10.1128/mmbr.64.4.655-671.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref010] 10.Tinh NT, Dierckens K, Sorgeloos P, Bossier P. A review of the functionality of probiotics in the larviculture food chain. Mar Biotechnol (NY). 2008;10(1):1–12. 10.1007/s10126-007-9054-9 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref011] 11.Martínez Cruz P, Ibáñez AL, Monroy Hermosillo OA, Ramírez Saad HC. Use of probiotics in aquaculture. ISRN microbiology. 2012;2012:916845–. 10.5402/2012/916845 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref012] 12.Buffie CG, Pamer EG. Microbiota-mediated colonization resistance against intestinal pathogens. Nature Reviews Immunology. 2013;13:790 10.1038/nri3535 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref013] 13.Stecher B, Hardt W-D. Mechanisms controlling pathogen colonization of the gut. Current Opinion in Microbiology. 2011;14(1):82–91. 10.1016/j.mib.2010.10.003 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref014] 14.de Bruijn I, Liu Y, Raaijmakers JM, Wiegertjes GF. Exploring fish microbial communities to mitigate emerging diseases in aquaculture. FEMS Microbiology Ecology. 2017;94(1). 10.1093/femsec/fix161 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref015] 15.Gomez-Gil B, Roque A, Turnbull JF. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture. 2000;191(1):259–70. 10.1016/S0044-8486(00)00431-2. [DOI] [Google Scholar]

[ppat.1009302.ref016] 16.Xiong JB, Nie L, Chen J. Current understanding on the roles of gut microbiota in fish disease and immunity. Zool Res. 2019;40(2):70–6. 10.24272/j.issn.2095-8137.2018.069 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref017] 17.Hai NV. The use of probiotics in aquaculture. Journal of Applied Microbiology. 2015;119(4):917–35. 10.1111/jam.12886 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref018] 18.Lazado CC, Caipang CMA. Mucosal immunity and probiotics in fish. Fish & Shellfish Immunology. 2014;39(1):78–89. 10.1016/j.fsi.2014.04.015 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref019] 19.Dierckens K, Rekecki A, Laureau S, Sorgeloos P, Boon N, Van den Broeck W, et al. Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae. Environmental Microbiology. 2009;11(2):526–33. 10.1111/j.1462-2920.2008.01794.x [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref020] 20.Fiebiger U, Bereswill S, Heimesaat MM. Dissecting the Interplay Between Intestinal Microbiota and Host Immunity in Health and Disease: Lessons Learned from Germfree and Gnotobiotic Animal Models. Eur J Microbiol Immunol (Bp). 2016;6(4):253–71. 10.1556/1886.2016.00036 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref021] 21.Yi P, Li L. The germfree murine animal: An important animal model for research on the relationship between gut microbiota and the host. Veterinary Microbiology. 2012;157(1):1–7. 10.1016/j.vetmic.2011.10.024 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref022] 22.Caruffo M, Navarrete NC, Salgado OA, Faundez NB, Gajardo MC, Feijoo CG, et al. Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae. Front Cell Infect Microbiol. 2016;6:127 10.3389/fcimb.2016.00127 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref023] 23.Qin C, Zhang Z, Wang Y, Li S, Ran C, Hu J, et al. EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish. Frontiers in Microbiology. 2017;8(2406). 10.3389/fmicb.2017.02406 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref024] 24.Rendueles O, Ferrieres L, Fretaud M, Begaud E, Herbomel P, Levraud JP, et al. A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria. PLoS Pathog. 2012;8(7):e1002815 10.1371/journal.ppat.1002815 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref025] 25.Forberg T, Arukwe A, Vadstein O. A protocol and cultivation system for gnotobiotic Atlantic cod larvae (Gadus morhua L.) as a tool to study host microbe interactions. Aquaculture. 2011;315(3):222–7. 10.1016/j.aquaculture.2011.02.047. [DOI] [Google Scholar]

