Review Reports - Bactericidal, Anti-Biofilm, and Stress-Response Modulatory Effects of Lacticaseibacillus rhamnosus ATCC 9595 Cell-Free Supernatant Against Listeria monocytogenes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript evaluated the bactericidal activity of Lactobacillus acidophilus ATCC 9595 against Listeria monocytogenes. Biocontrol technologies remain a hot topic in food research, making this subject both intriguing and appealing to readers. The experimental methods and results were sound, though certain modifications were still required.

1. Further details could be provided on specific studies applying LAB for bacterial eradication.
2. Figure 2: Please supply images for 1xMIC and 2xMIC concentrations. What effect did increased concentrations have on Listeria monocytogenes biofilms?
3. Figure 3: Please provide images at 0.25xMIC and 2xMIC concentrations. What effect did these concentrations have on Listeria monocytogenes biofilms? Why were different concentrations selected for Figures 2 and 3?
4. Figure 4: Please provide data for 0.25xMIC and 0.5xMIC concentrations. What effect did these concentrations have on reactive oxygen species (ROS)?
5. Figure 6: Why was gene expression assessed only at the 0.5xMIC concentration? Based on Figure 5, evaluating the 1xMIC concentration would be more meaningful.

Author Response

Further details could be provided on specific studies applying LAB for bacterial eradication.

We appreciate the reviewer’s comment. We agree that providing additional background on studies applying LAB for bacterial eradication would be beneficial. Accordingly, we have included an additional reference (now reference 24) to a review article that offers comprehensive information on this topic and enables readers to explore further these applications.

“24. Ibrahim, S. A.; Ayivi, R. D.; Zimmerman, T.; Siddiqui, S. A.; Altemimi, A. B.; Fidan, H.; et al. Lactic Acid Bacteria as Antimicrobial Agents: Food Safety and Microbial Food Spoilage Prevention. Foods. 2021, 10, 3131. https://doi.org/10.3390/foods10123131”

Figure 2: Please supply images for 1xMIC and 2xMIC concentrations. What effect did increased concentrations have on Listeria monocytogenes biofilms?

Figure 2 shows the optical microscopy images illustrating the effect of Lcr-CFS on biofilm formation. As indicated in Table 3, when Listeria monocytogenes is exposed to 1x MIC or 2x MIC concentrations, no microbial growth is observed due to the bactericidal activity of the Lcr-CFS. Consequently, as we do not have bacterial cells, no biofilm is formed under these conditions. Therefore, images for 1x MIC and 2x MIC were not included.

Figure 3: Please provide images at 0.25xMIC and 2xMIC concentrations. What effect did these concentrations have on Listeria monocytogenes biofilms? Why were different concentrations selected for Figures 2 and 3?

We appreciate the reviewer’s comment and have now included the images corresponding to the 0.25x MIC and 2x MIC concentrations to Figure 3, as requested. These additions allow a clearer visualization of the effects of these concentrations on Listeria monocytogenes biofilms. The use of different concentrations in Figures 2 and 3 reflects the distinct objectives of each experiment: Figure 2 evaluates biofilm formation, whereas Figure 3 assesses the viability of cells within pre-formed biofilms.

Figure 4: Please provide data for 0.25xMIC and 0.5xMIC concentrations. What effect did these concentrations have on reactive oxygen species (ROS)?

We appreciate the reviewer’s valuable comment and have incorporated the data corresponding to the 0.25x MIC and 0.5x MIC concentrations in Figure 4. Treatment with Lcr-CFS at concentrations ranging from 0.5x MIC to 2x MIC significantly increased ROS production in Listeria monocytogenes 4455 compared to the control. Only the 0.25x MIC treatment did not show a statistically significant difference relative to the control condition.

As described in the revised manuscript, exposure to 2x MIC produced highest fluorescence intensity (2.26-fold increase), indicating substantial ROS accumulation and consequent induction of oxidative stress. The ROS generated by Lcr-CFS may damage L. monocytogenes cells either through direct interactions with biomolecules or through the indirect modulation of cellular signaling pathways.

Line 343-349:

“Treatment with Lcr-CFS at 0.5x to 2x MIC significantly enhanced ROS production in L. monocytogenes 4455 compared to the control condition (Figure 4). Notably, exposure to 2x MIC resulted in markedly higher fluorescence intensity (2.26-times fold), indicating substantial ROS generation and induction of oxidative stress. The accumulation of ROS induced by Lcr-CFS can lead to the damage of L. monocytogenes cells by either direct reaction with biomolecules such as proteins, lipids, and nucleic acids, or to indirect regulation of signaling pathways”

Figure 6: Why was gene expression assessed only at the 0.5xMIC concentration? Based on Figure 5, evaluating the 1xMIC concentration would be more meaningful.

