The novel bacteriocin romsacin from Staphylococcus haemolyticus inhibits Gram-positive WHO priority pathogens

ABSTRACT Staphylococcus haemolyticus is an increasingly relevant nosocomial pathogen. The combination of multi-drug resistance and ability to form biofilms makes S. haemolyticus infections difficult to treat. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria to inhibit growth of often closely related bacteria. Due to differences in the modes of action between bacteriocins and antibiotics, bacteriocins are normally equally potent against antibiotic-resistant and antibiotic-sensitive strains. To find bacteriocins able to inhibit S. haemolyticus and related species, clinical and commensal S. haemolyticus isolates (n = 174) were assayed for bacteriocin production. One commensal isolate produced an antimicrobial substance inhibiting S. haemolyticus and Staphylococcus aureus. The substance had physicochemical properties that are characteristic of bacteriocins. Purification, whole-genome sequencing, and mass spectrometry identified the antimicrobial as a novel two-peptide lantibiotic, hereafter named romsacin. The bacteriocin was active against a broad range of Gram-positive bacteria, such as the World Health Organization priority pathogens S. aureus [methicillin-resistant S. aureus (MRSA)] and Enterococcus faecium [vancomycin-resistant E. faecium (VRE)]. Importantly, the bacteriocin also eradicated S. haemolyticus, Staphylococcus epidermidis, MRSA, and VRE biofilms. IMPORTANCE Bacteria produce bacteriocins to inhibit growth of other bacterial species. We have studied the antimicrobial activity of a new bacteriocin produced by the skin bacterium S. haemolyticus. The bacteriocin is effective against several types of Gram-positive bacteria, including highly virulent and antibiotic-resistant strains such as Staphylococcus aureus and Enterococcus faecium. Effective antimicrobials are important for the treatment of infections and the success of major surgery and chemotherapy. Bacteriocins can be part of the solution to the global concern of antimicrobial resistance.

Staphylococcus aureus and Enterococcus faecium, and food-borne pathogens Listeria monocytogenes and Bacillus cereus.However, its mode of action remains unclear as PI staining assay and SEM observation did not tell obvious trait of romsacin action.Overall, data carried out by a number of highly experienced techniques to investigate bacteriocin peptide so that the characteristic feature of romsacin was remarkedly described.The manuscript is scientifically sound and will provide impact on this field.Meanwhile, I still believe that several improvements are required to ensure rationality and importance of author's claim.
1.I could not find figure legends for figure 1, 2 and 3. Please provide legends for these figures.It will be great help for readers.
2. The authors are using the term "WHO priority pathogens" in the title.However, only some of the experiments actually examined about WHO priority pathogens such as S. aureus or E. faecium.For example, biofilm disruption assay, PI staining test, and SEM observations should be performed using any WHO priority bacteria.Alternatively, excluding "WHO priority pathogens" from the title could be an option.
3. Lines 89-91: This sentence seems appropriate to be included in the result section.Had this prediction already been done by previous study?If so, please provide citation.
4. Line 93, 180-181: "MRSA" does not stand just for S. aureus, but for "Methicillin-resistant" S. aureus as the author described in line 60 and elsewhere.Similar for VRE.I think it is not necessary to specify every time, and only abbreviations such as "VRE" and "MRSA" can be used only once after the first definition at line 60.

