Synergistic effects of commensals and phage predation in suppressing colonization by pathogenic Vibrio parahaemolyticus

Commensal intestinal bacteria can protect shrimp from the pathogenic Vibrio infection

To investigate the role of commensal bacteria in protecting shrimp from pathogenic Vibrio infections, we reconstructed a simplified, yet ecologically representative, version of the shrimp gut microbiota in vitro. Based on V3-V4 of 16S rRNA gene amplicon sequencing from healthy shrimp in our previous work¹⁹, we identified four dominant bacterial phyla including Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. From these groups, we curated a panel of twelve cultivable strains, each isolated from the healthy individual gut and taxonomically classified via whole-genome sequencing (see Methods) (Supplementary Table 1). These isolates formed a synthetic consortium, which we termed “Com12”, designed to capture key ecological and functional diversity of the native microbiota.

To examine interactions between commensal and pathogenic bacteria, we conducted in vitro co-culture experiments with Com12 consortium and two Vibrio strains: one pathogenic (Vibrio parahaemolyticus strain VP6) and one putatively beneficial (Vibrio spp. VA3). Initially, taxonomic characterization of these two Vibrio strains was performed via whole-genome sequencing, and phenotypic classification was based on the presence of virulence-associated genes and mortality assays. Genome annotation revealed that VP6 carries the pirAB toxin gene, whereas VA3 lacks this virulence determinant, a finding further confirmed by genetic analysis (Supplementary Fig. 1). Infection experiments demonstrated that VP6 induced significant mortality and vibriosis-specific symptoms, yet shrimp exposed to VA3 exhibited no detectable difference in survival relative to the control group (Fig. 1a, Supplementary Fig. 2). Co-culture experiments further revealed that both Vibrio strains significantly altered the structure of the Com12 consortium, with each strain becoming dominant within the microbial community (Supplementary Fig. 3). Notably, VA3 exhibited a distinct growth advantage over VP6 in Com12, suggesting it may competitively inhibit VP6 colonization.

To further assess the protective role of VA3, we pretreated shrimp with antibiotics to minimize the native microbiota and subsequently recolonized some individuals with the Com12 consortium, −+ VA3, at a concentration of 5 × 10^6 CFU/mL, a dosage informed by previous studies^20,21. All shrimp were then challenged with VP6 (5 × 10^6 CFU/mL). By day 5, the VP6-only group exhibited 100% mortality (Fig. 1b). In contrast, both the Com12 + VA3 and Com12-only groups showed increased survival, with the former achieving a significantly higher survival rate (69%) compared to the Com12-only group (49%, P < 0.05; Fig. 1b). These results highlight the role of VA3 in enhancing the Com12-based resistance to VP6 infection. However, further investigations are needed to determine whether VA3 alone is sufficient to confer protection in the absence of Com12 − an open question that warrants deeper exploration in future studies.

In addition, to explore the potential of phage therapy in augmenting microbiota-mediated colonization resistance, we isolated an obligate lytic myovirus, VP6phageC, using VP6 as the host (Supplementary Fig. 4a, b). The host range of VP6phageC was assessed against each monospecies within the Com12 consortium, including VA3, using phage infection assays²². As expected, VP6phageC failed to form plaques on any Com12 members and VA3 (Supplementary Table 2), confirming its strict specificity for VP6. This specificity makes VP6phageC as a promising candidate for investigating whether a combined approach—leveraging phage predation alongside microbiota modulation, can further enhance shrimp defenses against VP6 colonization.

