s2fieldsofstudy: Biology Medicine

text:
Isolation, Identification, and Investigation of Pathogenic Bacteria From Common Carp (Cyprinus carpio) Naturally Infected With Plesiomonas shigelloides

Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from Cyprinus carpio having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be Plesiomonas shigelloides. Subsequently, we comprehensively investigated the pathogenicity of P. shigelloides in C. carpio through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that P. shigelloides is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body’s immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of P. shigelloides, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of C. carpio after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of P. shigelloides.

INTRODUCTION
Cyprinus carpio is a valuable and unique fish resource that belongs to the order Cypriniformes and family Cyprinidae (1). As a dominant cyprinid species, C. carpio has been reported to account for almost 10% of the annual freshwater aquaculture production (1,2). However, with the deterioration of the natural environment and the increase in breeding density, the breeding environment of C. carpio has been destroyed. In particular, the increasing number of bacterial diseases has had a significant impact on the breeding industry of C. carpio and caused huge economic losses (3,4). The prevention and control of bacterial diseases are areas that need to be urgently addressed for the healthy development of the C. carpio industry (5). Plesiomonas shigelloides, a member of the Vibrionaceae family, is found in the intestinal tracts of fishes, mammals, and humans (6)(7)(8). P. shigelloides is widely distributed in the aquatic environment and is considered a new pathogen that causes digestive tract inflammation in many aquatic animals (8). P. shigelloides can infect Trionyx sinensis, Takifugu obscurus, and other aquatic animals alone or in combination; this has resulted in great economic losses to the aquatic aquaculture industry and posed a certain safety hazard to consumers (9,10). In recent years, P. shigelloides has been isolated and identified from several aquatic animals, such as Percocypris pingi, Lota, Mylopharyngodon piceus, and Carassius auratus (11)(12)(13); however, the pathogenicity of P. shigelloides in C. carpio remains unknown.
In humans, P. shigelloides has been associated with secondary challenges in immunocompromised states, including malignancy, blood disorders, and hepatobiliary diseases (14,15). In addition to infecting humans, P. shigelloides can be pathogenic to aquatic animals, such as Percocypris pingi (Tchang), Huso huso♀ × Acipenser ruthenus♂, Takifugu obscurus, Ctenopharyngodon idella, and Pelteobagrus fulvidraco; it has been reported to be infect these species and even cause death (16,17). The survival of aquatic animals as natural hosts are not affected under normal circumstances (18). The pathogenic bacteria obtained from Eriocheir sinensis H. milne Edwards were not found to be the cause of disease in a previous study (19). Moreover, symptoms were found to differ between cold-water fish and warm-water fish (20). Therefore, it can be said that P. shigelloides is an opportunistic pathogen that acts once the environment is suitable and has a high fatality rate after onset. After Ictalurus punctatus challenge, the mortality rate was found to be up to 60%-100% in a previous study (21). The median lethal dose for Nile tilapia was 1. 425 × 10 8 CFU/mL, while that for sturgeon was 1.0 × 10 5.8 CFU/mL (21,22). P. shigelloides is increasingly causing damages to aquaculture; however, its pathogenic mechanism remains unknown. At present, there are many studies on the immunity of carp after a bacterial challenge; the commonly used effective methods are transcriptome sequencing and gene function study. However, few studies have assessed the similarities and differences in immune responses in C. carpio after a bacterial challenge based on transcriptome analysis.
Comparative transcriptome analysis has been widely used in immunological studies of different fishes, providing reliable data on immune mechanisms (23). The potential mechanism of antibacterial immunity and the candidate genes has been identified using comparative transcriptome analysis in Epinephelus lanceolatus (24). Transcriptome analysis of intestinal epithelial cells of I. punctatus has revealed that the key to the invasion of Edwardsiella ictaluri is the actin bone in the intestinal barrier (25). In addition to adult tissues and organs, the transcriptome has been used to assess the immune response of fish embryos and primary cells. The innate host immune response of zebrafish embryos infected with Salmonella Typhi has been studied by RNA-seq and marker-based sequencing (26). RNA-seq has been used to study the expression changes in the transcriptome before and after Poly (I:C) stimulation of rainbow trout embryo cells (26). Several differentially expressed genes (DEGs) have been found to be involved in multiple physiological systems, including endocrinal, reproductive, and immune systems. In a previous study, after the intraperitoneal injection of P. shigelloides solution, transcriptome sequencing was performed using the intestinal tissue samples of sturgeon. The results revealed that DEGs in the intestinal tissues were significantly enriched in biological processes, including the immune system process, immune effect process, and stimulus response (13). In the present study, a bacterium referred to as Cc2021 was isolated and purified as the key pathogenic bacterium from infected C. carpio. The pathogenic bacterium causing septicemia was identified through morphological observation, a regression challenge, biochemical identification, and 16S rRNA and gyrB gene sequence analysis. The mortality rate and effects of Cc2021 in C. carpio were analyzed on the basis of the tissue load, tissue edema, and histopathological findings. Comparative transcriptome analysis was performed to assess the immune response mechanism following Cc2021 challenge in both normal intestinal tissues and challenged intestinal tissues. The present study provides reference data regarding the pathogenic mechanism of P. shigelloides in C. carpio.

Fish
The experimental fish C. carpio (mean weight, 50 -100 g) was obtained from the Guanqiao Experimental Station (Wuhan City, Hubei Province, China) and acclimatized at 25 ± 1°C. All the fish were fed twice daily with commercial pellets for 2 weeks before the experiments.

Isolation and Cultivation of Bacterial
Pathogens From Challenged C. carpio Naturally infected C. carpio was obtained from the temperaturecontrolled Fish Culture Center of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan City, Hubei Province, China). The body surface of C. carpio was disinfected with 70% alcohol and placed in a biosafety container for dissection. A sterilized inoculum ring was inserted deep into the lesion of the fish, the needle was gently rotated for sampling, and streaked inoculation was performed on the surface of sterile brain heart infusion (BHI) broth, followed by incubation at a constant temperature of 28°C for 48 h. A single dominant colony in the plate was selected and inoculated on another BHI plate thrice, and the purified pathogenic strain was obtained after pure culture. Finally, one part of the purified strain was used for subsequent experiments and the other part was stored in 50% glycerol at −80°C for future use.

Biochemical Analysis of Pathogenic Bacteria
Bacteria were analyzed using bacterial biochemical microidentification tubes and test papers (Hangzhou Microbial Reagent. Co., Ltd. Hangzhou, China). A single colony was selected, inoculated in a microbiological reaction tube, and cultured at 28°C for 48 h. Following this, the biochemical indicators were tested ( Table 1). The bacterial strains were preliminarily identified according to Bergey's Manual of Systematic Bacteriology and Common Bacterial System Identification Manual (27).

Phylogenetic Analysis of 16Sr RNA and gyrB Genes From Pathogenic Bacteria
The 16S rRNA of the pathogenic bacterial strain was amplified using the 16S rRNA primers 27F (AGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The PCR cycling conditions were as follows: initial denaturation at 95°C for 5 min; followed by 35 cycles at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The two primers used for PCR amplification of the gyrB gene sequence were as follows: UP1 (forward): 5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAART TYGA-3′ and UP2r (reverse): 5′-AGCAGGGTACGGATGTGCG AGCCRTCNACRTCNGCRTCNGTCAT-3′. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, renaturation at 60°C for 1 min, and extension at 72°C for 1 min. After 30 cycles, incubation was performed at 72°C for 7 min. The reaction process was ended at 16°C, followed by preservation at 4°C. Amplification products were placed in 1% agarose gel for electrophoresis. Positive amplification products were recovered and sequenced by TSINGKE Biotechnology Co., Ltd (Wuhan, Hubei, China). The obtained 16S rRNA and gyrB gene sequences were analyzed by the NCBI BLAST retrieval system for sequence homology, and multiple alignments were made with the sequences of strains with high sequence similarity obtained from the GenBank database using ClustalX software. The neighbor-joining method was used to construct phylogenetic trees using Molecular Evolutionary Genetics Analysis (MEGA) 7.0 software. A bootstrap test was performed with 1000 repetitions.

