Safety and Effects of Intravaginal Administration of Lacticaseibacillus rhamnosus CRL1332 Immobilized on Nanofibers in a Murine Experimental Model

Round 1

Reviewer 1 Report

Silva et. al. evaluated the effects of a vaginal probiotic, Lacticaseibacillus rhamnosus CRL 1332, and its immobilization in nanofibers, on vagina of murine models. They analyzed the cultivable lactobacilli, cytology of vaginal washing, vaginal histology and morphology of vaginal epithelium. The authors claimed that lactobacilli-nanofibers was safe to use intra-vaginally, causing little adverse effects.  While this manuscript is interesting, there are some issues need to be addressed to support the conclusion they claimed.

1.       The authors claimed that PVA nanofibers could help probiotic stick to mucus. However, they did not provide clear data about the benefits of using nanofiber immobilization. Figure 3 shows somehow the opposite result; there was a slight decrease of lactobacilli.

2.       The authors may need to comment on the validity of the CFU method they used. Plate count method only provides very limit information about the overall microbiota. It would be much more comprehensive to use 16S rRNA gene amplicon sequencing to show how the microbiota change among different treatments.

3.       It would be better to include an inflammation control in Figures 4 and 5, showing the inflammation compared to the healthy smear and H&E staining. It is also recommended to do cytokine test (eg, C-reactive protein, IL-6 or other biomarkers) as indicator of inflammation. Otherwise, it is not convincing to claim the absence of inflammatory response.   

4.       Please indicate the scale bars in Figure 6.

5.       Is there any change in vaginal pH upon the treatments?

 

The overall English needs to be polished to improve the readability of the manuscript.  Try to shorten sentences and make them concise. 

Author Response

San Miguel de Tucumán, August 15th, 2023

 

Reviewers' comments:

Reviewer #1: Silva et. al. evaluated the effects of a vaginal probiotic, Lacticaseibacillus rhamnosus CRL 1332, and its immobilization in nanofibers, on vagina of murine models. They analyzed the cultivable lactobacilli, cytology of vaginal washing, vaginal histology and morphology of vaginal epithelium. The authors claimed that lactobacilli-nanofibers was safe to use intra-vaginally, causing little adverse effects While this manuscript is interesting, there are some issues need to be addressed to support the conclusion they claimed.

We appreciate your comments. We have reformulated the conclusion section for a better understanding of the findings obtained.

Lines 362-366 (revised highlighted manuscript): ‘The intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers in the murine experimental model demonstrated that is safe related to the absence of inflammatory response at the cytological and histological level, and without modification at the ultrastructural level, and in the normal cultivable vaginal microbiota. The animals models are suggested by many scientific organizations as the first step to demonstrate the safety of formulas or products to be applied later in humans.

 

1. The authors claimed that PVA nanofibers could help probiotic stick to mucus. However, they did not provide clear data about the benefits of using nanofiber immobilization. Figure 3 shows somehow the opposite result; there was a slight decrease of lactobacilli.

Lines 194-202 (original manuscript): ‘No viable lactobacilli were detected after 7 days of administration in control mice (SS) and in those that received nanofibers without lactobacilli (N and N-SM-L). However, a significant increase in the number of lactobacilli was observed in the v.w. of mice inoculated with free lactobacilli (CRL1332) and immobilized in nanofibers (N+CRL1332 and N-SM-L+CRL1332) after 7 days of administration. The values of CFU/ml of lactobacilli cultivable in the vagina of the different EG inoculated with lactobacilli (CRL1332, N+CRL1332 and N-SM-L+CRL1332) did not show significant differences between them, reaching approximately 10^4-5 CFU/ ml v.w. one day after the last i.va. lactobacilli administration.’

According to these sentences in the manuscript, there was a slight decrease in lactobacilli related to the lactobacilli contained in the nanofibers (approximately 10^7-8 CFU L. rhamnosus CRL1332), but the same reduction was observed for the control group of lactic acid bacteria administered with peptone agar (~10^7-8 CFU of L. rhamnosus CRL1332).