Commensal bacteria and phage can supress Vibrio pathogen growth

To investigate the interplay between commensal bacteria and phage in resisting Vibrio pathogen invasion, we employed the Com12 consortium as a model system to simulate pathogen invasion in vitro and evaluate the combined effects of commensal-derived colonization resistance and phage predation. These experiments were performed on a 96-well microplate platform, using time-resolved measurements of mono-species to track the dynamic changes over 48-h period (Fig. 2a, see “Methods”).

a Schematic of the in vitro experimental design to stimulate pathogen invasion. The phylogenetically diverse Com12 consortium (12 species) was constructed with approximatedly equal initial optical densities (OD600). Relative abundance was assessed via V4-16S rRNA gene sequencing (see “Methods”). In invasion assays, VP6 culture was added at 1:1 ratio to Com12. Samples were collected at 0 h, 2 h, 6 h, 12 h, 24 h, 36 h, and 48 h post-invasion for sequencing. b Temporal dynamics of the Com12 strains, unexposed (left) versus exposed (right) to VP6. Stacked plots show strain-level relative abundance over time (y-axis: % abundance, x-axis: hours). Unless otherwise noted, data represent the mean of three biological replicates per condition (also apply to the panel c). c Temporal dynamics of Com12 strains exposed to pathogen VP6 and additional treatments: +VA3 (left), +Phage (center), and +VA3 and Phage (right). Plot shows strains-level relative abundance over time induced by individual or combined interventions. d Correlation between the community diversity (Shannon index) and VP6 abundance over time. Relationship between temporal changes in community diversity dynamics within the Com12 consortium and VP6 abundance at 2, 6, 12, 24, 36, and 48 h. Linear regression analysis was used to evaluation correlations between the dynamics of overall community diversity within the Com12 consortium and VP6 abundance, with R-square and P values (from two-sided ttests on regression coefficients) provided for each treatment: +VA3 (left), +Phage (center), and +VA3 and Phage (right). Colored circles represent data from different time points. Solid gray lines represent fitted correlations from linear regression (VP6 abundance ~ Shannon diversity + group), see “Methods”). Statistics: two-sided ttest.

Commensal-mediated resistance was evaluated by co-culturing Com12 and Com12 + VA3 with VP6 and monitoring strain abundance over time. VP6 rapidly dominated Com12, reaching 70% relative abundance within 6 h before stabilizing (Fig. 2b). However, the presence of VA3 significantly restricted VP6 expansion, limiting its abundance to 15%. Phage addition further suppressed VP6 to 5%, and the combination of VA3 and phage nearly eliminated VP6, reducing its abundance to less than 1% (Fig. 2c).

Further analysis of the co-culture dynamics revealed significant shifts in both bacterial abundance and overall community diversity. The introduction of VA3, phage, or their combination led to a marked reduced in VP6 abundance, accompanied by increased abundance of other strains within the Com12 consortium (Fig. 2d). To explore how VP6 suppression relates to overall community structure and community diversity, we performed linear regression analyses between the relative abundance of VP6 and community diversity, measured using the Shannon index. Importantly, the diversity metric included all community members, including VA3 and VP6, to reflect the total ecological outcome under each treatment condition. In the VA3-only treatment, the correlation between VP6 abundance and diversity was weak and statistically non-significant (R-square = 0.03, F-statistics = 0.51, P = 0.486). In contrast, phage treatment showed a positive correlation with diversity (R-square = 0.60, F-statistics = 24.30, P < 0.001). When VA3 and phage were combined, while the correlation between VP6 abundance and diversity was reduced, the positive relationship between diversity and pathogen suppression was maintained (R-square = 0.68, F-statistics = 33.30, P < 0.001) (Fig. 2d).

These results demonstrate that while Com12 alone impose a threshold on VP6 colonization, VA3 and phage act as potent inhibitors, with their combined application synergistically enhance colonization resistance. Importantly, phage contributed to increase microbial diversity, whereas VA3 appears to influence pathogen abundance without directly altering diversity, suggesting complementary mechanisms in pathogen exclusion. Together, these results underscore the potential of integrating commensals and phages as strategy to fortify maintaining microbiome stability and prevent pathogen invasion.