Mortality Rate and Regression Challenge
The pathogenic bacterial strain was inoculated in BHI broth and cultured at 28°C under 200 rpm for 48 h. To determine the 50% lethal concentration of the pathogenic bacterial strain, four groups of C. carpio (n = 30) were intraperitoneally injected 100 mL of the bacterial strain (diluted in physiological saline) at concentrations of 1.0 × 10 7 CFU/mL, 5.0 × 10 7 CFU/mL, 1.0 × 10 8 CFU/mL, and 5.0 ×10 8 CFU/mL, while the control group was injected with sterile physiological saline (0.65% NaCl). Following this, mortality was monitored for the next 7 days.
After infecting the experimental fish (n = 100) with the measured half lethal concentration, the surviving fish were sampled from 0 to 7 days after the challenge. The blood, liver, head kidney, spleen, trunk kidney, and intestine (hindgut) of the challenged fish were randomly collected for the subsequent experiment (n = 5). Fish tissues injected with the same dose of saline were used as controls.

Tissue Bacterial Load and Edema Detection
In total, 0.5 g of ground viscera (liver, head kidney, spleen, and intestine) was weighed and homogenized in BHI medium under sterile conditions using a Dounce tissue grinder (Sigma, USA). Serial dilutions (10 −4 , 10 −5 , and 10 −6 ) were performed, 100 mL of the diluted sample was plated on BHI plates and incubated at 28°C for 48 h. Finally, the colony morphology was identified when the colonies were counted. CFU/g (log10) was calculated taking the dilution and total weight of the fish into consideration. The whole target tissue was gathered and rinsed, and the immediate weight was recorded as the wet weight. Subsequently, the tissues were air dried for 2 days at 60°C, and their weight was recorded as the dry weight. The wet/dry weight ratio for each fish was calculated for evaluating tissue edema.

Histological Analysis by Hematoxylin and Eosin Staining
Tissue samples from the head kidney, spleen, liver, and intestine were dissected and immediately fixed in 10% neutral buffered formalin at 4°C, dehydrated in a graded ethanol series, leaned in xylene, embedded in paraffin, and cut into 4 mm sections using a rotary microtome (Leica, Germany). The sections were stained with HE staining for routine histological examination and observed using a light microscope (Olympus BX51, Japan).

Serum Biochemical Indices
Experimentally infected C. carpio was anesthetized with 3aminobenzoic acid ethyl ester methanesulfonate (MS-222). Blood samples were collected from the caudal vein and placed for 1 h at room temperature. After centrifugation under 4500 rpm at 4°C for 15 min, the serum was collected and stored at −80°C. The serum biochemical indices of complement C3, superoxide dismutase (SOD), and lysozyme (LZM) were assessed using corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The detection method refers to the kit instructions and previous reports (28).

RNA Isolation, cDNA Library Preparation, and Sequencing
Total RNA was extracted from six tissue samples (three from the control groups and three from the challenge groups, collected from the intestinal tissue after 3 days of P. shigelloides challenge) using TRIzol Reagent (Invitrogen, USA) and then treated with RNase-free DNase I (Thermo Scientific, USA) at 30°C for 30 min to remove genomic DNA contaminants. Subsequently, the purity, quantity, and integrity of the extracted total RNA samples were assessed using a NanoDropND-2000 spectrophotometer (Thermo, Waltham, USA), by 1.2% (w/v) agarose gel electrophoresis, and using an Agilent 2100 Bioanalyzer (Agilent Technologies, Richardson, USA), respectively. RNA samples with an RNA integrity number (RIN) > 8, 28S/18S > 0.7, and A260/280 of approximately 2.0 were used to construct the RNA-seq library. Poly (A) mRNA was isolated from total RNA using poly (dT) oligo-attached magnetic beads, and cDNA libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, USA). In total, six cDNA libraries were sequenced using the Illumina Nova-Seq (BGI) sequencing platform to generate 150-bp paired-end reads.

Differential Gene Expression Analysis and Enrichment Analysis
DEGs between the control and challenge groups were identified using Baggerly's test to calculate the fragments per kilobase of exon per million fragments mapped (FPKM) values. Benjamini-Hochberg correction was used to adjust the original P-values in Baggerly's test in order to minimize the false discovery rate (FDR) (35). DEGs were identified if the associated PFDR was less than 0.05, and an absolute value of log2 (fold change) > 1 was regarded as the cut-off criterion. Clustering analysis was performed using the pheatmap package in R based on the FPKM values of DEGs. Subsequently, DEGs were enriched and subjected to GO term (36) and KEGG pathway analyses using clusterProfiler (37). A P-value < 0.05 indicated statistical significance, and the top 10 terms related to immunity were selected for visualization.

qRT-PCR Validation of DEG Expression
In total, six genes associated with immune-related gene testis development were randomly selected for qRT-PCR to validate the RNA-seq results. Reversed-transcribed cDNA obtained from total RNA used for transcriptome sequencing was synthesized using the PrimeScript ™ RT Reagent Kit with gDNA Eraser (Takara, Shanghai, China), according to the manufacturer's protocol. All cDNA samples were diluted to 5 ng/µL and stored at −80°C until use. Specific primers were designed on the basis of NCBI Primer-BLAST (NCBI, USA), as listed in Table S1. All qRT-PCR reactions were performed in triplicate, and target specificity was determined on the basis of dissociation curve analysis. b-actin was selected as the internal control to normalize the expression level of each gene. The relative expression level of the target gene versus the b-actin gene was calculated using the 2 −DDCT method. The obtained data were statistically analyzed using GraphPad Prism 7.0 software.

Statistical Analysis
The results have been reported as the means ± SEs after data preparation and statistical analysis using GraphPad Prism 7.0 software. Statistical significance of the findings in each experimental group relative to the control group was assessed using Student's two-tailed t-test. Significance (P-value) has been indicated as follows: *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Isolation and Identification of Pathogenic Bacteria From Infected C. carpio
The symptoms of infected fish are shown in Figure 1; the symptoms mainly manifested as a mild floating head, outgroup swimming, slow swimming, and reduced feeding. In the middle stage of the disease, the fish stopped feeding completely, with slight eyeball protrusion, slight bleeding in the jaw and abdomen, and redness of the head ( Figures 1A, B). In the later stage, death, abdominal bleeding, and swelling were noted ( Figures 1C, D). After dissection, it was found that the dead fish had fluid accumulation (ascites) in the abdominal cavity. Moreover, the liver was swollen, with light yellow and irregular red spots distributed on it. Furthermore, the tissue felt slightly brittle, like bean paste ( Figures 1E, F).
The dominant strain of pathogenic bacteria was isolated from the lesions of C. carpio (ulceration, ascites, hepatopancreas, and intestinal tract) and named Cc2021 (27). The bacterium grew well on BHI agar, and the colonies were round, beige opaque, smooth, moist, and slightly raised but with neat edges (Figure 2Aa). Gram staining results were microscopically observed. The bacterial cells stained red and appeared as short rods, which were arranged singly or in pairs, indicating that the strain was gram negative (Figure 2Ab). Negative stain electron microscopy revealed a straight rod with a terminal round shape, no spore or capsule, and two fascicular flagella at both ends. The size of the bacterium (short diameter × long diameter) was 0.8-1.0 mm × 3.0 mm (Figure 2Ac). These characteristics were consistent with the description provided in Bergey's Manual of Systematic Bacteriology.
The physiological and biochemical characteristics of the pathogen are provided in Table 1. According to Bergey's Manual of Determinative Bacteriology (9th ed.), the bacterium could hydrolyze arginine, D-fructose, fucose, glucose, and myo-inositol but not adipic acid, citric acid, D-mannitol, phenylalanine aminotransferase, phenylacetic acid, sucrose, salicin, sorbitol, and urease. The results of methyl red and malic acid tests were also positive.
To further identify the strain, its 16S rRNA and gyrB genes were sequenced. NCBI BLAST was used for homology analysis based on the 16S rRNA and gyrB gene sequences of the strain; the results revealed that the strain was 99% similar to P. shigelloides. Phylogenetic analysis was performed after screening sequences with high similarity in terms of sequence alignment; the results (Figures 2B, C) revealed that both 16S rRNA and gyrB gene sequences of P. shigelloides formed a single cluster. Therefore, based on its morphological, physiological, biochemical, and molecular characteristics, the strain was identified to be P. shigelloides.

