Review Reports - Probiotic-Fermented Foods and Antimicrobial Stewardship: Mechanisms, Evidence, and Translational Pathways Against AMR

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal: Fermentation

Manuscript ID: fermentation-4010237

Title: Probiotic-Fermented Foods and Antimicrobial Stewardship:Mechanisms, Evidence, and Translational Pathways against AMR

Dear Author,

The manuscript entitled “Probiotic-Fermented Foods and Antimicrobial Stewardship: Mechanisms, Evidence, and Translational Pathways against AMR” mechanistic insights, clinical evidence, and translational frameworks linking PFFs to antimicrobial stewardship. The manuscript is engaging; however, a few sections could benefit from improvement. As a result, a minor revision is recommended. Please refer to the comments below for further details:

Line 38-44: While the manuscript addresses an interesting and timely topic, the introduction would benefit from a clearer and more detailed explanation of the public-health importance of antimicrobial resistance (AMR). AMR is widely recognized as one of the most urgent global health threats, as it compromises the effectiveness of essential treatments, increases morbidity and mortality, and places a substantial burden on healthcare systems. Resistant infections lead to longer hospital stays, higher medical costs, and reduced treatment options, particularly for vulnerable populations. By more explicitly framing the study within this broader public-health context, the authors can better highlight the relevance and potential impact of their work. Strengthening this section would help readers understand why the problem warrants investigation and how the present study contributes to addressing it.
Line 67-74: This section would benefit from a clearer explanation of the public-health relevance of detecting antimicrobial-resistance genes in probiotic and starter cultures. Because AMR is a major global health threat, the potential spread of mobile resistance elements through the food chain warrants stronger emphasis and context, particularly within a One-Health framework. The authors should also more clearly identify the key knowledge gaps that justify the need for critical synthesis and for defined regulatory and research pathways.
Line 106: please add reference.
Line 107: Please write short information or explanation about figure 1
Line 168: What is Box 1? Box 1. Bacteriocins as Precision Anti-Pathogen Tools. This title is a bit mixing.
Line 219-231: please give information a bit more about antimicrobial resistance in fermented foods. Suggested articles below.

https://dergipark.org.tr/tr/download/article-file/900637

https://pmc.ncbi.nlm.nih.gov/articles/PMC5960774/pdf/ijvr-19-053.pdf

Line 327: please explain figure 3 a bit more.
Line 435: please write explanation about figure 4.
Line 628-629, line 694-695, line 723-724: please, first letters are uppercase.
Line 629, 633, 636, 639, 642……. etc. : please check all references and be careful about the spelling of journal name. Please write full name.
Please be careful about spelling rules in reference section. For example, the use of uppercase or lowercase about title, Journal name. Also abbreviated journal name or not, please use the same rule in the whole references. Please italic for microorganism name.

Author Response

Reviewer 1

I sincerely thank the reviewer for the careful evaluation and constructive comments provided. Your insights have greatly contributed to improving the clarity, scientific rigor, and overall quality of this manuscript. All suggestions were carefully considered and incorporated into the revised version.

Comments and Suggestions for Authors

Journal: Fermentation

Manuscript ID: fermentation-4010237

Title: Probiotic-Fermented Foods and Antimicrobial Stewardship: Mechanisms, Evidence, and Translational Pathways against AMR

Dear Author,

Line 38-44: While the manuscript addresses an interesting and timely topic, the introduction would benefit from a clearer and more detailed explanation of the public-health importance of antimicrobial resistance (AMR). AMR is widely recognized as one of the most urgent global health threats, as it compromises the effectiveness of essential treatments, increases morbidity and mortality, and places a substantial burden on healthcare systems. Resistant infections lead to longer hospital stays, higher medical costs, and reduced treatment options, particularly for vulnerable populations. By more explicitly framing the study within this broader public-health context, the authors can better highlight the relevance and potential impact of their work. Strengthening this section would help readers understand why the problem warrants investigation and how the present study contributes to addressing it.