Timing of commensal and phage administration is critial for effective colonization resistance

In our study of the Com12 consortium, we identified a priority effect that influenced pathogen invasion, particularly when VP6 was introduced at different stages of Com12 growth. To evaluate how phage treatment could restore the consortium’s resistance following VP6 invasion, we reconducted co-culture experiments where Com12 was exposed to VP6, and VP6phageC (MOI = 1) was introduced at various time points (see “Methods”). Our findings indicated that the timing of phage introduction significantly affected its efficacy (Fig. 3a). More specifically, when VP6 was co-cultured with Com12 for 6 h or more before the addition of phage, the suppressive effect of the phage was notably diminished, with VP6 relative abundance surged to 70% of the community, suggesting that phage-mediated suppression was less effective after this time window (Fig. 3b). A comparison of absolute VP6 concentrations using copy numbers (unless otherwise specified) in the consortium further corroborated this observation, showing a marginal but not statistically significant reduction in VP6 when phage was added after 6 h (P > 0.05) (Fig. 3c). These results suggested that the efficacy of phage treatment is compromised once VP6 has had a chance to establish itself within the consortium for an extended period.

a Schematic of the in vitro experimental design modeling pathogen invasion in commensal consortium. Phage lysate (1:1 ratio, MOI = 1) was introduced to the Com12 consortium at specified timepoints. Samples were collected at 0 h, 2 h, 6 h, 12 h, 24 h, 36 h, and 48 h for sequencing. E.coli MG1655 (3.65 × 10^6 CFU) served as an interior marker (see “Methods”). b Temporal abundance dynamics of Com12 strains following pathogen VP6 exposure, with phage introduced at specific time points. Phage addition times are indicated above each subplot (also apply to panels d and f). Unless otherwise noted, data represent the mean of three biological replicates per condition (also apply to panels d and f). c Temporal quantification dynamics of Com12 strains following pathogen VP6 exposure, with phage introduced at specific time points. Data points represent Com12 strains (salmon) and VP6 (cyan) concentrations at indicated intervals (also applies to panels e and g). Box plots show the interquartile range with the median indicated by in line. Individual data points represent biological replicates measured at multiple time points (n = 3 per time point). Statistics: Tukey’s HSD test (NS, P > 0.05; *P < 0.01; ***P < 0.0001); only non-significant group comparison are shown. This format also applies to panels (e and g). d Temporal abundance dynamics of Com12 strains following pathogen VP6 exposure, with VA3 pretreatment and timed phage addition. e Temporal quantification dynamics of Com12 strains following pathogen VP6 exposure, with VA3 pretreatment and timed phage addition. Data points represent Com12 strains (salmon) and VP6 (cyan) concentrations at indicated intervals. f Temporal abundance dynamics of Com12 strains with timed pathogen VP6 introduction. g Temporal quantification dynamics of Com12 strains with timed pathogen VP6 introduction. Data points represent Com12 strains (salmon) and VP6 concentrations (cyan) at indicated intervals.

Next, we explored the potential for combining certain commensal species with phage-specific predation to further bolster colonization resistance. Specifically, we assessed the effects of introducing commensal VA3, alongside the lytic phage VP6phageC. In these experiments, VA3 was introduced into Com12 consortium following VP6 invasion, with concurrent VP6phageC treatment. Monitoring of the consortium dynamics revealed significant inhibition of VP6 by this combined treatment. While VA3 alone significantly reduced the relative abundance of VP6 to 15% and its biomass to 5×10^7 CFU/mL within 48 h (Fig. 2c), the combined treatment of VA3 and VP6phageC further amplified this inhibitory effect. In this dual intervention, VP6 proliferation was almost entirely eradicated, with its relative abundance dropping below 1% and biomass reduced to less than 1 × 10^6 CFU/mL (Fig. 3d, e). Even when phage was introduced after a 6-h delay, both VP6 relative abundance and biomass remained significantly lower compared to treatments using either VA3 or phage alone (P < 0.001) (Fig. 3d, e). These results underscore that although phage-mediated pathogen suppression is highly time-dependent, its effectiveness can be synergistically enhanced when combined with commensal bacteria such as VA3, which together provide robust colonization resistance and protect the microbiota from pathogen invasion.