Pathogenicity of P. shigelloides in C. carpio
To assess the pathogenicity of P. shigelloides in C. carpio, one group of C. carpio was injected with physiological saline (65% NaCl) (control group), while four groups were injected with four different concentrations (1.0 × 10 7 CFU/mL, 5.0 × 10 7 CFU/mL, 1.0 × 10 8 CFU/mL, and 5.0 × 10 8 CFU/mL) of the pathogenic bacterial strain (experimental groups). Subsequently, deaths were recorded ( Table 2). Both high and low concentrations of the bacterial suspension resulted in typical symptoms, similar to those observed in naturally infected fish. After the injection of a high concentration (5.0 × 10 8 CFU/mL) of the bacterial suspension, a large number of fish died within 2 days (73.3%), with the mortality reaching 100% within 7 days. The mortality rates of C. carpio were 80%, 53.3%, and 23.3% after the injection of 1.0 × 10 8 CFU/mL, 5.0 × 10 7 CFU/mL, and 1.0 × 10 7 CFU/mL concentrations, respectively. On the other hand, no death was noted in the control group. These mortality results served as the basis for the follow-up regression challenge experiment. Tissue edema and bacterial loads in C. carpio were assessed after P. shigelloides challenge. The dry/wet weight ratios of the liver, spleen, head kidney, and body kidney of C. carpio showed an increasing trend with an increase in the challenge time. After 2 days post-challenge, the dry/wet weight ratios of the liver and head kidney gradually increased, while those of the spleen and head kidney tended to decrease ( Figures 3A-D). The bacterial load in the tissues from the intestine, hepatopancreas, head kidney, body kidney, and spleen of C. carpio infected with P. shigelloides and the blood bacterial concentrations increased with an increase in the challenge time. The bacterial load in the intestinal tissues was the highest, followed by that in the tissues from the hepatopancreas, head kidney, and spleen. The lowest bacterial load was noted in the blood and body kidney tissues ( Figure 3E).   Histopathological Changes in C. carpio After P. shigelloides Challenge To observe the histopathological state of P. shigelloides-infected C. carpio, HE staining was performed using tissues from the liver, head kidney, spleen, and intestine at each period. The typical pathological states are shown in Figure 4. In the control group, the hepatic cells were rich in cytoplasm, had an obvious nucleus and cytoplasm, had a clear cell boundary, and were evenly dispersed (Figure 4Aa). After the artificial challenge, the inflammatory cells in the liver of infected C. carpio showed focal aggregation, with an unclear structure and a fuzzy boundary. The hepatic cells were denatured and necrotic, and the nucleus and cytoplasm were concentrated. Some hepatic cells were vacuolated and had dissolved nuclei. The tissue was loose and had edema, and degeneration and necrosis were noted around the sinuses. Moreover, the tissue structure was destroyed and several hepatocytes were vacuolated (Figure 4Ab). The renal structure of infected C. carpio was disorganized, with swelling and degeneration of cells, a narrow renal cavity, and a transparent tube type. The renal tissue was filled with lymphocytes, accompanied by multiple bleeding spots and an obvious inflammatory reaction (Figure 4Bb). Moreover, ferriflavin deposition in the splenic tissue, atrophy of the white pulp, and a reduced area occupied by splenic pulp hyperemia were noted (Figure 4Cb). The intestinal structure of C. carpio in the control group was intact; the inner wall of the tissue was not damaged, intestinal epithelial villi were arranged tightly and in an orderly manner, and basal epithelial tissues were obviously stratified (Figure 4Da). Villus epithelial cells in the intestinal tissues of infected C. carpio fell off, with the infiltration of local lymphocytes and the swelling of and increase in goblet cells in the mucosal epithelium being noted (Figure 4Db).

Changes in Serum Biochemical Indices of C. carpio After P. shigelloides Challenge
To assess the degree of activation of antibacterial immune responses in the key immune organs and bloodstream of C. carpio during P. shigelloides challenge, C3, SOD, and LZM activities were assessed at different stages after the challenge.
The expression level of C3 in the serum increased from 1 to 7 days and reached its maximum on the 5th day post-challenge, becoming nearly three times the normal expression level ( Figure 5A). The expression levels of LZM in all the tissues of healthy common carp were similar. The LZM levels in the blood, head kidney, liver, and intestine significantly increased after the challenge. Moreover, the LZM levels in all the tissues continued to increase from 1 to 7 days post-challenge. LZM activity was the highest in the intestine, followed by the liver and blood, while it was the lowest in the head kidney after the challenge ( Figure 5B). The SOD levels in each tissue of C. carpio showed a trend of first increasing and then decreasing after the challenge; the increase was most obvious on 1 and 3 days, followed by a decrease ( Figure 5C). The highest SOD activity was noted in the intestinal tissue after the challenge; this may be related to enteritis caused by the bacterium.

Transcriptome Sequencing and Annotation
All raw data generated in the present study have been uploaded to the NCBI Sequence Read Archive (SRA) under the registration number PRJNA793911. The original sequencing data included reads having low quality, joint contamination, and a high unknown base N content, which needed to be removed before data analysis in order to ensure the reliability of the results. After sequence filtration, a comparison of all genomes was performed, as shown in Figure S1A. Expression profiles of all the genes were obtained in both the control and challenge groups, with half reaching at least one FPKM ( Figure S1B). Correlation analysis of the samples revealed that the correlation coefficients of Challenge 2 and Control 1 within the group were far less than 0.7; thus, these two samples were removed from subsequent analysis ( Figure S1C). Analysis of FPKM expression levels for all the genes in the visualized heatmap revealed that the expression levels of Control 2 and Control 3 genes were consistent and those of Challenge 1 and Challenge 3 genes were consistent ( Figure S1D).
To further assess the immune response mechanism of C. carpio during P. shigelloides challenge, comparative transcriptome analysis was performed for comparing the gene expression profiles in the intestinal tract of C. carpio between the control and challenge groups. For this analysis, three control group samples and three challenge group samples, collected on the 3rd day after the infection, were included. Based on sequence homology, GO enrichment analysis of DEGs in the intestinal tract was performed after P. shigelloides challenge, and the similarities and differences in these terms were analyzed. To further understand the functions of DEGs and the signaling pathways that they participate in, all DEGs were classified into three GO categories: biological process (BP; 20 subclasses), molecular function (MF; 20 subclasses), and cellular component (CC; 20 subclasses) ( Figure S2A). KEGG pathway analysis of the intestinal tissue after P. shigelloides challenge revealed that there were 21 KEGG metabolic pathways involving different genes, mainly including the complement and coagulation cascade pathway, TNF signaling pathway, interleukin (IL)-17 signaling pathway, and toxoplasmosis and systemic lupus erythematosus pathway ( Figure S2B).

Identification of Candidate Genes Responding to P. shigelloides Infection
The identification of DEGs can provide a deeper understanding of the changes in the immune mechanism of P. shigelloidesinfected C. carpio. The heatmap in Figure 6A shows the expression of DEGs in each group. Intestinal gene expression was higher in the challenge group. Importantly, there was no significant difference in DEGs between the control and challenge groups. In total, 1704 genes were differentially expressed in the control and challenge groups, including 876 upregulated genes and 828 downregulated genes ( Figure 6B). The upregulation level of genes was thus significantly higher than the downregulation level, which may be attributed to the cascade amplification of immune signal transmission.
To further assess the immune response after P. shigelloides challenge, we focused on immune-related DEGs classified into BP, MF, and CC categories, as shown in Figure 6C. The top 10 KEGG pathway terms are shown in Figure 6D. GO enrichment analysis of this module was dominated by functional categories related to immune system. In the BP category, acute inflammatory response, regulation of acute inflammatory response, and leukocyte chemotaxis were the top three terms. In the CC category, platelet alpha granule lumen, platelet alpha granule, and plasma lipoprotein particle were the top three GO terms. In the MF category, CXCR3 chemokine receptor binding, chemokine activity, and C3HC4-type RING finger domain binding were the top three terms. Furthermore, KEGG The immune-related signaling pathways of all enriched immunerelated DEGs were selected as the top 10 ones, e.g., the TNF signaling pathway, IL-17 signaling pathway, antigen processing and presentation pathway, Chagas disease pathway, and Toll-like receptor (TLR) signaling pathway ( Figure 6D). Some immunerelated DEGs are shown in Table 3. These results suggested that both the innate and adaptive immune responses of C. carpio played vital roles in its response to P. shigelloides challenge. The discovery of the immune-related pathways and genes could provide a theoretical basis to understand the molecular mechanism of C. carpio infected with P. shigelloides or other bacteria.