Answers: This portion of the introduction has been substantially improved as shown below:

Antimicrobial resistance (AMR) is recognized as one of the most urgent public-health challenges of the twenty-first century. According to the World Health Organization’s Global Antimicrobial Resistance and Use Surveillance System (GLASS), nearly one in six bacterial infections worldwide now exhibit resistance to first-line therapies, leading to prolonged illness, increased morbidity and mortality, and significant economic strain on healthcare systems. The global rise of resistant pathogens not only undermines the effectiveness of essential medical treatments but also threatens routine procedures, such as surgeries, cancer therapies, and care of immunocompromised patients, that depend on reliable antimicrobial prophylaxis. While conventional antimicrobial stewardship has traditionally focused on optimizing antibiotic prescribing practices and strengthening infection-control measures, emerging scientific and policy frameworks now highlight the importance of preventive, nutritional, and microbiome-centered approaches to reduce infection risk and mitigate antibiotic demand [1–4]. Within this broader public-health context, probiotic-fermented foods (PFFs) have gained growing attention as sustainable, widely accessible tools capable of supporting microbiome resilience and potentially contributing to reduced infectious disease burden across populations [1–4].

Beyond its clinical impact, AMR also reflects broader ecological and societal pressures that demand integrated, multisectoral responses. The spread of resistance determinants across human, animal, and environmental interfaces, accelerated by global travel, intensive food production systems, and inadequate wastewater management demonstrates the limitations of antibiotic-centered interventions alone [1–4]. As resistant bacteria and mobile genetic elements circulate through food chains, agricultural systems, and natural ecosystems, there is increasing recognition that novel mitigation strategies must reinforce microbial ecosystem stability rather than rely solely on therapeutic control. This One Health perspective has catalyzed interest in interventions capable of modulating microbial communities in ways that reduce pathogen colonization and limit antibiotic use. Within this context, probiotic-fermented foods (PFFs) represent a promising, low-cost avenue for population-level resilience, supporting immune function, competitive exclusion of pathogens, and restoration of beneficial taxa disrupted by diet, stress, or infections [1–4].

Line 67-74: This section would benefit from a clearer explanation of the public-health relevance of detecting antimicrobial-resistance genes in probiotic and starter cultures. Because AMR is a major global health threat, the potential spread of mobile resistance elements through the food chain warrants stronger emphasis and context, particularly within a One-Health framework. The authors should also more clearly identify the key knowledge gaps that justify the need for critical synthesis and for defined regulatory and research pathways.

Answers: This portion of the introduction has been substantially improved as shown below:

However, despite promising evidence, the translational bridge between PFF consumption and measurable AMR outcomes remains underdeveloped. The diversity of fermentation processes, microbial consortia, and product quality poses challenges to standardization, safety assurance, and mechanistic interpretation [1–4,10]. From a public-health perspective, the detection of antimicrobial-resistance genes (ARGs) in certain starter cultures and commercial probiotic formulations is particularly relevant, as these elements, especially when associated with mobile genetic platforms, may contribute to the unintended dissemination of resistance within the food chain. This concern extends beyond the food sector, given that mobile ARGs can circulate across human, animal, and environmental compartments, reinforcing AMR as a multisectoral One-Health threat [1–4].

Strengthening genomic monitoring and functional characterization of PFF-associated microorganisms is therefore essential to distinguish intrinsic, non-transferable resistance traits from potentially mobilizable determinants that pose public-health risks. At present, key knowledge gaps limit regulatory clarity and hinder evidence-based implementation of PFFs in antimicrobial stewardship programs. These gaps include: (i) insufficient genome-resolved surveillance for starter and adjunct cultures; (ii) limited understanding of horizontal gene transfer potential during fermentation and gastrointestinal transit; (iii) variability in clinical and preclinical endpoints used to assess AMR-related benefits; and (iv) the absence of harmonized guidelines to define safety thresholds and quality criteria for PFFs [1–4,10].

Therefore, a critical synthesis of available data is required to delineate the mechanisms by which PFFs may support stewardship, to evaluate the strength and limitations of current clinical and preclinical evidence, and to outline regulatory and research pathways for their safe integration into public-health and One-Health frameworks [1–4,10].

Line 106: please add reference.

Answers: Thank you for the observation. I have added the appropriate references.