To further investigate whether the Com12 consortium itself possesses intrinsic, self-regulated resistance to pathogen invasion, we conducted an additional experiment by introducing VP6 at various stages of Com12 growth. Relative abundance analyses revealed a notable difference in outcomes depending on the timing of VP6 introduction. When VP6 was co-cultured with Com12 from the start (0 h), it quickly dominated the consortium as reflected in its high relative abundance and biomass (Fig. 2b, upper right). In contrast, when VP6 was introduced after 2 h of Com12 growth, its proliferation was irreversibly inhibited, with VP6’s absolute concentration falling to less than 1 × 10^5 CFU/mL, and its relative abundance dropped below 1% (Fig. 3f, g).

Overall, these findings underscore a crucial, timing-dependent trait of colonization resistance within the consortium, suggesting that early establishment of the commensal consortium provided a robust barrier against pathogen invasion, emphasizing the importance of microbial community maturation. Conversely, when the pathogen was allowed to establish dominance before the consortium had fully matured, the protective capacity was significantly compromised.

Commensal bacteria supress pathogen by nutrient competition and prophage induction

To explore the potential mechanisms underlying the observed colonization resistance in above co-culturing experiments (Figs. 2, 3), we investigated pairwise interactions among members of the consortium Com12, including V. parahaemolyticus (VP6) and Vibrio spp.(VA3), using a conditional coculturing approach²³. In this experiment, each species was grown in cell-free spent media collected from other species, supplemented with 60% full-nutrient Marine Broth (2216MB). This experimental design eliminated direct cell-cell contact as a potential mechanism for colonization resistance, allowing us to focus solely on metabolic-mediated interactions.

Analysis of the growth rate and maximum biomass (OD600) of each species grown in spent media from other community members versus self-derived media revealed a negative correlation, although the absolute correlation index was less than 1 (Estimate = -0.51, R-square = 0.12, F-statistic = 13.13, P = 2.57e–06) (Fig. 4a, left; Supplementary Fig. 5). This suggests that most interspecies interaction were inhibitory, albeit the extent of inhibition primarily affected total biomass rather than growth rate. When examining the effects of other species in Com12 on VP6, we observed a similar inhibitory trend, but with a stronger negative correlation (Estimate = –1.31, R-square = 0.54, F-statistic = 23.60, P = 0.004) (Fig. 4a, right). Ranking the effects of others on VP6 showed that the inhibitory effects were largely mediated by VA3, particularly when considering growth rate independently of biomass (Fig. 4a, right).