[ppat.1009302.ref026] 26.Verner-Jeffreys DW, Shields RJ, Birkbeck TH. Bacterial influences on Atlantic halibut Hippoglossus hippoglossus yolk-sac larval survival and start-feed response. Dis Aquat Organ. 2003;56(2):105–13. 10.3354/dao056105 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref027] 27.Vestrum R.I., Luef B., Forberg T., Bakke I., Emerging O. V. Issues in Fish Larvae Research. In: M. Y, editor.: Springer; 2018. [Google Scholar]

[ppat.1009302.ref028] 28.Quesada SP, Paschoal JAR, Reyes FGR. Considerations on the Aquaculture Development and on the Use of Veterinary Drugs: Special Issue for Fluoroquinolones—A Review. Journal of Food Science. 2013;78(9):R1321–R33. 10.1111/1750-3841.12222 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref029] 29.Declercq AM, Haesebrouck F, Van den Broeck W, Bossier P, Decostere A. Columnaris disease in fish: a review with emphasis on bacterium-host interactions. Veterinary Research. 2013;44(1):27 10.1186/1297-9716-44-27 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref030] 30.Butt RL, Volkoff H. Gut Microbiota and Energy Homeostasis in Fish. Frontiers in endocrinology. 2019;10:9–. 10.3389/fendo.2019.00009 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref031] 31.Austin B, Austin DA. Bacterial fish pathogens: disease of farmed and wild fish. 4th ed Dordrecht Chichester: Springer; Published in association with Praxis Pub.; 2007. xxvi, 552 p. p. [Google Scholar]

[ppat.1009302.ref032] 32.Russell Alistair B, Wexler Aaron G, Harding Brittany N, Whitney John C, Bohn Alan J, Goo Young A, et al. A Type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism. Cell Host & Microbe. 2014;16(2):227–36. 10.1016/j.chom.2014.07.007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref033] 33.Konstantinidis KT, Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(7):2567–72. 10.1073/pnas.0409727102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref034] 34.Stressmann FA, Bernal-Bayard J, Perez-Pascual D, Audrain B, Rendueles O, Briolat V, et al. Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection. The ISME Journal 2020. Epub 2020/10/21. 10.1038/s41396-020-00807-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref035] 35.Banerjee G, Ray AK. The advancement of probiotics research and its application in fish farming industries. Research in Veterinary Science. 2017;115:66–77. 10.1016/j.rvsc.2017.01.016 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref036] 36.Hoseinifar SH, Sun Y-Z, Wang A, Zhou Z. Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives. Frontiers in Microbiology. 2018;9(2429). 10.3389/fmicb.2018.02429 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref037] 37.Rawls JF, Samuel BS, Gordon JI. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(13):4596–601. 10.1073/pnas.0400706101 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref038] 38.Milligan-Myhre K, Small CM, Mittge EK, Agarwal M, Currey M, Cresko WA, et al. Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations. Disease Models & Mechanisms. 2016;9(2):187–98. 10.1242/dmm.021881 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref039] 39.Forberg T, Milligan-Myhre K. Chapter 6—Gnotobiotic Fish as Models to Study Host–Microbe Interactions. In: Schoeb TR, Eaton KA, editors. Gnotobiotics: Academic Press; 2017. p. 369–83. [Google Scholar]

[ppat.1009302.ref040] 40.Rekecki A, Dierckens K, Laureau S, Boon N, Bossier P, Van den Broeck W. Effect of germ-free rearing environment on gut development of larval sea bass (Dicentrarchus labrax L.). Aquaculture. 2009;293(1):8–15. 10.1016/j.aquaculture.2009.04.001. [DOI] [Google Scholar]

[ppat.1009302.ref041] 41.Olivares-Fuster O, Baker JL, Terhune JS, Shoemaker CA, Klesius PH, Arias CR. Host-specific association between Flavobacterium columnare genomovars and fish species. Systematic and Applied Microbiology. 2007;30(8):624–33. 10.1016/j.syapm.2007.07.003 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref042] 42.Elsabagh M, Mohamed R, Moustafa EM, Hamza A, Farrag F, Decamp O, et al. Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquaculture Nutrition. 2018;24(6):1613–22. 10.1111/anu.12797. [DOI] [Google Scholar]