Verification of Immune Response Candidates Activated by P. shigelloides
To verify significant immune response candidates activated by P. shigelloides, qRT-PCR was performed on several candidates selected from the comparative transcriptome. The expression levels of IL-1b, Hsp70, TLR5a, MCH II, CD22, and IL-8 were verified in the blood, spleen, head kidney, and intestine at 0, 1, 3, 5, and 7 days after the challenge (Figure 7). The mRNA expression level of IL-1b dramatically increased and immediately reached the highest level on the 1st day after the challenge, following which it began to decrease gradually. The expression level of IL-1b was the highest in the intestine ( Figure 7A). The mRNA expression level of Hsp70 gradually increased in all immune tissues from 1 to 7 days after the challenge; the expression level was higher in the head kidney and spleen ( Figure 7B). The mRNA expression level of TLR5a was upregulated to a similar extent in all the tissues after the challenge, with the increase being the highest at 3 and 5 days ( Figure 7C). Moreover, the mRNA expression level of MHC II was continuously upregulated after the challenge, with the most significant increase being noted in the intestine ( Figure 7D). In contrast, the mRNA expression levels of CD22 and IL-8 were downregulated to varying degrees after the challenge in all the tissues ( Figures 7E, F).

DISCUSSION
P. shigelloides, belonging to the order Eerobacterales, is widely distributed in nature. it is a kind of global distribution bacteria, its detection rate has obvious seasonal, high detection rate in summer, which may be related to the optimal growth temperature of the bacteria is higher. And P. shigelloides is a new pathogen of digestive tract inflammation in many aquatic animals, as well as a zoonotic pathogen, which is a pathogen of gastrointestinal infection outbreak transmitted by water and food (7). In recent years, there have been increasing reports on the pathogenicity of P. shigelloides in fishes. The pathogenic symptoms of P. shigelloides are slightly different in different fishes; this may be caused by the differences in fish species, culture environments, and strains (38,39). However, few studies have been conducted on the pathogenic mechanism of P. shigelloides in fishes (40). Studies on the isolation and identification of bacterial species are essential for understanding the exact incidence of a disease in a given region and for planning epidemiological studies and control and eradication programs (41). Moreover, understanding how bacteria cause pathological symptoms can form the basis of preventing and treating bacterial diseases (42,43). In the present study, we isolated a bacterium from C. carpio and identified it to be P. shigelloides by morphological, physiological, and biochemical methods; gene sequence analysis; and PCR. In addition, we used the isolated P. shigelloides strain to infect C. carpio and studied the changes in histopathology, tissue bacterial load, tissue edema, and immune gene expression to understand its pathogenic mechanism. Finally, and most importantly, through high-throughput sequencing technology, we explored the pathogenic mechanism of P. shigelloides and the immune response mechanism of C. carpio after P. shigelloides challenge. Our findings can serve as a reference for drug designing and vaccine adjuvant screening against pathogens. P. shigelloides is widely distributed and found in different water bodies, animals, and humans (7). It can cause human acute gastroenteritis and other toxic symptoms. In recent years, it has been detected in different aquatic animals and has been found to be highly pathogenic to fishes. It can multiply in the intestines of fishes, damaging their organs and ultimately causing death (37). Grass carp mainly presents with redness and swelling of the body surface, liver, intestine, and anus (38,39). Garra rufa infected with Streptococcus agalactiae presents with surface ulceration and intestinal and hepatic lesions (40). Pathological changes in the liver, stomach, intestine, kidney, throat, and other organs have been reported to be caused in Trionyx sinensis by Aeromonas hydrophila, with the findings in male and female gonads also being inconsistent (39). However, acute diarrhea and food poisoning have been found to be the main symptoms in humans after P. shigelloides challenge (41). In the present study, the disease in C. carpio was characterized by an abdominal cavity, the emergence of a few different levels of fish ascites, hepatomegaly, yellowish color, and an irregular distribution of red dots. Anatomic assessment revealed a crispy organization, light inflammation of the gills, erosion and rotting of the gills, bleeding of the gills, and significant intestinal inflammation. P. shigelloides is a pathogen that causes zoonotic diseases. It also causes a wide range of clinical symptoms in humans, such as acute gastroenteritis and diarrhea (7). Therefore, it is an important pathogen that can pose a risk to public health, especially to people who directly touch any infected fish.
Complement C3, SOD, and LZM in the serum are powerful indicators of the non-specific immune response of aquatic animals, which can identify pathogens, inactivate viruses, and reflect the degree of somatic cell damage to a certain extent (18). Moreover, they play an important role in the non-specific immunity of fishes (44). The complement system is an important component involved in the resistance of fishes to challenge and is involved in target cell lysis and conditioning reactions after activation. Complement C3 is the main component of the complement system (45). A previous study reported an increase in the complement C3 level of hybrid tilapia fed different doses of the commercial product DVAQUA (46). The serum complement C3 level of yellow catfish was found to significantly increase at 7, 14, 21, and 28 days after the injection of inactivated E. ictaluri vaccine, further providing that the vaccine could improve the non-specific immunity of yellow catfish (47). However, in the present study, the complement C3 level in the serum of P. shigelloides-infected C. carpio significantly increased, reached its maximum after 5 days postchallenge, and then significantly decreased. The results revealed that the non-specific immune mechanism was activated in C. carpio after P. shigelloides challenge and was maintained for a long time. SOD activity has been found to be closely related to the immune level of an organism. The SOD activity of Channa argus increased from 9 days after the injection of inactivated Nacardia seriolea, reaching its peak on the 12 th day (48). In the present study, the SOD activity in the serum of C. carpio first increased after the injection of P. shigelloides and then decreased after reaching its maximum on day 3. This result indicated that C. carpio could produce a large number of free radicals, such as superoxide anions, at an early stage of the challenge and improve its antioxidant and immune capacity to resist the challenge. Moreover, LZM activity was found to increase in A. hydrophila- , and IL-8 (F) in the blood, spleen, head kidney, and intestine of C carpio at 0, 1, 3, 5, and 7 days after P. shigelloides challenge. The b-actin gene was used as a reference gene, and the data of day 0 were used for comparison with those of days 1, 3, 5, and 7 in order to analyze the differences. Data are presented as the means ± SEs (n = 4). Statistical analysis was performed using unpaired Student's t-test (*P < 0.05 and **P < 0.01).
infected Chinese sturgeon (49). This result was consistent with that of the present study. In conclusion, the isolated bacterial strain could stimulate the body to produce an immune response after a challenge, which was manifested as increased C3, SOD, and LZM activities in the serum of C. carpio; this in turn stimulated the nonspecific immunity of C. carpio to a certain extent.
Intestinal mucosal immunity is an important mechanism by which a host can resist a pathogen; it plays an important role in natural immunity (50). In previous studies, P. shigelloides challenge was found to destroy the intestinal tissue structure, reduce the number of mucosal immune-related cells, cause intestinal digestion and absorption dysfunction, and activate the mucosal immune response in hybrid sturgeon (12,17). In the present study, the intestinal transcriptome of C. carpio was altered by P. shigelloides challenge. The results revealed that there were 1704 DEGs in the intestinal tissues of C. carpio after P. shigelloides challenge, including 876 upregulated DEGs and 828 downregulated DEGs. This may be related to extensive bacterial replication in C. carpio and the resistance of C. carpio to bacterial challenge. These results indicate that bacterial invasion into the digestive mucosa of C. carpio can induce a specific immune response through a non-specific immune function of the body, i.e., the phagocytosis of immune cells. Some pathogens, such as Legionella pneumophila, Listeria monocytogenes, and S. Typhi, recognize cytoplasmic pathogen-associated molecular patterns (PAMPs). The RIG-1-like receptor signaling pathway is activated to promote the expression of type 1 interferons and proinflammatory cytokines, thereby protecting against bacterial challenge (51,52). Fish TLRs (such as TLR3) are responsible for recognizing not only nucleic acids produced by viruses but also bacterial components, thereby acting as antibacterial agents (53). Intracellular parasitic bacteria, such as L. monocytogenes and Mycobacterium tuberculosis, can activate the cellular solute DNA sensing pathway by recognizing and activating the cGAS-STING pathway. Following this, the TBK1/IRF3 signal cascade is activated to activate the type I IFN transcriptional response and exert antibacterial effects (54). In the present study, the main immune-related signaling pathways in the intestine of P.
shigelloides-infected C. carpio were the TNF signaling pathway, IL-17 signaling pathway, antigen processing and presentation pathway, Chagas disease pathway, and TLR signaling pathway. These signaling pathways play an important role in immune cell activation, inflammatory response, DNA damage, lipid metabolism, and the inhibition of pathogenic bacteria.
To further understand the antibacterial mechanisms triggered by the innate immune response in C. carpio after P. shigelloides challenge, we analyzed the expression levels of six DEGs related to immunity in different tissues and at different times. The IL family is one of the largest cytokine families that plays an important role in the innate and acquired immune responses of the host (55). IL-1b was the first IL to be identified. It plays a critical role in the initiation and regulation of immune and inflammatory responses in many economically important teleost fishes (55). IL-1b in teleost fishes also plays an important role in host resistance to pathogens (56). Recent research has revealed that IL-1b plays an important role in the immune response of large yellow crocea against Vibrio alginolyticus (55). In the present study, after the challenge, the mRNA level of IL-1b rapidly increased and reached the highest level on day 1, following which it began to gradually decrease. The expression level in the intestine was the highest. IL-8, a CXC-type chemokine, plays a key role in acute inflammation by recruiting neutrophils in mammals. Moreover, fish IL-8 is a functional homolog of mammalian IL-8 (57). IL-8 is a wellknown cytokine that plays a crucial role in inflammatory responses and is activated in response to various diseases (57). IL-8 was found to be expressed in various tissues of unchallenged olive flounder. In particular, its expression was the highest in the gills (58). The same results have been reported in rainbow trout, tongue sole (Cynoglossus semilaevis), and common carp (59). In the present study, the mRNA expression levels of IL-8 were downregulated to varying degrees after the challenge in all the tissues. These results suggest that IL-8 is closely related to inflammation and immune regulation in olive flounders and may be used as a basis for research on the immune systems of other fishes. HSP70 is an important member of the heat-shock protein superfamily; it appears in almost all species. HSP70 proteins also play a role in improving disease resistance (60). In the present study, the mRNA expression level of Hsp70 gradually increased in all the immune tissues 1-7 days after the challenge. This upregulation was most obvious in the head kidney and spleen. These results suggest that HSP70 is involved in the immune response against bacterial attacks and heat stress. TLR5 is associated with flagellin detection (61). Moreover, significant upregulation of TLR pathway genes has been reported after A. hydrophila challenge in other fish species (61). Increased expression levels of tlr5a and tlr5b genes were noted in turbot (Scophthalmus maximus L.) mucosal tissues (i.e., intestine and gills) in response to infection by the gram-positive, non-flagellated pathogen S. iniae (62). In the present study, the mRNA expression level of TLR5a was upregulated to a similar extent in all the tissues after the challenge, with the increase being the highest at 3 and 5 days. A similar upregulation of tlr5 in response to alive and formalin-killed R. salmoninarum has been reported (63,64). Therefore, the role of TLR5 beyond the recognition of flagellin, particularly after exposure to nonflagellated bacteria in teleosts, warrants further investigation.
In conclusion, in the present study, a comprehensive and accurate method was used to identify pathogenic bacteria in C. carpio. Changes in the tissue bacterial load, tissue edema, histopathological parameters, innate immunity, and survival rates of P. shigelloides-infected C. carpio during the septicemia process were systemically analyzed. The results indicated that the host response to P. shigelloides challenge involved an inflammatory reaction. To further assess the pathogenesis, comparative transcriptome analysis was performed between the challenge and control groups at 3 days post-challenge. The analysis revealed 1704 genes that were differentially expressed. Further analysis revealed that about 10 KEGG pathways were involved in the host immune response to bacterial challenge; these included the TNF signaling pathway, IL-17 signaling pathway, antigen processing and presentation pathway, Chagas disease pathway, and TLR signaling pathway, among others. The present findings can serve as a reference for understanding the pathogenic mechanism of P. shigelloides in C. carpio and broaden the understanding of the systemic immune response of C. carpio.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/, PRJNA793911.