The 1x MIC concentration corresponds to the minimum inhibitory concentration (MIC), and when L. monocytogenes was grown in the presence of this concentration of Lcr-CFS no bacterial growth has been detected, since the Lcr-CFS exhibit bactericidal activity against L. monocytogenes. Therefore, sufficient growth to yield genetic material for rtPCR analysis could only be assessed at 0.5x MIC, which allowed adequate bacterial viability and biomass for RNA extraction.

A clarification has been added to the manuscript to explain why gene expression analysis was performed only at the 0.5x MIC concentration.

Lines 368-369

“Taking into account the bactericidal activity of the Lcr-CFS at MIC-level concentrations or above…”

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-designed study that provides valuable insights into the antimicrobial potential of L. rhamnosus CFS against L. monocytogenes. Here are my comments for improving this study.

1. The introduction could be enhanced by including background information on oxidative stress, as it is a key mechanism investigated in this study and listed among the keywords.

2. A more fundamental characterization of the L. rhamnosus CFS would be beneficial.

3. Figure 3: Scale bars are missing.

4. Line 391: The abbreviation "CBM" is not defined.

5. The relative contribution of the anti-biofilm effects versus ROS induction to the overall antimicrobial activity could be discussed more.

6. Figure 2 and 3: The biofilm structures could be more clearly indicated.

Author Response

The introduction could be enhanced by including background information on oxidative stress, as it is a key mechanism investigated in this study and listed among the keywords.

We agree that oxidative stress represents one of the mechanisms investigated in our study. However, to ensure that the introduction section remained concise and focused, the oxidative stress was addressed in more details in the Discussion section, where the role of reactive oxygen species (ROS) in the observed effects was discussed.

A more fundamental characterization of the L. rhamnosus CFS would be beneficial.

Thank you for this valuable suggestion. We agree that a more in-depth characterization of the Lc. rhamnosus CFS would enhance the study. These analyses are currently underway in ongoing investigations conducted by our research group, and the results will be reported in future publications.

Figure 3: Scale bars are missing.

Thank you for pointing this out. Scale bars were included to the microscopy images in Figures 2 and 3.

Line 391: The abbreviation "CBM" is not defined.

We apologize for the oversight. The abbreviation was mistakenly included in Portuguese instead of English. The correct English abbreviation was included (MBC – Minimal Bactericidal concentration) (line 410).

The relative contribution of the anti-biofilm effects versus ROS induction to the overall antimicrobial activity could be discussed more.

Thank you for this thoughtful comment. We agree that the antimicrobial activity of the Lr-CFS likely results from the combined action of multiple bioactive compounds, which exert different biological effects. Although the anti-biofilm activity and ROS induction effects were individually explored and discussed in this study, the overall bactericidal activity is probably the result of the cumulative contribution of these mechanisms, as stated in the Discussion section.

A brief clarification has been added to the Conclusion section to emphasize that the antimicrobial activity of Lcr-CFS is not driven by a single predominant mechanism, but rather by the combined action of multiple bioactive compounds.

Lines 517-520:

“As the supernatant contains a complex mixture of bioactive compounds, the anti-Listeria effect of Lcr-CFS is likely the result of synergistic action of multiple mechanisms rather than a single dominant pathway.”

Figure 2 and 3: The biofilm structures could be more clearly indicated.

Figure 2 assess the ability of L. monocytogenes cells to form biofilms in glass surfaces in the presence of Lcr-CFS. In contrast, the purpose of Figure 3 was to evaluate the viability of L. monocytogenes cells in mature biofilms after Lcr-CFS treatment, rather than the dismantling or structural analysis of the mature biofilm. We agree that assessing the structure of a mature biofilm would be highly relevant, and this will be addressed in future projects, which are currently planned as part of forthcoming studies.

A clarification has been added to the Materials and Methods section to specify the distinct objectives of Figures 2 and 3 (lines 180-190).

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the antagonistic activity of a lyophilized cell-free supernatant (Lcr-CFS) from Lacticaseibacillus rhamnosus ATCC 9595 against multiple Listeria monocytogenes strains, presenting the minimum inhibitory concentration (MIC) / minimum bactericidal concentration (MBC), time-kill kinetics, biofilm inhibition/disruption, TEM evidence of cell damage, reactive oxygen species (ROS) induction, and RT-qPCR of stress/virulence genes. The results showed that Lcr-CFS not only killed the pathogen in a dose- and time-dependent manner but also significantly reduced and disrupted its biofilms. The research identified the mechanism of action as causing physical damage to the bacterial cell wall and inducing lethal oxidative stress inside the cells. Remarkably, while sub-lethal doses triggered the bacteria's stress and virulence responses, these defenses were ultimately overwhelmed. The study concluded that Lcr-CFS was a highly promising natural solution for controlling L. monocytogenes and improving food safety.

