Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.
We gratefully appreciate your positive and professional comments.
Strengths:
The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability to move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.
We gratefully appreciate your positive and professional comments.
Weaknesses:
Although there is a sizeable amount of data reported in the manuscript, there seems to be a chronic issue of lack of details of how some experiments were performed. This is particularly true in the figure legends, which for the most part lack enough details to allow comprehension without constant return to the text. Additionally, 2 figures are missing. Figure 6 is a repetition of Figure 5, and Figure S4 is an identical replicate of Figure S3.
We gratefully appreciate your professional comments. Additional details to perform the related experiments had been added in Materials and methods section and figure legends (e.g., see Line 478-487, Line 996-1001, Line 1010-1012, Line 1019-1020, Line 1031-1033, Line 1041-1042, Line 1051-1053, Line 1082-1083, Line 1087-1088, Line 1093-1094, Line 1105-1107, Line 1113-1114,). Furthermore, we sincerely apologize for the mistakes and the inconvenience in the evaluating process of your review, and we have added the correct Figure 6 (see Line 1043-1053) and Figure S4 (see Line 1084-1088). We will carefully and thoroughly check the whole submitted manuscript along with supplementary information to avoid such mistakes in the future.
Reviewer #2 (Public review):
In this work, the investigators isolated one Lacticaseibacillus rhamnosus strain (P118), and determined this strain worked well against Salmonella Typhimurium infection. Then, further studies were performed to identify the mechanism of bacterial resistance, and a list of confirmatory assays was carried out to test the hypothesis.
We gratefully appreciate your positive and professional comments.
Strengths:
The authors provided details regarding all assays performed in this work, and this reviewer trusted that the conclusion in this manuscript is solid. I appreciate the efforts of the authors to perform different types of in vivo and in vitro studies to confirm the hypothesis.
We gratefully appreciate your positive and professional comments.
Weaknesses:
I have two main questions about this work.
(1) The authors provided the below information about the sources from which Lacticaseibacillus rhamnosus was isolated. More details are needed. What are the criteria to choose these samples? Where did these samples originate from? How many strains of bacteria were obtained from which types of samples?
Sorry for the ambiguous and limited information, more details had been added in Materials and methods section (see Line 480-496). We gratefully appreciate your professional comments.
Lines 486-488: Lactic acid bacteria (LAB) and Enterococcus strains were isolated from the fermented yoghurts collected from families in multiple cities of China and the intestinal contents from healthy piglets without pathogen infection and diarrhoea by our lab.
Sorry for the ambiguous and limited information, we had carefully revised this section and more details had been added in Materials and methods section (see Line 480-496). We gratefully appreciate your professional comments.
Lines 129-133: A total of 290 bacterial strains were isolated and identified from 32 samples of the fermented yoghurt and piglet rectal contents collected across diverse regions within China using MRS and BHI medium, which consist s of 63 Streptococcus strains, 158 Lactobacillus/ Lacticaseibacillus Limosilactobacillus strains, and 69 Enterococcus strains.
Sorry for the ambiguous information, we had carefully revised this section and more details had been added in this section (see Line 129-132). We gratefully appreciate your professional comments.
(2) As a probiotic, Lacticaseibacillus rhamnosus has been widely studied. In fact, there are many commercially available products, and Lacticaseibacillus rhamnosus is the main bacteria in these products. There are also ATCC type strains such as 53103.
I am sure the authors are also interested to know whether P118 is better as a probiotic candidate than other commercially available strains. Also, would the mechanism described for P118 apply to other Lacticaseibacillus rhamnosus strains?
It would be ideal if the authors could include one or two Lacticaseibacillus rhamnosus which are currently commercially used, or from the ATCC. Then, the authors can compare the efficacy and antibacterial mechanisms of their P118 with other strains. This would open the windows for future work.