ETHICS STATEMENT
The animal study was reviewed and approved by Institute of Hydrobiology, Chinese Academy of Sciences. Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
DL conceived the project. DL and HC designed the study. HC and YZ performed most of the experiment and analyses. KC, YW, HL, and FL helped in experiment, data curation and analyses. YL helped in breeding and sampling. HC, YZ, DL, WH and ZZ prepared the draft and final version of the manuscript. All authors read and approved the final manuscript.


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The early-life gut microbiome and vaccine efficacy

Vaccines are one of the greatest successes of public health, preventing millions of cases of disease and death in children each year. However, the efficacy of many vaccines can vary greatly between infants from geographically and socioeconomically distinct locations. Differences in the composition of the intestinal microbiome have emerged as one of the main factors that can account for variations in immunisation outcomes. In this Review, we assess the influence of the gut microbiota upon early life immunity, focusing on two important members of the microbiota with health - promoting and immunomodulatory properties: Bifidobacterium and Bacteroides . Additionally, we discuss their immune stimulatory microbial properties, interactions with the host, and their effect on vaccine responses and efficacy in infants. We also provide an overview of current microbiota-based approaches to enhance vaccine outcomes, and describe novel microbe-derived components that could lead to safer, more effective vaccines and vaccine adjuvants.

Introduction
The efficacy of many licensed vaccines varies within and between populations, 1 and in some cases, translates to little effectiveness. 2 Many intrinsic factors contribute to this variability in vaccine responses, including age, genetics (accounting for 20-40% of the variation between recipients), anaemia, 3 and gender. 4 In addition, a wide range of external factors can greatly affect vaccination outcomes, including vaccine composition and immun isation regimen, 5 preexposure to pathogens and chronic inflammation, 6 exposure to maternal antibodies, 7 nutritional status, and geographical location. 3 Crucially, those at most risk of morbidity and mortality from infectious diseases are infants (aged 0 -5 years) residing in lowincome and middleincome countries. Low immunisation rates, coupled with poor immune responses to vaccines in these regions, are of concern, particularly for mucosaldelivered vaccines. 1 For instance, although 98% of Finnish children (aged 0·5-2 years) respond to oral rotavirus vaccination, only 58% of children from Nicaragua and 46% of infants from Bangladesh develop protective immunity. 8 For parenterally delivered vaccines, such as the BCG vaccine, protection ranges between 0-51% and 88-100% when comparing responses in African and European children. 9 There is therefore a pressing need to understand how vaccine responses in infants, particularly for those most at risk, can be improved.
The gastrointestinal tract microbiota in early life is key for the development and maturation of the infant mucosal and systemic immune system. 2 Intestinal microbial perturbation caused by hygiene, diet, socio economic, and environmental circumstances of both mothers and newborn babies is associated with distinct microbiota profiles in infants from Africa versus those in western Europe. 2 These differences in microbiota profiles in children (or patients) from lowincome and middle income countries are associated with decreased antibody specific immune responses after vaccination, potentially explaining geographic and individual variability in vaccine efficacy. 2 However, other factors contributing to this discrepancy cannot be excluded.
Adjuvants can boost immunogenicity and vaccine efficacy. However, many adjuvants are considered unsafe, 10 with adverse effects that could contribute to vaccination hesitancy. Additionally, different types of adjuvants are required for parenteral and mucosal (oral) vaccines, driving vaccine costs higher and necessitating further research. A 2021 study by Yakabe and colleagues 10 reviewed the advantages and disadvantages of commonly used adjuvants, highlighting potential alternatives originating from the interaction between nutrition and the gastrointestinal tract microbiome.