We added the following sentence in order to clarify the interpretation of the viability results (lines 335-342 in the revised highlighted manuscript): ‘In this study, the viability of lactobacilli on murine vagina could be maintained in similar values by applying a nanosystem delivery based on PVA-nanofiber containing immobilized lactobacilli that could be applied to coat feminine hygiene products, instead the technique with no development of a delivery system. However, more studies are required to evaluate if a longer administration time will show differences between the administration systems (peptone agar and PVA nanofiber) in the lactobacilli viability and colonization in the murine vagina’.

 

2. The authors may need to comment on the validity of the CFU method they used. Plate count method only provides very limit information about the overall microbiota. It would be much more comprehensive to use 16S rRNA gene amplicon sequencing to show how the microbiota change among different treatments.

We appreciate your suggestion for a more complete study through the application of molecular techniques, however, is not possible to apply at this time in our lab. We were limited, and only the viability method by CFU was the only one we applied to show the modifications of cultivable microorganisms among the different treatments.

We added the following statement in the Discussion Section (lines 318-323 in the revised highlighted manuscript): ‘In this study, the microbiologically analysis by viable bacteria was applied to demonstrate the modifications of cultivable microorganisms among the different treatments. However, since this technique does not allow the identification of all the microorganisms of the vaginal microbiota, further studied needs to be applied by using molecular techniques in a way to demonstrate the effect of the exogenous administration of lactobacilli on the murine vaginal microbiota.’

 

3. It would be better to include an inflammation control in Figures 4 and 5, showing the inflammation compared to the healthy smear and H&E staining. It is also recommended to do cytokine test (eg, C-reactive protein, IL-6 or other biomarkers) as indicator of inflammation. Otherwise, it is not convincing to claim the absence of inflammatory response.

In the revised manuscript, the absence of inflammatory response was observed at the cytological and histological level, was added to the text, when possible. We can not perform at this time the cytokine tests or other inflammation indicators, as suggested.

Lines 24-27: ‘The results demonstrated that the i.va. administration of lactobacilli-functional nanofibers did not produce adverse effects in the murine vaginal tract evidenced by the absence of inflammatory response at the cytological and histological level, without modifications of the vaginal tract at the structural level.’

Lines 362-366: ‘The intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers in the murine experimental model demonstrated that is safe related to the absence of inflammatory response at the cytological and histological level, and without modification at the ultrastructural level, and in the normal cultivable vaginal microbiota.’

The following sentence in lines 282-284 (revised highlighted manuscript) was added: ‘Although no cytological and histological changes were found in the vaginal tract, complementary assays need to be performed to confirm the absence of adverse effects, and also the interaction with immune inflammatory cells.’

Regarding inflammation control, in the Results section the following sentence was added with the reference for a better comparison and comprehension of the results obtained in this work.

Lines 227-230 (revised highlighted manuscript): ‘The response to the i.va. lactobacilli inoculation observed in this work is not comparable with the positive inflammation response observed in previous work by our group in those models of challenge with pathogens [18].’

 

4. Please indicate the scale bars in Figure 6.

Scale bars are indicated at the left bottom of the SEM images. However, we have emphasized the scale bar in a black box for better visualization.

 

5. Is there any change in vaginal pH upon the treatments?

The changes in pH values was not included in this study, because this type of parameter will be further studied in the next protocols directed to evaluate the administration of nanofibers with immobilized lactobacillus against the challenge of urogenital pathogens. The determination of the decreased pH values could be an important factor to support the direct antimicrobial effect of the organic acids produced by lactic acid bacteria.

Reviewer 2 Report

1. The literature review is adequate. The authors have justified the objectives being in concordat with the conclusions. The laboratory basis of the research component is scientifically correct. However, a microbiologist and a histologist with expertise in the research processes should review this section.

2.      The murine vaginal tract is different from those of women, and it is difficult to predict behavior expected in humans and to extrapolate the results using just five mice for each group. 

3. The limitations of the study need to be specified better. 
4. The study aimed to evaluate the effects and safety of L. rhamnosus administration. Although some studies have shown its effectiveness in urogenital infections, it is specific to the gastrointestinal tract. The results show that it is safe to administer. Still, it does not influence the flora present, so it cannot justify an improvement of the microbiota, but only an increase of this type of lactobacillus. The effect on the pathogenic flora does not emerge from the study.