We gratefully appreciate your professional comments and valuable suggestions. We deeply agree that it will be better and make more sense to include well-known/recognized/commercial probiotics as a positive control to comprehensively evaluate the isolated P118 strain as a probiotic candidate, particularly in comparison to other well-established probiotics, and also help assess whether the mechanisms described for P118 are applicable to other L. rhamnosus strains or lactic acid bacteria in general. Those issues will be fully taken into consideration and included in the further works.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Line 28 - The sentence "with great probiotic properties" suggests that this strain was already known to have probiotic properties. Is that the case?
We gratefully appreciate your professional comments. This sentence "with great probiotic properties" in this context was intended as a summary of our findings, emphasizing that L. rhamnosus P118 exerts great probiotic properties after evaluating by traditional and C. elegans-infection screening strategies. We had revised this sentence (see Line27-30).
(2) Line 30 - What exactly do authors mean by "traditional"? They should add a bit more information here as to what these methods would be.
We gratefully appreciate your professional comments. By "traditional" methods, we refer to time-consuming and labor-intensive strategies for screening probiotic candidates with heavy works, which include bacterial isolation, culturing, phenotypic characterization, randomized controlled trials, and various in vitro and in vivo tests to assess probiotic properties (Sun et al., 2022). We had indicated this strategy in Line 91-94.
Reference:
Sun Y, Li HC, Zheng L, Li JZ, Hong Y, Liang PF, Kwok LY, Zuo YC, Zhang WY, Zhang HP. Iprobiotics: A machine learning platform for rapid identification of probiotic properties from whole-genome primary sequences. Briefings in Bioinformatics 2022;23.
(3) Line 37 - I believe "harmful microbes" is not the correct term here. I suggest authors use "potentially harmful".
Done as requested (see Line 36, 209, 212, 217, 381). We gratefully appreciate your valuable suggestions.
(4) Line 75 - What exactly do authors mean by "irregular dietary consumption"?
"irregular dietary consumption" means "irregular dietary habits" or " eating irregularly " or "abnormal eating behaviors". We had change to "irregular dietary habits" (see Line 76). We gratefully appreciate your professional comments.
(5) Line 85 - What exactly do authors mean by "without residues in raw food products"?
Here, "without residues in raw food products" means that probiotics barely remain in food animal products (e.g., meat, eggs, dairy) after dietary with probiotics in feeds by livestock and poultry. We gratefully appreciate your professional comments.
(6) Line 86 - Please, give a specific example of yeast.
Done as requested (see Line 85-86), “yeast (e.g., Saccharomyces boulardii, S. cerevisiae)”. We gratefully appreciate your valuable suggestions.
(7) Line 112 - Lactobacillus reuteri should be written out, since this is the first time the species name appears in the main text.
Done as requested (see Line 112). We gratefully appreciate your valuable suggestions.
(8) Lines 115-118 - Please, rewrite for clarity.
Done as requested (see Line 115-118). We gratefully appreciate your valuable suggestions.
(9) Line 118 -Lacticaseibacillus rhamnosus should be written out, since this is the first time the species name appears in the main text.
Done as requested (see Line 118). We gratefully appreciate your valuable suggestions.
(10) Line 119 - Throughout the text authors make it seem like strain P118 was previously known. Is that the case? If yes, how was it isolated again? This should be briefly mentioned in the introduction.
Sorry for the misunderstand caused by this statement, P118 strain was isolated and its probiotic properties were evaluated by our lab, not previously known, and we have revised this sentence (see Line 118-120). We gratefully appreciate your professional comments.
(11) Line 131 - How were strains identified?
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) method was employed to identify of bacterial species (He et al., 2022). This information was indicated in Materials and methods section (see Line 485-489). We gratefully appreciate your professional comments.
Reference
He D, Zeng W, Wang Y, Xing Y, Xiong K, Su N, Zhang C, Lu Y, Xing X. Isolation and characterization of novel peptides from fermented products of lactobacillus for ulcerative colitis prevention and treatment. Food Science and Human Wellness 2022;11:1464-74.