a Growth interference analysis. Scatter plots correlate maximum biomass and growth rate ratios for strains grown in self- versus cross-spent media. More specifically, a strain grown in the spent medium of b exhibited growth rate (Rs) and maximum biomass (Kms), while growth in its own spent medium resulted in growth rate (Ro) and maximum biomass (Kmo). Data are plotted as [ln (Kmo/Kms)] on the x-axis and [ln (Ro/Rs)] on the y-axis. Left: Interactions among commensals, VP6 and VA3 (two-sided t-test; regression line: y = -0.51x+b). Right: Effects of individual commensals within the Com12 consortium and VA3 on VP6 growth (two-sided t-test; regression line: y = -1.3x+b). Solid blue lines indicate linear regression fits; dashed gray lines represent 1:1 reference lines. Linear correlations between the maximum biomass ratio [ln (Kmo/Kms)] and growth rate ratio [ln (Ro/Rs)] are shown with corresponding P values. The position of VA3 is indicated with a red arrow. Statistics: two-sided ttest. b Metabolic pathway-level genomic redundancy. Heatmap shows gene family similarity across Com12, VA3 and VP6. Values are scaled to total genes per family. c Growth competition assay. VA3 and VP6 were co-cultured at equal initial OD600 concentrations. Strain concentration was quantified via morphology-discriminant plating on selective agar plates. Box plots show the interquartile range with the median indicated by in line. Individual data points represent biological replicates (n = 3). d Prophage induction under nutrient stress (Minimal Medium, SM condition). The prophage Vpp2 genome is shown. Transmission electron microscopy (TEM) of Vpp2 virion reveals a filamentous inoviridae phage. Scale bar: 200 nm. Prophage Vpp2 excision quantification by qPCR in SM condition medium (e) or spent medium (f). In panel (e), “Full” refers to VP6 cultured in 2216 marine broth (2216MB). Dashed lines (in e and f) indicated the baseline(y = 1). Statistics (e and f): two-tailed Student’s ttest (NS, P > 0.05; ***P < 0.0001). Bar plots show the mean relative level of prophage excision, with individual data points representing biological replicates. g In vivo protection efficacy of Com12 strains and VA3 on shrimp survival against VP6 exposure. Antibiotic-pretreated shrimp (n = 20 per group) were immersed in cultures of individual strain (5 × 10^6 CFU/mL) for 2 days, prior to VP6 exposure. Survival rates were assessed on day 5. NC (negative control): shrimp treated with antibiotics only. PC (positive control): shrimp exposed to VP6 following antibiotic treatment. The dashed line represents the survival rate of shrimp in the positive control group. Bar plots show the mean survival rate for each treatment group, with individual data points representing biological replicates. Statistics: two-tailed Student’s ttest (NS, P > 0.05; *P < 0.01; ***P < 0.0001).

To further explore the interactions underlying these observations, we employed genome-scale metabolic modeling to assess the functional similarity between VP6 and each member of Com12, including VA3, based on protein composition overlap. Specifically, we quantified the proportion of protein families carried by VP6 that were also shared in each commensal (see “Methods”). Our results highlight that VA3, Rueg, and Tena as key contributors to protein-family overlap with VP6, suggesting their potential role in shaping VP6’s growth dynamics by nutrient competition (Fig. 4b). Notably, VA3, belonging to the same bacterial genus as VP6, exhibited the highest degree of overlap, reinforcing its potential for strong competitive interactions.

To experimentally validate the pairwise interaction between VA3 and VP6, we conducted direct growth competition assays, in which a 1:1 mixture of VA3 and VP6 cells was co-cultured in nutrient-rich medium (2216MB) for 48 h, alongside monoculture controls. VA3 displayed robust growth, achieving cell densities comparable to its monoculture controls (P = 0.52) (Fig. 4c). In contrast, VP6 growth was severely impaired in the presence of VA3, with its cell density significantly reduced by 2- to 8-fold (P = 0.004) during the 48-h competition period (Fig. 4c). Furthermore, VA3 consistently outcompeted VP6 in both total biomass (0.97 vs 0.75) and growth rate (0.13 vs 0.10, by hour) (Fig. 4c, Supplementary Fig. 6). Together, these results align with the suppression observed in Com12 upon the introduction of VA3 (Figs. 2, 3), reinforcing the notion that VA3 inhibits VP6 proliferation. The observed suppression appears to be primarily due to nutrient competition, particularly the overlap in nutrient utilization between the two strains.

A recent study revealed that prophages in Vibrio strains are inducible and play critical roles in strain competition within marine environments²⁴. Inspired by this, we identified two intact prophages in the genome of Vibrio VP6. Under nutrient-limited conditions, one prophage was highly induced, as evidenced by increased read depth in its corresponding genomic region (Fig. 4d). Transmission electron microscopy (TEM) of the filtered supernatant confirmed the presence of filamentous phage particles characteristic of Inoviruses, measuring approximately 1,800–2,000 nm in length and 5 nm in width (Fig. 4d, upper panel). The genome of this phage, termed Vpp2, closely resembled that of filamentous phages based on its size (10,298 bp) and gene annotation (Fig. 4d, lower panel).