[ppat.1009302.ref043] 43.Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE, Guillemin K. Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation. Developmental Biology. 2006;297(2):374–86. 10.1016/j.ydbio.2006.05.006 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref044] 44.De Schryver P, Vadstein O. Ecological theory as a foundation to control pathogenic invasion in aquaculture. The Isme Journal. 2014;8:2360 10.1038/ismej.2014.84 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref045] 45.Zhu J, Dai W, Qiu Q, Dong C, Zhang J, Xiong J. Contrasting Ecological Processes and Functional Compositions Between Intestinal Bacterial Community in Healthy and Diseased Shrimp. Microbial Ecology. 2016;72(4):975–85. 10.1007/s00248-016-0831-8 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref046] 46.He S, Wang Q, Li S, Ran C, Guo X, Zhang Z, et al. Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis. Science China Life Sciences. 2017;60(11):1260–70. 10.1007/s11427-016-9072-6 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref047] 47.Brugiroux S, Beutler M, Pfann C, Garzetti D, Ruscheweyh HJ, Ring D, et al. Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium. Nat Microbiol. 2016;2:16215 Epub 2016/11/22. 10.1038/nmicrobiol.2016.215 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref048] 48.Kim YG, Sakamoto K, Seo SU, Pickard JM, Gillilland MG 3rd, Pudlo NA, et al. Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens. Science. 2017;356(6335):315–9. Epub 2017/04/22. 10.1126/science.aag2029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref049] 49.Little AEF, Robinson CJ, Peterson SB, Raffa KF, Handelsman J. Rules of Engagement: Interspecies Interactions that Regulate Microbial Communities. Annual Review of Microbiology. 2008;62(1):375–401. 10.1146/annurev.micro.030608.101423 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref050] 50.Stecher B, Chaffron S, Kappeli R, Hapfelmeier S, Freedrich S, Weber TC, et al. Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog. 2010;6(1):e1000711 Epub 2010/01/12. 10.1371/journal.ppat.1000711 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref051] 51.Boutin S, Bernatchez L, Audet C, Derôme N. Antagonistic effect of indigenous skin bacteria of brook charr (Salvelinus fontinalis) against Flavobacterium columnare and F. psychrophilum. Veterinary Microbiology. 2012;155(2):355–61. 10.1016/j.vetmic.2011.09.002 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref052] 52.Seghouani H, Garcia-Rangel C-E, Füller J, Gauthier J, Derome N. Walleye Autochthonous Bacteria as Promising Probiotic Candidates against Flavobacterium columnare. Frontiers in Microbiology. 2017;8(1349). 10.3389/fmicb.2017.01349 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref053] 53.Chatzidaki-Livanis M, Geva-Zatorsky N, Comstock LE. Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species. Proceedings of the National Academy of Sciences. 2016;113(13):3627 10.1073/pnas.1522510113 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref054] 54.Bernardet J-F, Bowman JP. The Genus Flavobacterium. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass New York, NY: Springer New York; 2006. p. 481–531. [Google Scholar]

[ppat.1009302.ref055] 55.Loch TP, Faisal M. Emerging flavobacterial infections in fish: A review. Journal of Advanced Research. 2015;6(3):283–300. 10.1016/j.jare.2014.10.009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref056] 56.Lowrey L, Woodhams DC, Tacchi L, Salinas I. Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin. Appl Environ Microbiol. 2015;81(19):6915–25. Epub 2015/07/26. 10.1128/AEM.01826-15 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref057] 57.Boirivant M, Strober W. The mechanism of action of probiotics. Curr Opin Gastroenterol. 2007;23(6):679–92. 10.1097/MOG.0b013e3282f0cffc . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref058] 58.Wang C, Chuprom J, Wang Y, Fu L. Beneficial bacteria for aquaculture: nutrition, bacteriostasis and immunoregulation. J Appl Microbiol. 2019. Epub 2019/07/16. 10.1111/jam.14383 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref059] 59.Pérez-Sánchez T, Ruiz-Zarzuela I, de Blas I, Balcázar JL. Probiotics in aquaculture: a current assessment. Reviews in Aquaculture. 2014;6(3):133–46. 10.1111/raq.12033 [DOI] [Google Scholar]