(12) Figure 1 - Legend needs a lot more info. Where are legends to panels PQ? Also, some of the text is too small to read.
Sorry for the limited info, we have revised Figure 1 legend and added more info (see Line 1000-1019), and we also provide vector graphic of Figure 1. We gratefully appreciate your professional comments.
(13) Line 136 - All strains were screened and 27 strains were positive, right?
Yes, all strains were screened and 27 strains were positive. We gratefully appreciate your professional comments.
(14) Figure 2 - What do authors mean by "spleen index" and "liver index"? This should be described in more detail. Also, p values for 'a', 'b', 'ab' should be given.
The organ index (spleen index, liver index) were calculated according to the formula: organ index = organ weight (g) / body weight (g) *1000, indicating in Materials and methods section (see Line 587-588). “Different lowercase letters ('a', 'b') indicate a significant difference (P < 0.05)” had been added in Line 1020-1029. We gratefully appreciate your professional comments.
(15) Line 212-214 - Again, I suggest authors use "potentially harmful" and "potentially beneficial".
Done as requested (see Line 36, 210, 213, 218, 383). We gratefully appreciate your valuable suggestions.
(16) Figure 3 - Which groups were tested in panels CD? Is this based on color? Legends should be restated in panels or clearly marked in the legend.
Sorry for this mistake, we have revised and added group info in Figure 3C-D (see Line 1013-1020). We gratefully appreciate your professional comments.
(17) Figure 4 - Lacks details.
Sorry for the mistakes, we have revised and added group info in Figure 4D-E and legend (see Line 1031-1037). We gratefully appreciate your professional comments.
(18) Figure 6 - This is a repetition of Figure 5.
Sorry for the mistakes, we have added the correct Figure 6 (see Line 1060-1070). We gratefully appreciate your professional comments.
(19) Lines 329-330 - C. elegans does not "mimic" animal intestinal physiology.
Sorry for the mistakes, we have revised this statement (see Line 139-142, 324-325). We gratefully appreciate your professional comments.
(20) Lines 358 and 418 - What do authors mean by "metabolic dysfunction" and "metabolic disorder"? I assume they mean changes in fecal metabolites. However, these are terms that may have different interpretations in the field of human metabolism. Therefore, I would suggest that the authors specify that they mean changes in fecal metabolite profiles when using these terms.
Sorry for the mistakes caused by this statement, we have revised this statement in the revised version (see Line 34-35, 122, 353-354, 413). We gratefully appreciate your professional comments.
(21) Line 475 - What do authors mean by "superficial effects"?
Sorry for the mistakes, we had change to “beneficial/protective effects” (see Line 469, Line 1074). We gratefully appreciate your professional comments.
(22) Line 486 - Were all yogurts artisanal? Where were piglets from? How were samples collected? Feces, rectal swabs? Does the ethics statement at the end of the manuscript also cover work with piglets?
Yes, all yogurts were artisanal. The 6 healthy piglet rectal content samples without pathogen infection and diarrhea were from a pig farm of Zhejiang province. Yes, the ethics statement at the end of the manuscript also cover the work with piglets.
(23) Line 490 - Which MALDI platform was used? The database used can have important implications for strain identification. What was the confidence of ID? This should be included.
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS, Bruker Daltonik GmbH, Bremen, Germany) was employed to identify of bacterial species with a confidence level > 90%. This information was indicated in Materials and methods section (see Line 487-489). We gratefully appreciate your professional comments.
(24) Line 501 - Is this a widely used method to characterize probiotics? Please, add a reference.
Done as requested (see Line 498). Many probiotics or microbes can produce milk clotting enzyme to clot milk. It's an important measurement in the dairy industry, especially when making cheese (Zhang et al., 2023; Arbita et al., 2024; Shieh et al., 2009). The milk-clotting activity analysis is usually used for evaluating the potential ability of candidate probiotic isolates in clotting milk into cheeses.