Early-life colonisation and development of a beneficial gut microbiota-immune relationship The gastrointestinal tract microbiome
The human gastrointestinal tract microbiome comprises a complex community of bacteria, viruses, archaea, and fungi, which varies in composition along the length of the gastrointestinal tract. 11 Microbial colonisation is initiated during birth, and changes continuously across the life course, with the first 1000 days of life representing the most vulnerable and unstable period with respect to ecosystem structure. 2,10 The gastrointestinal tract microbiota is essential for health, contributing to the maturation of the mucosal and systemic immune system, 12 maintaining tolerance, 13 resisting pathogen colonisation, 2 digesting dietary components, and providing micronutrients. 2 Structural and functional perturbations of the gastrointestinal tract microbiota are associated with various diseases, including metabolic disorders, 14 neurodegenerative conditions, 15 allergies, 16 autoimmune disorders, 17 and cancers. 18 Notably, factors influencing the microbiome are similar to those impacting vaccine immune responses, underlining the mutual relationship between immunity and the gastrointestinal tract microbiota. During early life, additional factors that affect the develop ing gastrointestinal tract microbiota are the method of delivery, 19 preterm birth, 20 nutrition (breastmilk vs formula milk), 19 early use of antibiotics or probiotics, 21 and hygiene, 22 as discussed in the following sections.

Establishment of a microbial profile in early life
During and after birth, the infant is exposed to environmental antigens and microbes that promote the maturation of the immune system, the nature of which is dependent on the method of delivery. 19 Vaginally delivered infants are exposed to the maternal vaginal and faecal microbiota, leading to microbial profiles dominated by Escherichia, Lactobacillus, Bacteroides, and Bifidobacterium. 10 By contrast, newborn babies delivered by caesarean section have more contact with maternal skin and hospitalderived microbes, and are often colonised by Streptococcus, Staphylococcus, and Enterococcus species. 10 Early treatment with antibiotics negatively impacts this initial colonisation and subsequent immune system development. These early-life disturbances can lead to an increased risk of infections in the short term, and in the longer term are associated with increased risk of developing diseases associated with immunity and metabolism, such as atopy. 14 Thus, colonisation by the right microbes at the right time is crucial for effective establishment of immune defences and homoeostasis. 19 Breastfeeding provides a supply of antimicrobial peptides, maternal antibodies, and innate immunity factors (eg, soluble TLR2 and 4, CD14, and MD2), which promote passive protection of the newborn baby and provide key dietary components that shape the infant's microbiota. 19 Human milk oligosaccharides enhance Bifidobacterium colonisation and persistence (making up to 80% of the total microbial community), which is less pronounced in infants fed by formula milk (5-30%). 23 Microbial metabolism of human milk oligosaccharides leads to the production of shortchain fatty acids (SCFAs), which are recognised by specific Gproteincoupled receptors bound to membranes, expressed by immune cells both systemically and in the gastrointestinal tract, and are key for developing immune tolerance. 19 Breastmilk also introduces microbes directly via the breastmilk microbiome, allowing further seeding of the infant intestine. 19,23 Weaning and transitioning from breastmilk or formula based nutrition to solid foods drives major changes in the gastrointestinal tract microbiota, with reductions in Bifidobacterium species, and introduction of Ruminococcus, Akkermansia, and Prevotella species. 24 Zmora and colleagues 25 provide a detailed overview of the influence of nutrition on the gut microbiota and its composition.

Influence of the infant gastrointestinal tract microbiome on immunity and vaccination
There is a growing appreciation for the role of the infant gastrointestinal tract microbiota in immunity from vaccines. 26 The gastrointestinal tract microbiota has been shown to promote the efficient stimulation of both humoral and cellular immune responses to vaccines via a variety of mechanisms (figure). 27 Microbiota stimulation is essential for the development and maturation of B cells that produce immunoglobulin (IgA), memory plasma cells through promotion of IgA class switching, and the development of germinal centres within the Peyer's patches in the lumen. 2 Additionally, antigenspecific T-cell responses are enhanced by the microbiota via mediation of type I interferon production by plasmacytoid dendritic cells. 28 The mutualistic relationship between the gut microbiota and the immune system, together with genetic and environmental influences, could explain the variability of individual immune responses to vaccines. 2 Antibiotic induced disturbances of the neonatal mouse microbiome, and use of immunedeficient germfree pups, resulted in impairment of humoral responses to different adjuvanted and liveattenuated vaccines, characterised by a reduction in Th1 and Th17 response, and lower IgG and IgM production. Notably, this impairment was reversible after restoration of the microbiota via administration of specific flagellated Escherichia coli strains or faecal microbiota transfer. 29 The abundance of certain bacterial families, genera, and species have been linked to differences in immune responses to vaccines in humans, both positively and negatively. Huda and colleagues 30 observed a positive effect of Actinobacteria, but a negative correlation if Enterobacteriaceae predominated in response to systemic BCG, tetanus, and hepatitis B vaccines, and oral polio vaccine, in Bangladeshi infants. A followup study of Bangladeshi infants at 2 years of age showed that a high abundance of Bifidobacterium species in early life was positively associated to BCG, tetanus, and polio vaccines, with CD4 + T-cell responses and detectable IgG and IgA at both 15 weeks and 2 years. 31 Comparative studies of microbial profiles in responding and non responding children given the rotavirus vaccine, from Ghana, Pakistan, Bangladesh, and the Netherlands, revealed that the microbiota composition of responders from different lowincome and middleincome countries showed more similarities to Dutch infant microbiota (ie, higher abundance of Clostridium cluster XI and Proteobacteria, and lowered amounts of the Bacteroidetes phylum) than to nonresponders from the same cohort. 32 As the gastrointestinal tract microbiome has a multifactorial role in terms of activation and suppression of immune responses and subsequent effects on vaccine immunity, different microbiotamodulating interventions have been investigated to maximise vaccine efficacy.

Use of prebiotics, probiotics, and antibiotics in vaccine trials
Studies of influenza and cholera vaccination in mice reported a positive association between different prebiotics (defined by Gibson and colleagues 33 for the International Scientific Association for Probiotics and Prebiotics as "a substrate that is selectively utilized by host microorganisms conferring a health benefit") and systemic immune responses after vaccination, linked to increases in the www.thelancet.com/microbe Vol 3 October 2022 e789 Review abundance of Bifidobacterium and Lactobacillus, and to SCFA production. 34 As these bacterial genera are often used in commercial probiotic foods (defined by WHO 35 as "live microorganisms, which when consumed in adequate amounts, confer a health effect on the host"), their effects on vaccination responses have been investigated. A systematic review by Zimmerman and Curtis 36 in 2018 summarised the results of 26 interventional studies in humans using probiotics to enhance efficacy of 17 different vaccines, revealing positive outcomes in half of the studies. Trials of the use of probiotic Lactobacillus and Bifidobacterium strains in neonates and young children (aged 0-16 weeks) showed an elevated success rate when compared with adults, with the highest effects on humoral immunity occurring after vaccination against influenza, diphtheria, rotavirus, and polio. However, the lack of conformity in the design of the different studies (including bacterial strain used) makes it difficult to draw robust conclusions. 36 Human studies have investigated the effect of antibiotic mediated microbial depletion on the immune response to influenza, polio, rotavirus, tetanus, and BCG vaccination (due to ethical reasons, only the polio trial was performed in infants). Either no improvement or markedly reduced vaccine immunogenicity was observed, which links to the negative associations of antibiotic usage, microbiota disturbances, and an increase of immunemediated diseases. 32 Taken together, certain bacterial species are positively correlated with, or directly induce, enhanced humoral or cellular immunity in infant vaccine responders. Bifidobacterium is a good candidate for augmenting vaccination responses, and is also a key member of the healthy earlylife gut microbiota. Other core gut microbiota members, such as Bacteroides, that can stimulate innate and adaptive immune responses, are emerging as novel microbiota therapeutics in vaccine research.