Fig 6:
I cannot see scientific reason not to perform the biofilm experiment with S. aureus.S. aureus and E. faecium can also form biofilm in laboratory condition.Test with S. aureus or E. faecium strain (ideally MRSA or VRE) will ensure that romsacin can be effective agent against WHO priority pathogen infection.
6. Fig 7 and 8: It is little bit confusing that pore forming assay and SEM observation were carried out only with L. lactis.Since Fig. 6 shows that romzacin destroys staphylococcal biofilms (suggesting lytic activity against these bacteria rather than only growth inhibition), the mechanism should be investigated using this staphylococcus, not other model bacteria.One cannot exclude possibility that romsacin can form pore on Staphylococcus cell membrane even if it does not pore form on L. lactis.7. I am curious the mode of action of romsacin is bacteriostatic or bacteriolytic.To address this issue, the author could try some simple test where bacteria is exposed with romsacin in liquid phase and then measure changes of CFU or turbidity of culture over incubation time.If CFU or culture turbidity decreases over time, it suggests that romsacin has bacteriolytic activity.Also, Fig 4 displays several bacteriocins similar to romsacin.Are there any previous report about the mode of action of these bacteriocins?If there are literatures, the manuscript can include further discussion about the mode of action of romsacin.
8. Lines 222-223: Please describe a little bit more detail and examples about "bacteriocin effective against MRSA and VRE".Then, please compare them with romsacin, so that characteristic feature and possible advantage of romsacin highlights novelty and impactfulness of this manuscript.9. Lines 244-247: Effect of PMBN or EDTA on romsacin activity against Gram-negative bacteria should be addressed here in this study.
10. Lines 286-287: To clarify relationship between purified romsacin and the genetic element, constructing of deletion mutant of romA1/romA2 would be better evidence.

Table 2:
The list of same names for distinct strain rows is not very informative for reader.Please specify at least strain name or any unique identifier (for example, Freeze no. in Supplementary table 1 can be used) within species.
Reviewer #2 (Comments for the Author): The paper from R. Wolden et al. describes the isolation, purification and partial characterization of an antimicrobial peptide (a bacteriocin) produced by a strain of Staphylococcus haemolyticus that was selected among 174 clinical and commensal isolates.This bacteriocin was named romsacin by the authors and it was identified as a two-peptide lantibiotic.The authors characterized the producing gene cluster, isolated romsacin using a classical purification protocol, and performed MALDI-TOF MS that identified the molecular mass of romsacin.From these data, they predicted that romsacin was a novel two-peptide lantibiotic.Romsacin was shown to inhibit a panel of Gram positive bacteria among which S. haemolyticus, and the WHO priority pathogens vancomycin-resistant Enterococcus faecium (VRE) and methycilin resistant S. aureus (MRSA).It also destroyed S. epidermidis and S. haemolyticus biofilms.In addition, the authors performed preliminary experiments to tackle the mechanism of action of romsacin, and concluded that the formation of pores could not explain the romsacin antibacterial activity.The paper is well written, the study could be interesting and could open interesting perspectives, but many aspects are very preliminary.The points detailed below need being taken into account.
Specific comments 1-The study appears preliminary as regard both the structural characterization, and its spectrum of activity and mechanism of action.The MALDI-TOF measurements only allowed obtaining the molecular masses of the two components of romsacin RomA1, RomA2.Paragraph lines 164-173 is only speculative.The mechanism of action of RomA1-A2 was determined for a part on S. haemolyticus and S. epidermidis (confocal microscopy) and on L. lactis IL1403 (propidium iodide, SEM).Please justify the choice of three different strains for these experiments.2-Perturbation of the biosynthesis of peptidoglycane is another mechanism of action demonstrated for other lantibiotics.The hypothesis should be tested here.3-In Table 2 that shows the antibacterial spectrum of romsacin, the references and origin of the strains tested and the units for the MICs should be added (the unit is provided for propidium iodide and SEM only).As L. lactis was used for the mechanism of action, the activity on this strain should be added in the Table 2, while it only appears in Table S1.4-Page 7, lines 151-173: it is desirable to separate sections MALDI-TOF and structure prediction in two different sections.5-As the authors argue for the activity of romsacin against VRE, MRSA pathogens, a toxicity assay should be included in the paper (at least hemolysis assay) to further identify its potential.6-Figure 2: antibacterial activity should be indicated on the figure; fraction numbers are too small to be read.Perhaps indicate only a few fractions in bigger letters...; time should appear on the figure 7-Legend to Figure 3 is too elusive: "of an active fraction"; specify which fraction 8-Legend to figure 4: the names of producing bacteria should be indicated 9-Literature references: The names of bacteria (genus and species) and if useful of other organism should be in italics (Refs 6, 8, 14,...) and the titles should not include systematic capital letters (ref 16).10-The sentence turns used in the materials and methods "We screened overnight cultures; line 332 "we tested a S.haemolyticus type strain" etc should be avoided and changed to the impersonal grammatical turn.11-Typing mistake: line 293 suppress "and"