 

 

Author Response

Reviewer #2:

1. The literature review is adequate. The authors have justified the objectives being in concordat with the conclusions. The laboratory basis of the research component is scientifically correct. However, a microbiologist and a histologist with expertise in the research processes should review this section.

We really appreciate your comments. Microbiological and histological protocols were applied, processed, and presented in numerous publications of our research group (Silva-Ruiz et al., 2001. Experimental administration of estradiol on the colonization of Lactobacillus fermentum and Escherichia coli in the urogenital tract of mice. Biol.Pharm.Bull; 2003. Structural and ultrastructural studies of the urinary tract of mice inoculated with Lactobacillus fermentum. BJU Int; Zárate et al., 2009. Protective effect of vaginal Lactobacillus paracasei CRL 1289 against urogenital infection produced by Staphylococcus aureus in a mouse animal model. Infect. Dis. Obstet. Gynecol; De Gregorio et al., 2012. Beneficial lactobacilli: effects on the vaginal tract in a murine experimental model. Antonie van Leeuwenhoek; 2015. Preventive effect of Lactobacillus reuteri CRL1324 on Group B Streptococcus vaginal colonization in an experimental mouse model. J. Appl. Microbiol.; 2016. Immunomodulation of Lactobacillus reuteri CRL1324 on group B Streptococcus vaginal colonization in a murine experimental model. Am. J. Reprod. Immunol.; 2019. Anti-Candida activity of beneficial vaginal lactobacilli in in vitro assays and in a murine experimental model. FEMS Yeast Res). In the revised manuscript some details related to microbiological and histological results (also suggested for another reviewer) were added in order to clarify the comprehension of the findings.

The following sentence was added in order to clarify the interpretation of the viability results (lines 325-331 in the revised highlighted manuscript): ‘In this study, the viability of lactobacilli on murine vagina could be maintained in similar values by applying a nanosystem delivery based on PVA-nanofiber containing immobilized lactobacilli that could be applied to coat feminine hygiene products, instead the technique with no development of a delivery system. However, more studies are required to evaluate the longer administration and define the differences between the administration systems (peptone agar and PVA nanofiber) in the viability and colonization of the lactobacilli in the murine vagina’.

 

The sentence in lines 24-27 (revised highlighted manuscript) was added in order to improve the interpretation: ‘The results obtained demonstrated that the i.va. administration of lactobacilli-functional nanofibers did not produce adverse effects in the murine vaginal tract evidenced by the absence of inflammatory response at the cytological and histological level, without modifications of the vaginal tract at the structural level.’

 

2. The murine vaginal tract is different from those of women, and it is difficult to predict behavior expected in humans and to extrapolate the results using just five mice for each group.

The number of mice included in this work was approved by the Institutional Laboratory Animal Care and Use Committee of the Centro de Referencia para Lactobacilos (n≤9 mice per experimental group in each sampling day), in compliance with recommendations to employ the lower and minimum required number of animals for experimentations. Animal models are requires by scientific organizations before performing phase I human assays. On the other hand, we have explained and justified the reason for the choice of the murine experimental model in lines 280-287: ‘At the moment to select the animal experimental model to use, and depending on the type of test that corresponds, the physiological characteristics must be taken into account. The murine vaginal tract is different from those of women, mainly in the vaginal pH, which is neutral, the autochthonous microbiota with a low number or absence of lactobacilli, and the type and duration of the sexual cycle [32]. However, based on the success of using the murine model to study the vaginal tract described by numerous researchers and fine-tuned in our work group, the experimental model in estrogenized mice is useful for predicting the behavior expected in humans [8,15,18,19, 27,32-34]’.   

Moreover, Spurbeck and Arvidson (Lactobacilli at the front line of defense against vaginally acquired infections. Future Microbiol. 2011;6(5):567-582. doi:10.2217/fmb.11.36) described: ‘Murine models are some of the most common animal models used for the study of pathogenesis. Mice are used to study UTIs, chlamydial patho­genesis and the efficacy of vaginal probiotic instal­lation’. We understand that murine animal model is still being using like a way to predict behavior expected in humans related to vaginal researches (Wang et al., 2021. Translocation of vaginal microbiota is involved in impairment and protection of uterine health. Nat Commun; McCracken et al., 2021. Animal Models and Alternatives in Vaginal Research: a Comparative Review. Reprod Sci.; Lebeau et al., 2022. HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources. Nat Commun.; Lee et al., 2023. Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract. Mol Hum Reprod).

