Probiotic Lactobacilli Ameliorate Antibiotic-Induced Cognitive and Behavioral Impairments in Mice
, Vera Novoselova 1, Anastasia Alexandrova 1, Alisa Arslanova 1, Olga Yakovleva 1, Ilnar Shaidullov 1, Yury Nikolaev 2, Galina El-Registan 2, Vladimir Kudrin 3 and Guzel Sitdikova 1Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript contains a section „O” that should be removed.
The work concerns the important issue of the impact of bacteria on mental health and the gut-brain axis. The introduction presents the definition of probiotics and a brief review of the current state of knowledge regarding their effects, including on the gut-brain axis, in line with the statement that over the last decade, there has been increasing evidence that probiotic supplementation can beneficially modulate the neurophysiology of mice.
The aim of the study was to investigate the effect of two probiotic strains of lactic acid bacteria on behavioral disorders in mice caused by parenteral treatment with broad-spectrum antibiotics. To determine their possible mechanisms of action, their ability to produce H2O2 and neurotransmitters was also examined.
Methodically, the work is done correctly. The subjects used classical microbiology methods to analyze the fecal microbiota. Bacteria were counted with the plate counting method using microbiological media that were non-selective for the total number of bacteria and selective for Lactobacillus spp and Enterobacteriaceae. The evaluation of grown bacteria was performed by analyzing bacterial colonies.
The research methods are selected and performed correctly, although the question arises why the authors did not also use qualitative taxonomic analysis based on sequencing. My second doubt is the duration of the experiment, which raises the question whether 14 days is not too short a period to observe the changes that were assumed for the purpose of the study. Typically, this type of research lasts from 6 to 12 weeks. The dose of probiotics was low 106. Taking into account the unfavorable conditions of the digestive system, which always underestimate the number of probiotic bacteria reaching the intestines, and the fact that the probiotics used in the experiment were sensitive to the administered antibiotics, changes in the intestinal microbiota could have been negligible.
Author Response
Comment 1: The manuscript contains a section „O” that should be removed.
Response 1: Thank you. We are sorry for this mistake. This section was removed.
The work concerns the important issue of the impact of bacteria on mental health and the gut-brain axis. The introduction presents the definition of probiotics and a brief review of the current state of knowledge regarding their effects, including on the gut-brain axis, in line with the statement that over the last decade, there has been increasing evidence that probiotic supplementation can beneficially modulate the neurophysiology of mice.
The aim of the study was to investigate the effect of two probiotic strains of lactic acid bacteria on behavioral disorders in mice caused by parenteral treatment with broad-spectrum antibiotics. To determine their possible mechanisms of action, their ability to produce H2O2 and neurotransmitters was also examined.
Methodically, the work is done correctly. The subjects used classical microbiology methods to analyze the fecal microbiota. Bacteria were counted with the plate counting method using microbiological media that were non-selective for the total number of bacteria and selective for Lactobacillus spp and Enterobacteriaceae. The evaluation of grown bacteria was performed by analyzing bacterial colonies.
Comment 2: The research methods are selected and performed correctly, although the question arises why the authors did not also use qualitative taxonomic analysis based on sequencing. My second doubt is the duration of the experiment, which raises the question whether 14 days is not too short a period to observe the changes that were assumed for the purpose of the study. Typically, this type of research lasts from 6 to 12 weeks. The dose of probiotics was low 106. Taking into account the unfavorable conditions of the digestive system, which always underestimate the number of probiotic bacteria reaching the intestines, and the fact that the probiotics used in the experiment were sensitive to the administered antibiotics, changes in the intestinal microbiota could have been negligible.
Response 2: Thank you. The comment is reasonable and we appreciate the opportunity to clarify our view. The 16S rRNA-based sequencing approach is indeed a very informative method to characterize the gut microbiota. But the conventional culture method is also able to demonstrate antibiotics-induced shift in the microbiota, if there is any. We used the duration of the experiment for two weeks as in the following works: (1) Fröhlich EE, Farzi A, Mayerhofer R, Reichmann F, Jačan A, Wagner B, Zinser E, Bordag N, Magnes C, Fröhlich E, Kashofer K, Gorkiewicz G, Holzer P. Cognitive impairment by antibiotic-induced gut dysbiosis: Analysis of gut microbiota-brain communication. Brain Behav Immun. 2016 Aug;56:140-55. doi: 10.1016/j.bbi.2016.02.020. (2) Kwon HJ, Mohammed AE, Eltom KH, Albrahim JS, Alburae NA. Evaluation of antibiotic-induced behavioral changes in mice. Physiol Behav. 2020 Sep 1;223:113015. doi: 10.1016/j.physbeh.2020.113015.