To investigate the role of prophage Vpp2 in microbiota interaction, we assessed its induction in conditioned media from 13 donor strains, using media from VP6 as a control. Vpp2 production increased in all conditioned media except that from Deme (Fig. 4e, f). When nutrient-deficient SM buffer was used, Vpp2 production also increased, indicating that nutrient limitation is a key trigger for its activation (Fig. 4e). Co-culturing VP6 with VA3 or Com12 separately revealed continuous induction of Vpp2 over 48 h with VA3, whereas the induction was less pronounced with Com12 (Supplementary Fig. 7). Together, these findings indicate that both Com12 and commensal VA3 promote prophage induction in VP6, with VA3 exhibiting the most robust effect. This induction likely involves nutrient competition, which activates a stress response in VP6, suggesting that prophage activation may be part of a broader ecological strategy that influences the growth dynamics of VP6.

Synergistic interaction of commensal microbes and phage confers colonization resistance against pathogenic Vibrio in shrimp

The dynamics of the Com12 consortium, −+VA3, revealed that individual strains contribute variably to colonization resistance against pathogen invasion, with some strains playing more pivotal roles. To evaluate the protective capacity of each commensal, we further assessed shrimp survival following exposure to pathogen VP6 (Fig. 4g). Shrimp were pretreated with antibiotics as before (Fig. 2g) to minimize the influence of indigenous bacteria and then immersed in cultures of each strain (5×10^6 CFU/mL) or combinations, including Com12 and VA3, prior to VP6 exposure. With this assay system, we could rank the strains based on their abilities to protect shrimp from VP6 infection. Shrimp survival rates were significantly higher when pretreated with VA3 ( ~ 69.0%), Psyc ( ~ 44.0%), Rueg ( ~ 43.0%), or Halo ( ~ 38.0%) compared to the positive control group exposed only to VP6 ( ~ 23.0%). Other strains showed insufficient or adverse effects on survival.

To evaluate whether the subset consortium comprising VA3, Psyc, Rueg, and Halo (Com4) could protect shrimp from VP6 infection and whether this protective effect could be enhanced by phage addition within the complex intestinal microbiome, we let shrimp be colonized with Com4 (5×10^6 CFU/mL per strain) before exposing to VP6 (5×10^6 CFU/mL) (Fig. 5a). Then, the shrimp were maintained under standard aquaculture conditions and fed daily with phage ( ~ 10^9 PFU/g) throughout the experiment. Successful colonization by VP6 causes an acute infection over a five-day period with white hepatopancreas and empty digestive tracts (Supplementary Fig. 8), which is a typical symptom of vibriosis^25,26, whereas shrimp with Com4 could rapidly succumb to the infection.

a Experimental diagram for in vivo intervention. Shrimp were pre-treated with Com4 (5×10^6 CFU/mL per strain) and phage for 2 days prior to VP6 challenge. Phage-supplemented feed was supplied daily( ~ 10^9 PFU/g). Shrimp samples were collected on days 1, 3, and 5 after VP6 exposure, and aquatic water samples were collected daily. b Survival rates of shrimp over 5 days. Groups: NC(untreated); PC (VP6 only); Phage (phage+VP6); Com4+phage (VP6+Com4+phage). Statistics: log-rank test (P < 0.0001). Group sizes were equal (n = 50). Median survival is indicated by black dashed lines. VP6 quantification in aquatic water (c) and in shrimp intestine (d) samples. VP6 quantification was measured by counting colonies on selective TCBS plates. Data are presented as log10 CFU per mL for aquatic water samples and log10 CFU per g for intestinal samples. Points represent the geometric means ± SD (n = 3 ~ 6) at different time points. Intestinal samples were collected on days 3 and 5 (n = 3 ~ 6). In panel (d), statistics: two-tailed Student’s ttest (NS, P > 0.05; ***P < 0.0001). Individual data points represent biological replicates. e Phage susceptibility of VP6 isolates from the shrimp intestine. Percentage of phage-sensitive VP6 colonies (n = 10) from shrimp samples (n = 3) at days 1, 3, 5, 7, and 9 post-teatment. Bars = mean ± SD. Individual data points represent biological replicates. Statitics: pairwise Wilcox test with adjusted P value (NS, not significant, P > 0.05; ***P < 0.0001). f Alpha diversity of the shrimp intestinal microbiome at days 1, 3 and 5 post-infections, assessed using the Shannon index based on bacterial OTUs ( > 97% similarity). Box plots show the interquartile range with the median indicated by in line. Individual data points represent biological replicates (n = 3 ~ 12). Statistics: pairwise Wilcox tests with adjusted P value (NS, not significant, P > 0.05; ***P < 0.0001). g Relative abundance of VP6 and the Com4 strains in the shrimp intestinal samples within different treatment groups based on 16S rRNA gene sequences.