Line 107: Please write short information or explanation about figure 1

Answers: This portion of the text has been substantially improved as shown below:

The bioactivity of probiotic-fermented foods (PFFs) arises from the combined action of their living microorganisms, structural cell-wall components, and diverse fermentation-derived metabolites, all of which synergistically modulate the gut microbial ecosystem and pathogen dynamics. Once consumed, these microorganisms and metabolites interact with the host intestinal environment by influencing microbial succession, strengthening mucosal barriers, and shaping competitive interactions between beneficial and pathogenic taxa. Such interactions operate through multiple and overlapping mechanisms that reinforce colonization resistance, inhibit pathogenic growth, regulate innate and adaptive immune responses, and alter metabolic niches in ways that are unfavorable to antibiotic-resistant organisms [1–4,10]. These effects are further supported by the production of organic acids, bioactive peptides, and short-chain fatty acids, which can directly impair pathogen viability while promoting a microbiome structure associated with greater resilience to infection and reduce reliance on antibiotics [10].

Given the complexity and interdependence of these processes, a visual synthesis is essential to clarify how microbial, metabolic, and immunological pathways interface within the broader concept of antimicrobial stewardship [1–4,10]. Figure 1 provides an integrated overview of these mechanistic pathways, illustrating how PFF-associated microorganisms and their metabolites collectively contribute to pathogen suppression, shaping of microbial communities, enhancement of host defenses, and mitigation of antimicrobial-resistance (AMR) risks [1–4,10]. By capturing these interactions in a unified framework, the figure helps contextualize the potential role of PFFs as complementary tools within public-health and One-Health strategies aimed at reducing the global burden of AMR [1–4,10].

Line 168: What is Box 1? Box 1. Bacteriocins as Precision Anti-Pathogen Tools. This title is a bit mixing.

Answers: Thank you for this comment. Box 1 is an informational box included to highlight and summarize a key concept relevant to the manuscript. To address your concern about the title being unclear or mixed, I have revised it to ensure greater precision and readability. The updated title now more clearly reflects the content presented in the box.

Line 219-231: please give information a bit more about antimicrobial resistance in fermented foods. Suggested articles below.

https://dergipark.org.tr/tr/download/article-file/900637

https://pmc.ncbi.nlm.nih.gov/articles/PMC5960774/pdf/ijvr-19-053.pdf

Answers: I improved the text and managed to insert one of the articles in the references:

Erginkaya, Z.; Yalanca, İ.; Ünal Turhan, E. Antibiotic Resistance Profile of Lactic Acid Bacteria from Traditional Meat Products. Pamukkale University Journal of Engineering Sciences 2019, 25(7), 834–838. https://doi.org/10.5505/pajes.2018.34466

The revised text follows below:

Dietary intervention studies have demonstrated that the habitual consumption of fermented foods can beneficially modulate host immunity and microbial ecology. A pivotal randomized controlled trial (RCT) conducted by Wastyk et al. [1] revealed that a 10-week fermented-food diet significantly increased gut microbial diversity and decreased a broad range of inflammatory cytokines in healthy adults. These findings indicate that fermented-food consumption supports resilience against dysbiosis caused by antibiotics and may strengthen the gut ecosystem’s capacity to resist colonization by opportunistic pathogens [14,21].

Beyond the Cell study, observational and experimental data reinforce that fermented diets enriched with lactic acid bacteria (LAB) can improve epithelial barrier integrity, increase immunoglobulin A (IgA) production, and modulate toll-like receptor signaling, all of which are relevant for AMR prevention through improved mucosal immunity [14,15,22]. LAB present in fermented foods also contribute to the maintenance of intestinal homeostasis through competitive exclusion, secretion of organic acids, and production of antimicrobial peptides, which together reduce the ecological niches available for resistant pathogens [14,15,22].

Additional evidence from food microbiology studies demonstrates that LAB naturally present in fermented foods frequently exhibit metabolic traits that favor a healthier microbial environment, such as lactic-acid–driven pH reduction, biofilm disruption, and inhibition of pathogen adhesion to epithelial surfaces [14,15,22]. These microbes also produce fermentation-derived metabolites that modulate immune pathways involved in infection control and may attenuate inflammatory processes that otherwise increase susceptibility to resistant infections. Importantly, fermented foods rich in LAB have been shown to enhance microbial community stability following antibiotic exposure, supporting recovery of beneficial taxa and limiting expansion of resistant strains [14,21].