It is important that probiotic microorganisms are introduced in appropriate amounts. The recommended dose for the majority of probiotic strains for animals is 109 CFU/kg of feed [Simon O. Microorganisms as feed additives—probiotics. Adv Pork Prod. 2005;16:161–7.]. In our work we used the dose of probiotics determined by Chen et al. [Chen YM, Wei L, Chiu YS, Hsu YJ, Tsai TY, Wang MF, Huang CC. Lactobacillus plantarum TWK10 Supplementation Improves Exercise Performance and Increases Muscle Mass in Mice. Nutrients. 2016 Apr 7;8(4):205. doi: 10.3390/nu8040205]. In that paper the mouse Lactobacillus plantarum dose (2.05 × 108 CFU/kg) was converted from a human equivalent dose (1 × 1010 CFU per day). In our work, we assumed the mouse mass of 20 g. Probiotics were administered via oral gavage of 1 mL. Therefore, we prepared the suspention of lactobacilli cells with density 4 × 106 CFU/mL.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis research focused on evaluating the impacts of two probiotic strains, Lacticaseibacillus rhamnosus 12L and Lactiplantibacillus plantarum 8PA3, on behavioral disruptions in mice caused by a two-week parenteral administration of broad-spectrum antibiotics. After the antibiotic regimen, the mice were exposed to various behavioral evaluations, including the open field test (OFT), novel object recognition test (ORT), and T-maze test. In addition, the fecal microbiota was examined to explain the mechanisms of how lactobacilli used their beneficial effect on the nervous system. The results showed that giving the probiotics with antibiotics mitigated the adverse effects of antibiotic treatment on behavioral and cognitive disturbances, reduced mortality, and overall toxicity in mice.
This study was well-designed and written, and the objectives were well-investigated and analyzed. However, there are a few minor issues that should be addressed.
Was the chi-square test used to analyze the mortality of the mice at (p < 0.05) or (p < 0.001)?
Tables 1 and 2 Data are expressed as mean ± SD. The number of readings is not mentioned.
The statistical analysis is not included in all the figures and tables. It should be included to show the statistical differences.
Author Response
This research focused on evaluating the impacts of two probiotic strains, Lacticaseibacillus rhamnosus 12L and Lactiplantibacillus plantarum 8PA3, on behavioral disruptions in mice caused by a two-week parenteral administration of broad-spectrum antibiotics. After the antibiotic regimen, the mice were exposed to various behavioral evaluations, including the open field test (OFT), novel object recognition test (ORT), and T-maze test. In addition, the fecal microbiota was examined to explain the mechanisms of how lactobacilli used their beneficial effect on the nervous system. The results showed that giving the probiotics with antibiotics mitigated the adverse effects of antibiotic treatment on behavioral and cognitive disturbances, reduced mortality, and overall toxicity in mice.
This study was well-designed and written, and the objectives were well-investigated and analyzed. However, there are a few minor issues that should be addressed.
Comment 1: Was the chi-square test used to analyze the mortality of the mice at (p < 0.05) or (p < 0.001)?
Response 1: The chi-square test was used to analyze the mortality of the mice (P < 0.05) (lines 236-237).
Comment 2: Tables 1 and 2 Data are expressed as mean ± SD. The number of readings is not mentioned.
Response 2: We added in section 2.4 of Materials and Methods: For a statistical analysis, we performed at least three seedings of five fecal samples harvested per caged group of mice (lines 121-122).
Comment 3: The statistical analysis is not included in all the figures and tables. It should be included to show the statistical differences.
Response 3: In Figure 2, we moved “* p < 0.05 compared to the control untreated group, # p < 0.05 compared to the initial values” to the end of the figure caption, as in Figures 1, 3 and 4.
We added the statistical analysis for differences where it is appropriate (Figs. 1-4, Tables 1 and 2), line 250:
Fig. 1: *p < 0.05 compared to the control untreated group, # p < 0.05 compared to the initial values.
Fig. 2:* p < 0.05 compared to the control untreated group, # p < 0.05 compared to the initial values (A and C), on panels B and D no significant difference can be observed.
Fig. 3:* p < 0.05 compared to the control untreated group, # p < 0.05 compared to the initial values (B and C), on panel A no significant difference can be observed.
Fig. 4:* p < 0.05 compared to the control untreated group, # p < 0.05 compared to the initial values (B), on panels A and C no significant difference can be observed.