Reference:
Zhang Y, Wang J, He J, Liu X, Sun J, Song X, Wu Y. Characteristics and application in cheese making of newly isolated milk-clotting enzyme from bacillus megaterium ly114. Food Res Int 2023;172:113202.
Arbita AA, Zhao J. Milk clotting enzymes from marine resources and their role in cheese-making: A mini review. Crit Rev Food Sci Nutr. 2024;64(27):10036-10047.
Chwen-Jen Shieh, Lan-Anh Phan Thi, Ing-Lung Shih. Milk-clotting enzymes produced by culture of Bacillus subtilis natto. Biochemical Engineering Journal. 2009;1(43): 85-91.
(25) Line 713 - How were fecal metabolites extracted?
Sorry for the missed information, the fecal metabolites extracted information had been added we have revised and added Materials and methods section (see Line 705-706). We gratefully appreciate your professional comments.
(26) Figure 7 - Please correct "macrophages".
Done as requested (see Figure 7, Line 1072). We gratefully appreciate your valuable suggestions.
(27) Table 1 - Should read "number of strains", not size.
Done as requested (see Line1084). We gratefully appreciate your valuable suggestions.
(28) Figure S1B - Is this data for P118?
Sorry for the mistakes, we have revised Figure S1 legend (see Line 1086-1088). We gratefully appreciate your professional comments.
(29) Figure S3 - Legends C, S, PS, P are not specified.
Sorry for the missed information, we have revised and added group info in Figure S3 legend (see Line 1095-1101). We gratefully appreciate your professional comments.
(30) Figure S3B - What is the "clinical symptom score"? How was this determined?
Sorry for the lack information, and the detailed information had been added in Materials and methods section (see Line 659-661, Table S7). We gratefully appreciate your professional comments.
(31) Figure S4 - This is an identical copy of Figure S3.
Sorry for the mistakes, we have added the correct Figure S4 (see Line 1103-1106). We gratefully appreciate your professional comments.
(32) Figure S5 - Legend lacks details.
Sorry for the missed information, we have revised and added group info in Figure S5 legend (see Line 1107-1112). We gratefully appreciate your professional comments.
(33) Figure S8 - What is "GM"? Since it inhibits growth to a greater extent than the highest metabolite concentration used, I imagine it must be an antibiotic (gentamycin?) as a positive control. This needs to be clearly stated.
Sorry for the missed information, GM: 100 μg/mL gentamicin (see Line 1134). We gratefully appreciate your professional comments.
(34) Figure S9 - Labels for panels are missing.
Sorry for the missed information, labels had been added (see Line 1135-1139). We gratefully appreciate your professional comments.
Reviewer #2 (Recommendations for the authors):
(1) This reviewer appreciates the efforts of the authors to provide the details related to this work. In the meantime, the manuscript shall be written in a way that is easy for the readers to follow.
We had tried our best to revise and make improve the whole manuscript to make it easy for the readers to follow (e.g., see Line 27-30, Line 115-120, Line 129-132, Line 480-496). We gratefully appreciate your valuable suggestions.
(2) For example, under the sections of Materials and Methods, there are 19 sub-titles. The authors could consider combining some sections, and/or citing other references for the standard procedures.
We gratefully appreciate your professional comments and valuable suggestions. Some sections had been combined according to the reviewer’s suggestions (see Line 497-530, Line 637-671).
(3) Another example: the figures have great resolution, but they are way too busy. Figures 1 and 2 have 14-18 panels. Figure 5 has 21 panels. Please consider separating into more figures, or condensing some panels.
We deeply agree with you that some submitted figures are way too busy, but it’s not easy to move some results into supplementary information sections, because all of them are essential for fully supporting our hypothesis and conclusions. Nonetheless, some panels had been combined or condensed according to the reviewer’s suggestions (see Line 1000-1020, Line 1052-1071). We gratefully appreciate your professional comments and valuable suggestions.
(4) Line 30: spell out "C." please.
Done as requested (see Line 31). We gratefully appreciate your valuable suggestions.