Moreover, findings from traditional fermented-food microbiota research suggest that the ecological interactions established during fermentation, such as cross-feeding, metabolite exchange, and in situ bacteriocin synthesis, contribute to shaping microbial assemblages with natural antagonistic activity against opportunistic pathogens [14,15,22]. Such mechanisms reinforce colonization resistance and reduce the likelihood that antibiotic-resistant organisms can proliferate or persist in the gut. These combined effects provide physiological and ecological justification for exploring probiotic-fermented foods as complementary tools in antimicrobial stewardship, especially within One-Health frameworks that integrate human, food, and environmental microbiomes [1–4,10].

Line 327: please explain figure 3 a bit more.

Answers: The revised text follows below:

The multifactorial nature of kombucha’s antimicrobial potential and its translational relevance to antimicrobial stewardship can be visualized through the integrated model presented in Figure 3, which summarizes the composition of the SCOBY microbial consortium, the key bioactive compounds produced during kombucha fermentation, their cellular and molecular targets, and the systemic outcomes associated with host resilience and reduced antibiotic dependence [8,27]. As shown in the figure, the SCOBY is composed primarily of acetic acid bacteria (AABs), lactic acid bacteria (LABs), and yeasts, which interact synergistically to generate a diverse repertoire of metabolites, including organic acids, ethanol, bacteriocin-like peptides, polyphenol derivatives, and short-chain fatty acids (SCFAs). These metabolites form the biochemical foundation of kombucha’s antimicrobial activity.

Figure 3 also illustrates how these fermentation-derived compounds act on specific cellular targets: (i) disruption of pathogenic bacteria through pH reduction, membrane destabilization, and metabolic inhibition; (ii) modulation of oxidative and inflammatory pathways, which may decrease host susceptibility to infection; and (iii) interference with quorum-sensing signaling, thereby impairing pathogen coordination, virulence, and biofilm formation. By integrating these microbial and molecular mechanisms, the model highlights how kombucha-derived metabolites create hostile conditions for antibiotic-resistant organisms while simultaneously supporting beneficial taxa and mucosal immunity.

Finally, the figure connects these cellular effects to broader systemic outcomes, such as enhanced gut homeostasis and modulation of immune responses, which contribute to lower infection risk and reduce the likelihood of antibiotic use [8,27]. Through this multilevel framework from the microbial ecology of the SCOBY to host-level physiological responses. Figure 3 underscores kombucha’s potential role as a complementary tool within antimicrobial stewardship strategies and within a One-Health perspective.

Line 435: please write explanation about figure 4.

Answers: The revised text follows below:

The multifactorial antimicrobial potential of kefir and its mechanistic relevance to antimicrobial-resistance mitigation are illustrated in Figure 4, which summarizes the microbial consortium, key bioactive compounds, molecular targets, and systemic outcomes that contribute to host resilience and reduced antibiotic demand [34–37,40,44]. As shown in the figure, the kefir grain microbiota is composed of a complex and stable consortium of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts, whose metabolic interactions during fermentation generate a wide array of functional compounds with recognized antimicrobial and immunomodulatory properties. This microbial synergy forms the foundation of kefir’s bioactivity and contributes to the high functional diversity observed across kefir beverages.

Figure 4 also highlights the principal bioactive metabolites produced during fermentation, including bacteriocins, organic acids (such as lactic and acetic acid), and kefiran, a unique exopolysaccharide associated with barrier protection and immunomodulatory effects. These compounds exert antimicrobial actions by acidifying the intestinal environment, destabilizing pathogenic cell membranes, and disrupting key cellular processes in antibiotic-resistant organisms. Kefiran has been associated with enhanced colonization resistance and improved epithelial integrity, which are crucial for limiting pathogen invasion and systemic inflammation.

The figure further illustrates how these metabolites influence cellular and molecular targets relevant to AMR prevention:

(i) inhibition of pathogenic bacteria through acidification, membrane disruption, and organic acid-mediated metabolic suppression;
(ii) modulation of cytokine responses, reducing inflammatory signaling pathways that otherwise predispose the host to infection; and
(iii) suppression of virulence gene expression, which diminishes pathogen aggressiveness, biofilm formation, and overall infective capacity.