While mortality occurred across all groups following VP6 exposure, the cumulative survival rate of shrimp in the Com4-phage treatment group significantly increased to 58% (P < 0.001) compared to >20% in the VP6-only challenge group (Positive control) (Fig. 5b). The survival rate in the Com4-phage group was also notably higher than in the phage-only treatment group, confirming in vitro findings (Fig. 3) that the combination of commensal bacteria and phage more effectively inhibits VP6 invasion.

In addition, both the phage and the Com4-phage treatments effectively suppressed VP6 colonization in the aquatic environment. Plate counting assays revealed that VP6 became nearly under detectable in water surrounding the shrimp after three days in the Com4-phage treatment group, whereas similar suppression was observed only on the fifth day in the phage-only group (Fig. 5c). Quantitative analysis of VP6 in shrimp intestinal samples collected on day 3 and 5 showed a similar trend: VP6 abundance significantly decreased by over 90% in both treatment groups compared to the positive group (Fig. 5d).

Interestingly, the phage (VP6phageC) and its susceptible Vibrio target (VP6) coexisted in the shrimp intestine of the phage-only treatment group. While most Vibrio strains isolated from gut samples remained susceptible to the wild type phage (Supplementary Fig. 9), the observed increased in phage particles alongside a decrease in Vibrio load suggests that phage-mediated suppression of VP6 was effective but limited within the intestinal environment.

To investigate the effects of the treatment on the shrimp gut microbiome, samples were collected at three time points for further analysis. Alpha diversity, as measured by the Shannon index, decreased in the positive control group but increased in the phage-only and Com4-phage treatment groups (Fig. 5f). Characterization of the microbiome revealed that both Com4 strains and VP6 successfully colonized the shrimp gut (Fig. 5g). Importantly, the relative abundance of VP6 was significantly lower in the Com4-phage treatment group compared to the positive control group and phage-only groups, demonstrating superior pathogen resistance and microbiome recovery in the Com4-phage treatment group.

A ternary plot of bacterial OTUs in shrimp gut samples demonstrated that the four commensal strains, in combination with phage predation, effectively suppressed VP6 colonization (Supplementary Fig. 10). These findings suggest that Com4 strains provide substantial resistance to pathogen colonization in the shrimp gut, complementing the inhibitory effects of phage. Co-occurrence network analysis of the gut microbiomes in the Com4-phage group revealed positive interactions between VP6 and other Vibrio species, including VA3 (Supplementary Fig. 11) This suggests that VP6, VA3, and indigenous Vibrio spp. occupy similar ecological niches within the shrimpgut, potentially contributing to complex competitive dynamics.

Together, these results highlight the synergistic effects of commensal bacteria and phage in enhancing colonization resistance against VP6. The combination of Com4 strains and phage not only improved shrimp survival rates but also restored microbiome diversity and reduced VP6 colonization more effectively than phage treatment alone. This underscores the potential of leveraging commensal-phage synergies to protect aquaculture species from pathogenic infections.