The manuscript is generally well written and methodologically organized; however, some revisions are necessary before it can be considered for publication.

Some technical details are unclear: how many mL of culture were harvested to make CFS, yield from lyophilization, conditions for resuspension, such as volume, and whether MIC tests used lyophilizate in PBS or filtered supernatant
25 mg/mL for MIC implies mass concentration, but it is unclear what mass was measured (total lyophilizate mass, protein mass, dry weight of CFS). Authors must report how the lyophilizate mass was determined.
Figures 2 and 3 lack scale bars and quantitative analysis.
It was claimed that Lcr-CFS is a "promising biocontrol agent" for food processing settings; however, all assays were in broth or on abiotic lab surfaces. No data in food matrices, stainless steel, or at refrigeration temperatures were shown.

Author Response

Some technical details are unclear: how many mL of culture were harvested to make CFS, yield from lyophilization, conditions for resuspension, such as volume, and whether MIC tests used lyophilizate in PBS or filtered supernatant

Thank you for highlighting the need for more detailed methodological information. We have now specified the volume used for CFS preparation, the resuspension procedure, and the nature of the material applied in MIC assays, in the Materials and Methods section. These details were incorporated to enhance clarity and reproducibility.

Lines 119-123

“The CFS was lyophilized (SP Scientific, USA) and subsequently rehydrated in phosphate-buffered saline (PBS, pH 7.4) to obtain a stock solution at a final concentration of 500 mg/mL (corresponding to the resuspension of lyophilizate mass). This 500 mg/mL solution derived from the lyophilized CFS was designated as Lcr-CFS, stored at −20 °C, and used in all subsequent experiments.”

25 mg/mL for MIC implies mass concentration, but it is unclear what mass was measured (total lyophilizate mass, protein mass, dry weight of CFS). Authors must report how the lyophilizate mass was determined.

We apologize for the lack of clarity. The concentration reported (25 mg/mL) refers to the lyophilizate mass, which was determined by weighing the lyophilized material and subsequently resuspending it in PBS. The description of the procedure for obtaining Lcr-CFS was revised in the Materials and Methods section to enhance clarity and ensure reproducibility.

Lines 126-129

“Aliquots of 50 μL of Lcr-CFS and its serial twofold dilutions (prepared from the 500 mg/mL stock solution) were dispensed into the wells of a sterile 96-well microplate containing 50 μL BHI broth (2x concentrated).”

Figures 2 and 3 lack scale bars and quantitative analysis.

Thank you for pointing this out. Scale bars have now been added to the figures.

We agree that a quantitative analysis would provide valuable insights into the mode of action of Lcr-CFS. However, this analysis could not be conducted at the time due to limitations in the available equipment. Nevertheless, we recognize its importance and plan to include quantitative imaging approaches in future studies to better elucidate the biological effects of Lcr-CFS.

It was claimed that Lcr-CFS is a "promising biocontrol agent" for food processing settings; however, all assays were in broth or on abiotic lab surfaces. No data in food matrices, stainless steel, or at refrigeration temperatures were shown.

Thank you for this valuable comment. The experiments conducted in the present study were intentionally restricted to in vitro models, which were designed to elucidate the overall effects of Lcr-CFS against Listeria monocytogenes. Despite the inherent limitations of in vitro assays, the current findings provide a strong basis for considering Lcr-CFS as a promising biocontrol agent, supporting the rationale for further research and potential application in food safety strategies.

We fully acknowledge that assays performed in food matrices, on stainless steel surfaces, or under refrigeration conditions would provide highly relevant insights. Accordingly, additional experiments are already in progress to evaluate the efficacy of Lcr-CFS under conditions that more closely reflect real food processing environments.

The Discussion section has been revised to clarify that the current results are based on in vitro models and that future studies will investigate the performance of Lcr-CFS in food-relevant environments.

Lines 506-512

“Although the experiments reported in this study were conducted exclusively using in vitro models, these conditions were strategically selected to elucidate the fundamental effects of Lcr-CFS against L. monocytogenes, and the findings reported provide essential preliminary evidence supporting the biocontrol potential of Lcr-CFS. The lack of experiments in more realistic food-relevant environments represents an important limitation and should be considered for validating Lcr-CFS applicability in food safety strategies.”