[ppat.1009302.ref060] 60.Huber I, Spanggaard B, Appel KF, Rossen L, Nielsen T, Gram L. Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum). Journal of Applied Microbiology. 2004;96(1):117–32. 10.1046/j.1365-2672.2003.02109.x [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref061] 61.Huyben D, Sun L, Moccia R, Kiessling A, Dicksved J, Lundh T. Dietary live yeast and increased water temperature influence the gut microbiota of rainbow trout. Journal of Applied Microbiology. 2018;124(6):1377–92. 10.1111/jam.13738 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref062] 62.Araújo C, Muñoz-Atienza E, Nahuelquín Y, Poeta P, Igrejas G, Hernández PE, et al. Inhibition of fish pathogens by the microbiota from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment. Anaerobe. 2015;32:7–14. 10.1016/j.anaerobe.2014.11.001 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref063] 63.Brunt J, Austin B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases. 2005;28(12):693–701. 10.1111/j.1365-2761.2005.00672.x [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref064] 64.Mohammadian T, Nasirpour M, Tabandeh MR, Heidary AA, Ghanei-Motlagh R, Hosseini SS. Administrations of autochthonous probiotics altered juvenile rainbow trout Oncorhynchus mykiss health status, growth performance and resistance to Lactococcus garvieae, an experimental infection. Fish & Shellfish Immunology. 2019;86:269–79. 10.1016/j.fsi.2018.11.052 [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref065] 65.Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I, et al. Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae. Journal of Fish Diseases. 2011;34(7):499–507. 10.1111/j.1365-2761.2011.01260.x [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref066] 66.Lemon KP, Armitage GC, Relman DA, Fischbach MA. Microbiota-targeted therapies: an ecological perspective. Sci Transl Med. 2012;4(137):137rv5 10.1126/scitranslmed.3004183 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref067] 67.Newaj-Fyzul A, Al-Harbi AH, Austin B. Review: Developments in the use of probiotics for disease control in aquaculture. Aquaculture. 2014;431:1–11. 10.1016/j.aquaculture.2013.08.026. [DOI] [Google Scholar]

[ppat.1009302.ref068] 68.Vergin KL, Urbach E, Stein JL, DeLong EF, Lanoil BD, Giovannoni SJ. Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl Environ Microbiol. 1998;64(8):3075–8. Epub 1998/08/04. 10.1128/AEM.64.8.3075-3078.1998 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref069] 69.Michel C, Messiaen S, Bernardet J-F. Muscle infections in imported neon tetra, Paracheirodon innesi Myers: limited occurrence of microsporidia and predominance of severe forms of columnaris disease caused by an Asian genomovar of Flavobacterium columnare. Journal of Fish Diseases. 2002;25(5):253–63. 10.1046/j.1365-2761.2002.00364.x [DOI] [Google Scholar]

[ppat.1009302.ref070] 70.Evenhuis JP, Mohammed H, LaPatra SE, Welch TJ, Arias CR. Virulence and molecular variation of Flavobacterium columnare affecting rainbow trout in Idaho, USA. Aquaculture. 2016;464:106–10. 10.1016/j.aquaculture.2016.06.017. [DOI] [Google Scholar]

[ppat.1009302.ref071] 71.LaFrentz BR, García JC, Waldbieser GC, Evenhuis JP, Loch TP, Liles MR, et al. Identification of Four Distinct Phylogenetic Groups in Flavobacterium columnare With Fish Host Associations. Frontiers in microbiology. 2018;9:452–. 10.3389/fmicb.2018.00452 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref072] 72.Olivares-Fuster O, Bullard SA, McElwain A, Llosa MJ, Arias CR. Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge. Diseases of aquatic organisms. 2011;96(3):221–7. 10.3354/dao02371 . [DOI] [PubMed] [Google Scholar]