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Response to reviewers
Thank you both for your valuable input regarding our manuscript.We have responded to the majority of your comments.We sincerely believe that the revisions made based on your feedback have improved the quality of the manuscript.Please see our detailed answers below.
Reviewer #1 (Comments for the Author): In this manuscript, the authors describe studies where they screeded out novel bacteriocin from Staphylococcus haemolyticus collection and successfully identified new bacteriocin named romsacin.
The genetic element encoding romsacin synthesis and function was identified.The structure of romsacin was also determined by MALDI-TOF Mass spectrometry and structural prediction.
Bacteriocin activity of romsacin was effective against broad range of Gram-positive bacterial species.
Especially, romsacin was also effective against clinically important pathogenic bacteria including WHO priority pathogens such as Staphylococcus aureus and Enterococcus faecium, and food-borne pathogens Listeria monocytogenes and Bacillus cereus.However, its mode of action remains unclear as PI staining assay and SEM observation did not tell obvious trait of romsacin action.
Overall, data carried out by a number of highly experienced techniques to investigate bacteriocin peptide so that the characteristic feature of romsacin was remarkedly described.The manuscript is scientifically sound and will provide impact on this field.Meanwhile, I still believe that several improvements are required to ensure rationality and importance of author's claim.
1.I could not find figure legends for figure 1, 2 and 3. Please provide legends for these figures.It will be great help for readers.
-Sorry for not making it clear enough.Legends for all figures are provided after the references.Please see line 813 to 858.
2. The authors are using the term "WHO priority pathogens" in the title.However, only some of the experiments actually examined about WHO priority pathogens such as S. aureus or E. faecium.For example, biofilm disruption assay, PI staining test, and SEM observations should be performed using any WHO priority bacteria.Alternatively, excluding "WHO priority pathogens" from the title could be an option.
-Thank you for your input.We have now included the WHO priority pathogens Staphylococcus aureus and Enterococcus faecium in the biofilm disruption assay, and S. aureus in SEM observations.Please also see answers under point 5 and 6 below.
3. Lines 89-91: This sentence seems appropriate to be included in the result section.Had this prediction already been done by previous study?If so, please provide citation.
-Thank you for the comment, we have edited the paragraph to improve clarification.This paragraph is a summary of what was done in this study.The genomes were sequenced previously, and the reference has now been provided.The gene cluster was discovered in this work and is further described in this paper.Please see lines 89-90.
4. Line 93, 180-181: "MRSA" does not stand just for S. aureus, but for "Methicillin-resistant" S. aureus as the author described in line 60 and elsewhere.Similar for VRE.I think it is not necessary to specify every time, and only abbreviations such as "VRE" and "MRSA" can be used only once after the first definition at line 60.
-We have now changed the manuscript according to your input.Changes are marked in yellow in the marked-up manuscript.-Thank you for your valuable feedback.We have now included biofilm producers of MRSA and VRE in the biofilm experiment.Please see updated Figure 6, and updated paragraphs in results, discussion and material and methods (marked in yellow in the marked-up manuscript).