Immune modulatory features of Bifidobacterium and Bacteroides
In a murine obesity model, administration of Bifidobacterium pseudocatenulatum CECT7765 reduces systemic inflammation via restoration of a balanced state of regulatory T cells (Treg) and B lymphocytes, and lowers concentrations of proinflammatory cytokines interleukin 17a (IL17A) and TNF (tumour necrosis factor). 37 Similar immune homoeostatic properties have been attributed to Bifidobacterium bifidum strains, which, via stimulation of dendritic cells in vitro, induce Th17 profiles and enhance the differentiation of Treg cells from naive lymphocytes. 12 Stimulation of peripheral T cells with lysates of B bifidum DSM 20082 increases CD8 + Tcell cytotoxic activity without any affect on CD4 + Tcell activity. 38 Although the mechanisms underpinning these immunomodulatory properties are unclear, several targets have been proposed. These include dietary fermentation products (eg, after metabolism of human milk oligosaccharides and other complex carbohydrates) leading to production of SCFAs (ie, acetate) and other metabolite byproducts that directly interact with host immunecell receptors and facilitate crossfeeding of other commensal bacteria such as Bacteroides and E coli. 39 Bifidobacterium longum subspecies longum produces immunomodulatory compounds and proteins, such as extracellular serpin that irreversibly inactivates proinflammatory proteases. B bifidum MIMBb75 has previously been shown to have peptidoglycan hydrolase TgaA on its surface, which, via initiation of IL2 production and monocytederived dendritic cell activation, promotes Treg expansion. 40 In neonatal piglets, B longum strain AH1206 enhanced production of intestinal IL10, whereas Bifidobacterium animalis subspecies lactis Bb12, Bifidobacterium infantis MCC12, and Bifidobacterium breve MCC1274 promoted immune maturation and immune homoeostasis. 41 Review T-cell responses, and higher titres of IgG after vaccination against BCG, tetanus, and hepatitis B. 30,31 Chinese infants with high abundance of B breve showed increased poliospecific IgA responses after oral polio vaccination. 44 In vaccine supplementation studies, B longum BB536 was shown to enhance Th1 responses in infants via induction of interferonγ (IFNγ) secretion. 37 Bacteroides is a major genus colonising the intestinal tract in infancy, and it is dominant throughout adult life. Certain species and strains have important mutualistic roles, from production of antimicrobial molecules to nutrient provision through breakdown of different glycans. 10 With respect to Bacteroides and vaccine responsiveness, contradictory results have been reported. Rotavirus trials in Ghana found a negative association between elevated prevalence of members of the Bacteroidetes phylum, 45 whereas a similar study of Pakistani infants revealed increased concentrations of this phyla in vaccine responders. 32 Fix and colleagues 46 observed differences in the abundances of different Bacteroides strains in vaccine responders and nonresponders in Nicaraguan infants. However, these findings were not statistically significant after multiple adjustments due to the small sample size, emphasising the need for larger studies to explore these associations.
A link between Bacteroides and vaccine responses can be expected given the prominent role they have in induction of homoeostatic immune priming. The unique lipoligosaccharide structure in Bacteroides thetaiotaomicron has adjuvant properties, inducing elevated concentrations of hepatitis B virus antigen specific antibodies initiated by the hepatitis B virus vaccine, in a manner dependent on CD4 + T cells, suggesting this lipoligosaccharide as an alternative adjuvant to the more toxic lipopolysaccharides of E coli. 47

Immune stimulatory properties of different microbial products SCFAs
SCFAs are produced by different members of the gut microbiota through fermentation of dietary complex carbohydrates, including those in breastmilk or prebiotics. 19 SCFAs contain less than six carbon atoms in their monocarboxylic acid carbon chain, with acetate (C2), propionate (C3), and butyrate (C4) being the most prevalent. 48 They are water soluble and can be directly absorbed by, transported into, or interact with, different cells including intestinal epithelial cells, sympathetic neurons, and immune cells. 49 They provide numerous beneficial health effects including an energy source for enterocytes, strengthening the epithelial barrier, changing metabolic processes, inhibiting enteric pathogen growth, mediators for ion absorption, and as signalling molecules in regulatory pathways of intestinal and systemic immunity. 49 Acetate produced by both Bifidobacterium and Bacteroides, and propionate production by Bacteroides, 49 will be discussed in more detail in the following sections.
Acetate can be enzymatically converted to acetyl coenzyme A (acetylcoA), and is used by many different microbiota members to produce butyrate, and as a major energy source in the tricarboxylic acid cycle. 49 Increased intracellular acetylcoA in T cells activates mTOR, which drives differentiation of Th1 and Th17 T cells. 50 Acetate can also activate Gprotein-coupled receptor 43 (GPR43) expressed on B cells, subsets of T cells, neutrophils, macrophages, dendritic cells, and intestinal epithelial cells, leading to proliferation of lamina propria Treg cells, 50 and regulation of autoantibody production and marginal zone B cells. 51 Activation of GPR43 influences neutrophil chemotaxis and degranulation, and TNF production by macrophages in adipose tissue. 52 Furthermore, dendritic cells lacking GPR43 are unable to induce class switching in B cells. 53 Mice with no SCFAproducing gastrointestinal tract bacteria have decreased plasma cell differentiation and deficiencies in homoeostatic and pathogenspecific antibody responses. 54 Acetate can potentiate vaccine responses through an enhanced production of antigenspecific IgA and IgG against cholera toxin in vitro, and stimulation of signalling molecules in dendritic cells essential for plasma cell differentiation. 55 Propionate induces the differentiation and proliferation of murine Treg cells and expression of IL10. 49 Activation of GPR15 and GPR43 by propionate also increases concentrations of colonic Treg cells via reduced expression of histone deacetylase 6 and 9 and inhibition of NFκB signalling. 49 GPR41 activation by propionate alters bone marrow haematopoiesis, resulting in elevated concentrations of macrophage and dendritic cell precursors, and skews Th2 differentiation. 10 A study investigating the affect of SCFAs on influenza A virus infection revealed an essential role for SCFAs, including propionate, via activation of GPR43 to limit infection severity and concomitant pneumococcal superinfection. 56 GPR43 was proposed to be a coreceptor for influenza A virus entry, meaning that binding of SCFAs to this receptor inhibited virus entry and replication, which indicates potential adjuvant properties for influenza vaccines.

Exopolysaccharides
Exopolysaccharides are clusters of monosaccharides or oligosaccharides, comprising glucose, fructose, galactose, fucose, and rhamnose, which form either homo polysaccharides or heteropolysaccharides. 39 These can be secreted into the intestinal environment or are associated with the cell wall of the parent bacteria. 57 Expression of exopolysaccharides enhances adhesion to host cells, provides protection from digestion and environmental stress, and facilitates biofilm formation and longerterm colonisation in the gastrointestinal www.thelancet.com/microbe Vol 3 October 2022 e791 Review tract. 57 Exopoly saccharides derived from different Bifidobacterium strains can be fermented by other microbes, altering the metabolite milieu and SCFA concentrations. 57 Exopolysaccharides can be recognised as microbeassociated molecular patterns via specific patternrecognition receptors, such as TLR1, TLR2, or TLR6, expressed on the surface of macrophages and dendritic cells. The activation of these receptors, depending on the chemophysical properties of exopolysaccharides (eg, molecular weight or charge 39 ), leads to the production of distinct proinflammatory and antiinflammatory cytokine profiles, and respective differentiation of naive T cells in a strainspecific manner. 12 The surface polysaccharide A of Bacteroides fragilis activates TLR2 on macrophages, and induces the Foxp3 Treg expansion and production of antiinflammatory IL10, which promotes robust antiinflammatory responses during virus infection. Moreover, poly saccha ride A activates TLR4 and secretion of TNF by colonic dendritic cells, providing increased natural resistance to viral infections. 58 Exopolysaccharides derived from B longum BCRC14634 were shown to enhance IL10 production by macrophages, 59 with exopolysaccharides from B longum subspecies longum 35624 shown to suppress expansion of proinflammatory Th17 cells through the dampening of proinflammatory cytokine production, compared with an isogenic exopoly saccharide negative mutant. 60 In some cases, the presence of exopolysaccharide has been associated with an immune silencing effect and an evasion of adaptive B-cell responses, as observed in B breve UCC2003. Moreover, this bifidobacterial strain and presence of exopoly saccha rides were also associated with a lower abundance of proinflammatory IFNγ, TNF, and IL12. 61 Yu and colleagues 12 showed that Bifidobacterium adolescentis IF1 03 increased antiinflam matory immune responses via increased IL10 secretion through macrophages, with enhanced Treg concen trations, which required activation of TLR2 and signal transduction via the extracellular signalregulated kinase, or p38 mitogenactivated protein kinase, and NFκB pathway. Notably, an opposite effect was seen in the case of exopolysaccharides produced by B adolescentis IF111, which simulated macrophages to secrete high concentrations of proinflammatory IL6, IL 17A, and TGFβ, and low amounts of IL10, subsequently skewing T cells to Th17 cells. These studies highlight the immunemodulatory abilities of exopolysaccharides, dependent on strain. Previous work using Lactobacillus derived exopolysaccharides has indicated they can act as novel vaccine adjuvants, 62 therefore further tests focusing on the role of Bacteroides and Bifidobacteriumassociated exopolysaccharides after specific vaccinations are required.

Bacterial extracellular vesicles (BEVs)
BEVs are spherical, membranederived structures, ranging in size from 10 nm to 400 nm, which contain various components from the membrane and periplasm of the parent cell. 63 They are distinguished according to their membrane composition and structure, with their contents being influenced by environmental factors (eg, culture conditions or nutrient stress). This influence can result in quantitative and qualitative differences in DNA, RNA, lipopolysaccharides, enzymes, peptidoglycan, toxins, signalling molecules, metabolites, and virulence factors. 63 BEVs are not restricted to the gastrointestinal tract and have been detected in the bloodstream, from where they can easily access different tissues, including the brain. 64 A study by Aytar Çelik and colleagues 65 gives a detailed overview about different bacterial membrane vesicles, their characteristics, functions, and potential applications. BEVs generated by commensal bacteria contribute to cooperation and syntrophic interactions between members of complex microbial communities, and as mediators of interkingdom crosstalk between members of the gastrointestinal tract microbiota and the host. 63 BEVs can activate immune cells and promote immune responses against the vesicles themselves and the parental cells. 66 The membrane lipids, proteins (including microbe associated molecular patterns), danger signals, and exopolysaccharides can bind to and activate pattern recognition receptors and tolllike receptors, triggering different immune responses beyond the epithelium. 63 Vesicles from B fragilis carry capsular polysaccharide A, which, as stated previously, activates TLR2 on submucosal dendritic cells, and the extracellular vesicles are subsequently internalised in an actindependent manner, leading to increased concentrations of IL10 and Tcell polarisation skewed towards Treg differentiation. 64 Findings by Durant and colleagues 67 showed that BEVs derived from Bacteroides thetaiotaomicron induce IL10 dependent immuno regulatory responses in human mucosal and blood dendritic cells in a speciesspecific manner.
The nonreplicative nature of BEVs and their inbuilt adjuvanticity, thermostability, and resistance to low pH and enzymatic degradation, offers multiple possibilities for vaccination design and delivery. 64 In addition, they can be administered without injections and directly to mucosal sites (eg, gastrointestinal and respiratory tract), reducing administration costs and reducing the risk of potential adverse effects linked to parental delivery. 64 Also, BEVs containing immunogenic components can promote strong innate and adaptive immune responses, and provide high amounts of protection against infectious diseases. 68 BEVs from pathogenic bacteria have been successfully used in vaccine formulations, and several extracellular vesicles vaccines against Vibrio cholerae and serogroup B Neisseria meningitidis are licensed, with those for serogroup B N meningitidis showing potential cross species protection against Neisseria gonorrhoeae. 69 Other studies have shown that BEVs produced by microbiota Review members, including bioengineered BEVs, have been used to deliver antigens from pathogens. Carvalho and colleagues 70 showed that BEVs deriving from Bacteroides thetaiotaomicron and expressing different Yersinia pestis antigens elicited specific and strong immune responses in vivo, including serum IgG and mucosal IgA, which were able to clear plague infection. Lee and colleagues 71 showed that BEVs from mutated, nonpathogenic E coli have adjuvant properties on antigenspecific T-cell responses with decreased toxicity.

Conclusion and future perspectives
The human gut microbiota is emerging as an important determinant of vaccine responsiveness, with two prominent members, Bifidobacterium and Bacteroides, being able to influence immunity and individual vaccine immune responses. This factor is particularly important in early life as these two genera are important constituents of the developing, healthy infant gut microbiota, but are highly susceptible to early-life disturbances, such as caesarean section birth, formula milk versus breastmilk, and antibiotic usage. 19 Focusing on these health-promoting genera and exploiting their immunomodulatory properties could lead to safer approaches to enhancing infant immunity and vaccine efficacy. Novel strategies, with either whole bacteria or their products and metabolites to modulate immune responses, are possible, such as observed in the augmen tation of responses to immune checkpoint inhibitors in cancer. 72 Given that some Bifidobacterium species are generally recognised as safe microorganisms, 73 preclinical studies can rapidly advance to the clinic. Focusing on specific microbial products, such as BEVs, represents a more refined approach. The success of the already established BEV vaccine against cholera and type B meningitis, 69 and the promising results with commensal BEV antigen carriers against plague and influenza, 70 could give rise to a novel vaccine generation based on immunomodulatory BEVs of commensal origin with high efficacy and biosafety standards on a global scale. Nevertheless, further studies will be required to identify which strains produce novel adjuvantlike BEVs from early-life microbiota members such as Bifidobacterium and Bacteroides. For instance, few studies have investigated BEVs from Bifidobacterium strains that might represent a promising avenue for future potent adjuvants. 74 Use of BEVs as a vaccine-antigen delivery vehicle that possesses inherent adjuvanticity could also potentially result in safe, effective mucosal vaccines. Thus, a greater understanding and characterisation of key strains from the healthy infant microbiome, and their byproducts and metabolites, could give rise to a new generation of safe, needlefree, and economical vaccine-boosting therapies, ideally suited to use in lowincome and middleincome countries. Key features, like dosage, vaccine design, and time of administration, will be important considerations, 2 with in depth studies to provide underlying immuneassociated mechanisms representing a substantial challenge because of their molecular complexity. With the launch of the Immunisation Agenda 2030 by WHO, 75 the need for full understanding has become even more important.

Contributors
AJ and LJH conceptualised the Review and methodology. AJ did the formal analysis (literature review) and generated the figure with BioRender. All authors prepared the original draft, and reviewed and edited it. SRC and LJH provided supervision and acquired funding.

Declaration of interests
We declare no competing interests.

Search strategy and selection criteria
Between May 5, 2021, and Sept 30, 2021 we searched PubMed (1996 to present) with the search terms: (microbiome OR microbiota OR Bacteroides OR Bifidobacterium OR short chain fatty acid OR acetate OR propionate OR exopolysaccharides OR extracellular membrane vesicles OR probiotics OR prebiotics OR antibiotics) AND (vaccine OR vaccination OR humoral, cellular, vaccine, immune, mucosal response OR immunisation OR humoral, cellular, mucosal immunity OR antibodies OR immunoglobulin OR adjuvants) AND/OR (breast milk OR delivery OR antibiotics OR nutrition), disregarding language limitations. A publication year filter of 2019 and onwards was applied at first, but was disregarded for fundamental publications and essential studies looking at the microbiota-vaccination relationship. 328 studies and reviews were identified and taken into consideration. We limited the number of reviews based on the focus of our Review and their respective publication year.