These mechanistic pathways are ultimately connected to broader systemic outcomes, such as the maintenance of microbiota homeostasis, reduced infection severity, and a lower need for antibiotic interventions. By supporting a resilient gut ecosystem and enhancing mucosal immunity, kefir-derived metabolites help limit the proliferation of resistant pathogens and promote conditions that reduce antibiotic demand. Together, these interconnected processes underscore kefir’s potential role as a complementary, food-based strategy in antimicrobial stewardship and within One-Health AMR mitigation frameworks [34–37,40,44].

Line 628-629, line 694-695, line 723-724: please, first letters are uppercase.

Answers: This was corrected in the manuscript.

Line 629, 633, 636, 639, 642……. etc. : please check all references and be careful about the spelling of journal name. Please write full name.

Answers: This was corrected in the manuscript.

Please be careful about spelling rules in reference section. For example, the use of uppercase or lowercase about title, Journal name. Also abbreviated journal name or not, please use the same rule in the whole references. Please italic for microorganism name.

Answers: I performed a check on the list of references.

Reviewer 2 Report

Comments and Suggestions for Authors

The topic of the article fits within the scope of the journal, and the article is well written. The review article analyses the possibility of application of Probiotic-Fermented Foods for mitigation of Antimicrobial resistance. The subject is highly relevant, especially since there is several propositions defined as specific objectives for EFSA guidelines regarding Fermented foods on the way. Conclusions and Further Perspectives were well articulated. Analysis was done to great extents in terms of kombucha and kefir. But, why were only kumbucha and kefir selected? What about Sauerkraut for example?

Please recheck the manuscript for spelling errors.

In general, the manuscript can be accepted after minor correction.

There are some comments that need to be addressed.

Comments:

Page 5, Line 155. and line 168 What is Box.1 please explain.

Page 11. Lines 367-369. Sentence: “For PFFs, we recommend that strain identity, genome sequence, and mobile-element context be part of product dossiers, with clear labelling (strain IDs, CFU/dose, sequencing accession where applicable) to align consumer use with stewardship goals.” Who recommends, who is “we”? Please rephrase.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Answers: Thank you for this insightful comment. Kefir and kombucha were selected because both have an extensive and well-established body of scientific literature supporting their probiotic, postbiotic, and functional bioactive properties, including documented microbial consortia, metabolic profiles, and health-related mechanisms. These characteristics allow for a more robust and evidence-based discussion in the context of antimicrobial resistance mitigation.

In contrast, sauerkraut is primarily fermented by lactic acid bacteria that may or may not exhibit probiotic traits, and its microbial composition and functional metabolites are less consistently characterized in the literature. For this reason, sauerkraut was not included as a representative PFF in this review.

Please recheck the manuscript for spelling errors.

Answers: Thank you for this comment. I have carefully rechecked the entire manuscript and corrected all spelling and typographical errors.

In general, the manuscript can be accepted after minor correction.

Answers: Thank you for your positive evaluation of my work and for indicating that the manuscript can be accepted after minor corrections. I sincerely appreciate your constructive feedback and the time dedicated to reviewing this manuscript.

There are some comments that need to be addressed.

Comments:

Page 5, Line 155. and line 168 What is Box.1 please explain.

Answers: Thank you for this observation. Box 1 refers to a highlighted informational box included in the manuscript to summarize a key concept specifically, “Bacteriocins as Precision Anti-Pathogen Tools.” This box provides a concise, standalone explanation of bacteriocins, their modes of action, and their relevance to probiotic-fermented foods in the context of antimicrobial resistance. I have clarified this in the manuscript to ensure that its purpose and placement are explicitly understood.

Answers: Thank you for this comment. I revised the sentence to remove the ambiguous use of “we” and to clearly attribute the recommendation to the appropriate sources. The updated text now reads:

“Beyond point assessments, agencies and research consortia advocate genome-resolved surveillance for starter and adjunct cultures, combining complete genome assemblies, mobile-element scanning, and periodic re-qualification, and leveraging WGS/metagenomics in food safety investigations to improve traceability and risk attribution [34,37]. In line with these recommendations, several regulatory and scientific bodies emphasize that, for PFFs, product dossiers should include strain identity, genome sequence, and mobile-element context, along with clear labeling (strain IDs, CFU/dose, sequencing accession numbers where applicable) to better align consumer use with stewardship and safety goals.”