Fig 7 and 8:
It is little bit confusing that pore forming assay and SEM observation were carried out only with L. lactis.Since Fig. 6 shows that romsacin destroys staphylococcal biofilms (suggesting lytic activity against these bacteria rather than only growth inhibition), the mechanism should be investigated using this staphylococcus, not other model bacteria.One cannot exclude possibility that romsacin can form pore on Staphylococcus cell membrane even if it does not pore form on L. lactis.
-We have included MRSA, S. haemolyticus, S. epidermidis and a B. subtilis strain in the SEM observations.Please see updated method, results, Figure 8 and discussion (marked in yellow in the marked-up manuscript).The cell surface of the staphylococci appeared the same both with and without romsacin treatment.The B. subtilis strain showed a severe disruption of the cell.We still don't know the mechanism in staphylococci, and perhaps the results would have been different with bacteriocin treatment longer than 30 minutes.However, that is for further investigation.-Thank you for your feedback.We have made a growth curve of MRSA and S. haemolyticus where we have compared untreated and romsacin treated growth.Please see Figure 9, and updated method, results and discussion (marked in yellow in the marked-up manuscript).
-We have now added information about the mode of action of vagococcin mentioned in -Thank you for pointing this out.We have added further details about other bacteriocins effective against MRSA and VRE, please see lines 274-277.Romsacin has broad antimicrobial activity and are also effective against biofilm.All clinically important strains don't have the same resistant profiles, and it's important to map out several alternatives (including romsacin) for future treatment options.9. Lines 244-247: Effect of PMBN or EDTA on romsacin activity against Gram-negative bacteria should be addressed here in this study.
-Investigating the synergistic properties of romsacin with other antimicrobial agents is of high clinical relevance.However, demonstrating the synergistic potential of romsacin would require inclusion of several agents and bacterial strains, which is outside the scope of this first descriptive paper.This is something we would like to pursue in a follow-up study.
10. Lines 286-287: To clarify relationship between purified romsacin and the genetic element, constructing of deletion mutant of romA1/romA2 would be better evidence.
-Thank you for your suggestion.Instead of creating a S. haemolyticus deletion mutant, we heterologously expressed the bacteriocin cluster by cloning the lanA-M2 genes into the inducible expression vector pRMC2 and transforming it into S. aureus RN4220.When induced, RN4220 carrying the plasmid was able to inhibit the growth of Lactococcus lactis as the original S. haemolyticus strain 57-27 was able to do.Neither the RN4220 wild type, nor uninduced RN4220+pRMC2_Romsacin, was able to produce clear zones in the L. lactis lawn.These additions can be found in the text in lines 136-143, 148-150, 268-269, and 404-449.We trust that this provides the supporting evidence for the activity of the bacteriocin cluster requested by the reviewer.
11. Table 2: The list of same names for distinct strain rows is not very informative for reader.Please specify at least strain name or any unique identifier (for example, Freeze no. in Supplementary table 1 can be used) within species.
-Thank you for your comment.We have now included more information in the table to guide the reader.

Reviewer #2 (Comments for the Author):
The paper from R. Wolden et al. describes the isolation, purification, and partial characterization of an antimicrobial peptide (a bacteriocin) produced by a strain of Staphylococcus haemolyticus that was selected among 174 clinical and commensal isolates.This bacteriocin was named romsacin by the authors and it was identified as a two-peptide lantibiotic.The authors characterized the producing gene cluster, isolated romsacin using a classical purification protocol, and performed MALDI-TOF MS that identified the molecular mass of romsacin.From these data, they predicted that romsacin was a novel two-peptide lantibiotic.Romsacin was shown to inhibit a panel of Grampositive bacteria among which S. haemolyticus, and the WHO priority pathogens vancomycinresistant Enterococcus faecium (VRE) and methicillin resistant S. aureus (MRSA).It also destroyed S. epidermidis and S. haemolyticus biofilms.In addition, the authors performed preliminary experiments to tackle the mechanism of action of romsacin and concluded that the formation of pores could not explain the romsacin antibacterial activity.
The paper is well written, the study could be interesting and could open interesting perspectives, but many aspects are very preliminary.The points detailed below need being taken into account.

Specific comments
1-The study appears preliminary as regard both the structural characterization, and its spectrum of activity and mechanism of action.The MALDI-TOF measurements only allowed obtaining the molecular masses of the two components of romsacin RomA1, RomA2.Paragraph lines 164-173 is only speculative.The mechanism of action of RomA1-A2 was determined for a part on S. haemolyticus and S. epidermidis (confocal microscopy) and on L. lactis IL1403 (propidium iodide, SEM).Please justify the choice of three different strains for these experiments.
-Thank you for you feedback.We agree that some of our work is preliminary.However, this is a first descriptive paper, and we will include more assays in follow-up studies.We agree that the structure prediction described in lines 164-173 is speculative, as the sequence-related lantibiotics lacticin 3147 and lichenicidin were used.A crystal structure would provide further confirmation of structure, but this will we pursued in follow-up work.
-We chose S. epidermidis and S. haemolyticus strains because they are good biofilm producers and often cause implant-associated infections.L. lactis was chosen because it is commonly used as a model organism in the lab when testing bacteriocin mode of action.We have now also included MRSA and VRE (WHO priority list) in the biofilm confocal microscopy analysis.S. haemolyticus, S. epidermidis, and S. aureus are now also included in the SEM analysis.
2-Perturbation of the biosynthesis of peptidoglycane is another mechanism of action demonstrated for other lantibiotics.The hypothesis should be tested here.

3-In Table
As L. lactis was used for the mechanism of action, the activity on this strain should be added in the Table 2, while it only appears in Table S1.
-Thank you for your comment.We have now added strain references and L. lactis in Table 2.
-We have added the BU/mL for the MIC units in Table 2 and Table S1.4-Page 7, lines 151-173: it is desirable to separate sections MALDI-TOF and structure prediction in two different sections.
-Thank you for your suggestion.We have now separated the sections, please see line 159 and 168.
5-As the authors argue for the activity of romsacin against VRE, MRSA pathogens, a toxicity assay should be included in the paper (at least hemolysis assay) to further identify its potential.
-Investigating the toxicity is highly relevant for further application.However, we believe this can be included in later studies, and is outside the scope of this first descriptive paper.-Thank you for your feedback.Due to lack of space in the figure, it was difficult to write the fractions in larger font.However, we have added shades of gray to make it easier to understand in which of the fractions we find antimicrobial activity.The elution peak is indicated by an arrow.
-The RPC software has not generated a time curve, so it does unfortunately not appear on the figure.However, we have written in the method section that we used 200 ml in each purification, and that the flow rate was 2-4 mL/min.-Thank you for your comment, we have now corrected the references.10-The sentence turns used in the materials and methods "We screened overnight cultures; line 332 "we tested a S. haemolyticus type strain" etc should be avoided and changed to the impersonal grammatical turn.
-Thank you for your comment.We believe that using impersonal or personal terms is subjective, so we choose to keep our style after consulting the journal regarding their guidelines and advice on this matter.You can find the journal's response below: Dear Dr. Wolden, Thank you for your interest in our journals.Either approach is fine: We screened, we tested or Cells were screened, strains were tested.The only caveat for the first approach is that if a paper has only one author, "I" would be used instead of "we."(In that case, some authors prefer to use the impersonal approach rather than sprinkle "I" throughout the paper.) We do not have style specific resources available, although our https://journals.asm.org/editorial-style?journal=spectrum page does list the references that underpin our style.
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Becky Zwadyk Spectrum 11-Typing mistake: line 293 suppress "and" -Thank you for your observation.We have now corrected the typing mistake, line 349.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Thank you for submitting your paper to Spectrum.Sincerely, Sacha Pidot Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

5.
Fig 6: I cannot see scientific reason not to perform the biofilm experiment with S. aureus.S. aureus and E. faecium can also form biofilm in laboratory condition.Test with S. aureus or E. faecium strain (ideally MRSA or VRE) will ensure that romsacin can be effective agent against WHO priority pathogen infection.
7. I am curious the mode of action of romsacin is bacteriostatic or bacteriolytic.To address this issue, the author could try some simple test where bacteria is exposed with romsacin in liquid phase and then measure changes of CFU or turbidity of culture over incubation time.If CFU or culture turbidity decreases over time, it suggests that romsacin has bacteriolytic activity.Also, Fig 4 displays several bacteriocins similar to romsacin.Are there any previous report about the mode of action of these bacteriocins?If there are literatures, the manuscript can include further discussion about the mode of action of romsacin.

6-
Figure 2: antibacterial activity should be indicated on the figure; fraction numbers are too small to be read.Perhaps indicate only a few fractions in bigger letters...; time should appear on the figure.

7-
Legend to Figure 3 is too elusive: "of an active fraction"; specify which fraction -We have now changed the figure legend to «pooled active fractions».The fractions with the highest antimicrobial activity were pooled and used for further testing.The area for the pooled fractions is now also indicated on Figure 2. 8-Legend to figure 4: the names of producing bacteria should be indicated -We have now indicated the names of the producing bacteria in the figure legend, lines 828-832.9-Literature references: The names of bacteria (genus and species) and if useful of other organism should be in italics (Refs 6, 8, 14,...) and the titles should not include systematic capital letters (ref 16).