[ppat.1009302.ref073] 73.Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology. 2017;67(5):1613–7. 10.1099/ijsem.0.001755 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref074] 74.Sung-Min H, Chang Ki K, Juhye R, Jung-Hyun B, Seung-Jo Y, Seon-Bin C, et al. Application of the Whole Genome-Based Bacterial Identification System, TrueBac ID, Using Clinical Isolates That Were Not Identified With Three Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Systems. Annals of Laboratory Medicine. 2019;39(6):530–6. Epub 2019/11/01. 10.3343/alm.2019.39.6.530 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref075] 75.Aronesty E. Command-line tools for processing biological sequencing data. ea-utils [Internet]. 2011. Available from: https://github.com/ExpressionAnalysis/ea-utils.

[ppat.1009302.ref076] 76.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology: a journal of computational molecular cell biology. 2012;19(5):455–77. Epub 2012/04/18. 10.1089/cmb.2012.0021 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref077] 77.Segata N, Bornigen D, Morgan XC, Huttenhower C. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nat Commun. 2013;4:2304 Epub 2013/08/15. 10.1038/ncomms3304 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref078] 78.Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. Epub 2014/01/24. 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009302.ref079] 79.Renier N, Wu Z, Simon David J, Yang J, Ariel P, Tessier-Lavigne M. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell. 2014;159(4):896–910. 10.1016/j.cell.2014.10.010 [DOI] [PubMed] [Google Scholar]

PERMALINK

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection

David Pérez-Pascual

Sol Vendrell-Fernández

Bianca Audrain

Joaquín Bernal-Bayard

Rafael Patiño-Navarrete

Vincent Petit

Dimitri Rigaudeau

Jean-Marc Ghigo

Roles

Abstract

Author summary

Introduction

Results

Germ-free trout show normal development and growth compared to conventional larvae

Fig 1. Protocol used in this study to raise and infect or re-conventionalize germ-free (GF) trout larvae.

Fig 2. Anatomical comparison of Conventional (Conv) and GF rainbow trout larvae.

Identification of susceptibility to fish pathogens in germ-free but not conventional trout larvae

Fig 3. Survival of GF and Conv rainbow trout larvae infected with different fish pathogens.

Fig 4. Survival of re-conventionalized trout larvae against F. columnare Fc7 infection.

Conventional rainbow trout microbiota protects against F. columnare infection

Table 1. The 11 strains isolated from Conv rainbow trout larvae.

Resistance to F. columnare infection is conferred by one member of the trout microbiota

Fig 5. Protection of GF trout larvae against F. columnare infection by individual species isolated from the Conv rainbow trout microbiota.

Fig 6. Representative images of in vitro growth-inhibition activity of Flavobacterium sp. strain 4466 against different virulent F. columnare strains.

Endogenous Flavobacterium sp. strain 4466 protects germ-free rainbow trout against infection by different strains of F. columnare

Fig 7. Strain 4466 provides full protection to gnotobiotic rainbow trout larvae against infection by four strains of F. columnare.

Use of germ-free trout model to validate exogenous probiotics protecting against F. columnare infection

Fig 8. C. massiliae provides trout protection against F. columnare infection.

Discussion

Materials and methods

Ethics statement

Handling of rainbow trout larvae

Sterilization and raising of germ-free rainbow trout

Bacterial strains and growth conditions

Fish infection challenge

Characterization of culturable conventional rainbow trout microbiota

Whole genome sequencing

Phylogenomic analysis

Germ free rainbow trout microbial re-conventionalization

Histological analyzes

Whole fish clearing and 3D imaging

Agar overlay assay for growth inhibition detection

Statistical methods

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Karen Guillemin

Nina R Salama

Roles

Author response to Decision Letter 0

Decision Letter 1

Karen Guillemin

Nina R Salama

Roles

Acceptance letter

Karen Guillemin

Nina R Salama

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases