Enterobacter asburiae E7, a Novel Potential Probiotic, Enhances Resistance to Aeromonas veronii Infection via Stimulating the Immune Response in Common Carp (Cyprinus carpio)

ABSTRACT Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1β, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-β at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls (54%; P < 0.01). Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic. IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety.

I have received the reviews of your manuscript entitled "Enterobacter asburiae E7, a novel potential probiotic, enhances resistance to Aeromonas veronii infection via stimulating the immune response in common carp ( Cyprinus carpio)", and I regret to inform you that we will not be able to publish it in Spectrum. Your submission was read by reviewers with expertise in the area addressed in your study and it was the consensus view of these reviewers that your paper did not meet the standards necessary for publication. Copies of the reviewers' comments are attached for your consideration.
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Sincerely, jianjun wang Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): After thoroughly reviewing the manuscript (Spectrum04273-22), I suggest that this manuscript should have a minor revision. The work of Jing Li et al. studied a novel potential probiotic Enterobacter asburiae E7 enhances resistance to Aeromonas veronii infection via stimulating the immune response in common carp. In general, the research described in this manuscript is quite interesting and of great importance for our understanding of the functional role of E. asburiae as feed supplementary, this study is of great value to develop ecologically efficient and environmentally friendly method to control disease. E. asburiae strain E7 was isolated from the intestinal tract of healthy common carp, and its probiotic potential was systematically evaluated in vitro and in vivo. E7 was antagonistic to major Aeromonas pathogens, and dietary supplementation enhanced the resistance to A. veronii via stimulating the immune response in common carp. Overall, the study is well carried out and gets valuable conclusions. However, there are several important issues that should be addressed before publication. I have the following overall suggestions: 1. The "INTRODUCTION" is lengthy and it is recommended to refine the language. 2. Please add a subheading. 3. Line 61: "simpler" should be changed to "simple". 4. Line 63: "already poses" should be changed to "already have posed". 5. Line 95: "Aeromonas" is not a specific disease name. 6. Line 205: "(P < 0.05, Fig. 7C)" should be "(P < 0.05, Fig. 7C)". 7. It is very hard to take blood from the small fish via caudal vein especially in cyprinids. Authors should explain how much volume blood they took in a fish. 8. Figure 1: The last picture should be "Shewanella sp." 9. The legend of Figure 4: It is suggested explain the abbreviation "ip" at the end of the note. 10. The legend of Figure 6: What does "n=45/n=18" mean? Why are there two quantities? 11. Table 2 shows the total size of the inhibition zone. It is suggested to add the original diameter of the paper discs in the table note to avoid confusion.
Probiotics can be an alternative strategy to antibiotics，and this paper described E. asburiae E7 is a promising new Gramnegative probiotic that can enhance the health condition and bacterial resistance of aquatic animals. But I still have some questions remaining to be clarified.
1)The English writing of this manuscript should be checked and improved by a native English speaker. 2)Based on Figure 1, authors stated that E. asburiae E7 against common aquatic pathogenic bacteria, So what are the criteria for E. asburiae E7 to be antibacterial? You can't define that E. asburiae E7 has antibacterial effect just according to a little bit of a bacteriostatic zone emergence. 3)Safety evaluation need more rigorous, for example, Whether the E7 strain causes damage to cells and animal tissues should be taken into account. 4)In this paper, authors concluded that E. asburiae E7 is a promising new Gram-negative probiotic. It should be added which resistance genes the strain carries and whether it will develop resistance to antibiotics commonly used in fish. 5)The picture is too fuzzy. 6)Pay attention to the reference case, italics and other problems, according to the requirements of the magazine.

Dear editors and reviewers:
On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript, and also for the reviewers' comments concerning our manuscript entitled "Enterobacter asburiae E7, a novel potential probiotic, enhances resistance to Aeromonas veronii infection via stimulating the immune response in common carp (Cyprinus carpio)". Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our study. We have studied comments carefully and have made correction which we hope meet with approval. Moreover, we carefully checked the tense and grammar of each sentence and corrected the mistakes in this manuscript. The corrected content was highlighted in red color. The main corrections in the paper and the responses to the reviewer's comments are as follows: Response to reviewer #1: 1. The "INTRODUCTION" is lengthy and it is recommended to refine the language.
Thanks for the reviewer's valuable and professional suggestion. We have refined the content of INTRODUCTION.

Please add a subheading.
Thanks for the reviewer's advice. We have added a running title called "Enterobacter asburiae E7 enhances host immunity".
Thanks for the reviewer's careful examination. These mistakes were carefully corrected in the manuscript. 7. It is very hard to take blood from the small fish via caudal vein especially in cyprinids.
Authors should explain how much volume blood they took in a fish.
Thanks for the reviewer's professional and careful review. We collected blood samples from the broken neck using pipette, not from the tail vein. The blood of each fish was about 20 μL. This method was described on line 489. 8. Figure 1: The last picture should be "Shewanella sp." 9. The legend of Figure 4: It is suggested explain the abbreviation "ip" at the end of the note.
Thanks for the reviewer's comment. We have modified Figure 1 and supplemented the full name of "ip" for Figure 4.
10. The legend of Figure 6: What does "n=45/n=18" mean? Why are there two quantities?
Thanks for the reviewer's professional and careful review. 45 fish in each group were randomly selected to measure body length and body weight, and 18 of them were measured carcass weight. In order for the test to proceed smoothly and to ensure sufficient numbers of fish, a smaller number of carcass weight are measured. We have added the meaning of "n=45/n=18" in Figure 6 legend. 11. Table 2 shows the total size of the inhibition zone. It is suggested to add the original diameter of the paper discs in the table note to avoid confusion.
Many thanks for the comments. We have supplemented the original diameter of the paper discs in the table note.
Response to reviewer #2: 1. The English writing of this manuscript should be checked and improved by a native English speaker.
Thanks for the reviewer's comments. This manuscript has been checked and improved by native English speaker. Figure 1, authors stated that E. asburiae E7 against common aquatic pathogenic bacteria, So what are the criteria for E. asburiae E7 to be antibacterial? You can't define that E. asburiae E7 has antibacterial effect just according to a little bit of a bacteriostatic zone emergence.

Based on
Thanks for the reviewer's professional and careful review. Screening criteria for probiotics is based on antibacterial activity. The antibacterial effect of probiotics can be directly judged by examining the presence or size of the inhibition zones of probiotics against pathological bacteria.
A large number of studies have used the method of antagonistic experiment to define antibacterial activity, so it is reasonable to define the antibacterial effect of E7 according to the inhibition zones.
3. Safety evaluation need more rigorous, for example, Whether the E7 strain causes damage to cells and animal tissues should be taken into account.
Thanks for the reviewer's valuable and professional suggestion. In the experiment, histopathological sections of liver, spleen, kidney and intestine of common carp after feeding E7 for 28 days were completed and observed, and no pathological changes were found. Due to the large number of pictures in this paper, we did not add histopathological pictures, and only showed the results of no hemolytic and no pathogenicity to the host, which proved that E7 was safe. We think the reviewer's consideration is reasonable, so we added the histopathological pictures in Figure 4C. 4. In this paper, authors concluded that E. asburiae E7 is a promising new Gram-negative probiotic. It should be added which resistance genes the strain carries and whether it will develop resistance to antibiotics commonly used in fish.
Thanks for the reviewer's professional comments. We accepted the reviewer's valuable suggestions, so we supplemented some tests. We examined whether E7 carries resistance genes and its sensitivity to common aquatic / veterinary antibiotics. These results are shown in Supplementary Fig. 3 and Table 4 Many thanks for the comments. We will upload a clear picture with high resolution when we submit again. 6. Pay attention to the reference case, italics and other problems, according to the requirements of the magazine.
Thanks for the reviewer's professional and careful review. We have carefully revised the references according to the format of the journal.