 

3. The limitations of the study need to be specified better.

We recognize the differences between the murine and human vaginal tracts (mainly in the vaginal pH, which is neutral, the native microbiota with a low number or absence of lactobacilli, and the type and duration of the murine sexual cycle), however, different evidences suggest that results obtained in animals can be extrapolated: Wang et al., 2021. Translocation of vaginal microbiota is involved in impairment and protection of uterine health. Nat Commun; McCracken et al., 2021. Animal Models and Alternatives in Vaginal Research: a Comparative Review. Reprod Sci.; Lebeau et al., 2022. HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources. Nat Commun.; Lee et al., 2023. Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract. Mol Hum Reprod.

 

4. The study aimed to evaluate the effects and safety of L. rhamnosus administration. Although some studies have shown its effectiveness in urogenital infections, it is specific to the gastrointestinal tract. The results show that it is safe to administer. Still, it does not influence the flora present, so it cannot justify an improvement of the microbiota, but only an increase of this type of lactobacillus. The effect on the pathogenic flora does not emerge from the study.

Lacticaseibacillus rhamnosus is described as part of the vaginal microbiota in healthy women in a lower degree than others species such as Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii. However, different L. rhamnosus strains were also described as an applicable species for the restoration of the female reproductive tract (Chenoll et al., 2019. Selection of New Probiotics for Endometrial Health. Front Cell Infect Microbiol.; Pino et al., 2019. Detection of vaginal lactobacilli as probiotic candidates. Sci Rep.; Pan et al., 2020.Comparative Analysis of Lactobacillus gasseri and Lactobacillus crispatus Isolated From Human Urogenital and Gastrointestinal Tracts. Front Microbiol.; Chen et al., 2021. The Female Vaginal Microbiome in Health and Bacterial Vaginosis. Front Cell Infect Microbiol; Silva et al., 2021. Immobilization of vaginal Lactobacillus in polymeric nanofibers for its incorporation in vaginal probiotic products. Eur. J. Pharm.; 2022. Functional and safety characterization of beneficial vaginal lactic acid bacteria for the design of vaginal hygiene products. J. Appl. Microbiol; Miquel et al., 2022. Lacticaseibacillus rhamnosus Lcr35 Stimulates Epithelial Vaginal Defenses upon Gardnerella vaginalis Infection. Infect Immun).

On the other hand, in this study, we aimed to determine the effect of the nanofibers with lactobacilli administration to assess their safety, in terms of observing whether modifications of the normal culturable murine microbiota are produced. We demonstrated the in vitro antimicrobial activity of this strain immobilized in nanofibers against urogenital pathogens was maintained, as indicated in lines 72-75 (original manuscript). However, future trials are necessary to determine the effect of the administration of nanofibers with lactobacilli and their antimicrobial activity in vivo against urogenital pathogens.

 

We hope that the modifications performed in the revised version of the manuscript are now adequate to be accepted for publication in the journal.

 

Hoping to hear from you soon. Sincerely,

Round 2

Reviewer 1 Report

The revised manuscript has largely addressed my concerns. I don't have other comments. 

 

Lines363-367:  The sentence can be improved. Consider this:  Our results of demonstrated that the intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers is safe in murine models, given the absence of inflammatory response at the cytological and histological level, and little modification of the normal cultivable vaginal microbiota at ultrastructural level. 

Line 25-26: The sentence has similar issue. 

Author Response

We appreciate comments. We have improved the following sentences, as required, but considering that our comments were related to the no modifications observed at the electronic microscopic evaluation, this is why we used the ultrastructural level.

Lines 362-366 (minor revisions highlighted manuscript): ‘Our results demonstrated that the intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers is safe in murine models, given the absence of inflammatory response at the cytological and histological level, and little modification of the normal cultivable vaginal microbiota and at ultrastructural level.’

Lines 24-27 (minor revisions highlighted manuscript): ‘Our results demonstrated that the intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers is safe in murine models, given the absence of inflammatory response at the cytological and histological level, with minor modification at ultrastructural level, and also related to the normal cultivable vaginal microbiota.’