Enterococcus faecium Isolates Present in Human Breast Milk Might Be Carriers of Multi-Antibiotic Resistance Genes
Round 1
Reviewer 1 Report
In the paper “Enterococcus Faecium Isolates Present in Human Breast Milk 2 might be Carriers of Multi-antibiotic Resistance Genes” the authors analize the antibiotic resistance profile of Enterococcus strains isolated from human breast milk. After a first phenotypic analysis, they evaluated the presence of genes responsible of the antimicrobial resistence on plasmids.
The aim of this study was to consider the safety of potential probiotic strains for human and animal use. Anyway they did not analyze the probiotic potential of these strains, and their possible use as probiotics only comes from their origin (human breast milk).
The main conclusion is that only one isolate met the criteria of EFSA being sensitive to all the tested antibiotics. About the other strains the presence of resistance to 1 to 7 antibiotics has been found. The authors analyzed the presence of genes potentially responsible to the resistance on plasmids. At the same time they say (line 20) that "in the majority of isolates antibiotic resistance genes were located on chromosomes". Condidering that even the presence of antibiotic resistance cassette on the chromosome is not raccomanded, it is not clear why the authors exclude the chromosomal genes from their analysis.
They also discuss about the origin of the antibiotic resistant strains but at the end they conclude that "the questionnaire that was given to donors in the current study was not exhaustive enough to provide this type of information" (lines 184-185). In conclusion the best way to evaluate the possibility to use a strain as probiotic is to directly analyze its sensitivity to antibiotics by phenotypic analysis.
In summary I think that this study does not provide new information about the isolation of new potential probiotic strains or about the diffusion of antimicrobial resistance genes.
Other comments:
lines 106-107 the authors said: "... strains did not demonstrate even growth on solid media (data not shown)". How the authors explain that the strains were able to grew in liquid medium and not on solid medium? How did they isolate them?
paragraph 2.2: Did the authors consider genes coding for efflux pumps, expecially for the multiresistant strains?
paragraph 2.3: it could be interesting to introduce information about the plasmids present in the strains. Are they contained in all of the analyzed strains? Are there information about the number or the dimension of the different plasmids? Did the used protocols able to even isolate big plasmids?
paragraph 2.4. Gene expression analysis
Did the author consider that the genes that were not expressed in the tested conditions could be expressed in other environmental conditions?
line 137: why the authors decided to analyze only the strains with 2 or more antibiotic resistance genes? if the aim of the study is to know if the strains are safe for their use as probiotics, even the presence and the expression of a single gene coding for 1 antibiotic resistance is important
line 172: why the authors decided to exclude the Lactobacillus strains, that are commonly used as probiotics, from their analysis?
line 221 the sentence is probably incomplete
Author Response
The main conclusion is that only one isolate met the criteria of EFSA being sensitive to all the tested antibiotics. About the other strains the presence of resistance to 1 to 7 antibiotics has been found. The authors analyzed the presence of genes potentially responsible to the resistance on plasmids. At the same time they say (line 20) that "in the majority of isolates antibiotic resistance genes were located on chromosomes". Condidering that even the presence of antibiotic resistance cassette on the chromosome is not raccomanded, it is not clear why the authors exclude the chromosomal genes from their analysis.
Response: The research that we described in our paper was the part of the project that aimed at developing dietary supplements based on probiotics. Based on the recommendations of the European Food Safety Authority (EFSA), if tested strain shows phenotypic antibiotic resistance, it could still be considered as safe for use, if it could be excluded that such resistance is not acquired. Therefore, we only considered the analysis of plasmids because within very limited timeframe that was predicted for the project and limited access to the proper equipment, we couldn’t investigate further if antibiotic resistance genes located on chromosomes were either acquired or intrinsic. The analysis of plasmids was the most time effective method to exclude strains that didn’t meet the criteria defined by EFSA.
They also discuss about the origin of the antibiotic resistant strains but at the end they conclude that "the questionnaire that was given to donors in the current study was not exhaustive enough to provide this type of information" (lines 184-185). In conclusion the best way to evaluate the possibility to use a strain as probiotic is to directly analyze its sensitivity to antibiotics by phenotypic analysis.
Response: The significance of the questionnaire is very prominent because it can deliver information about possible routes of the transmission of the antibiotic resistance. Also, it could potentially help to identify when in life of women involved in the study, their natural microbiome could have acquired resistance to certain antibiotics. In that case, collected data could be used for the proper education on the usage of antibiotics.
In summary I think that this study does not provide new information about the isolation of new potential probiotic strains or about the diffusion of antimicrobial resistance genes.
Other comments:
lines 106-107 the authors said: "... strains did not demonstrate even growth on solid media (data not shown)". How the authors explain that the strains were able to grew in liquid medium and not on solid medium? How did they isolate them?
Response: We added the following information in our revised manuscript in line 112-113: used for that analysis (Brain Heart Infusion agar, data not shown).
Media used for the isolation of analysed strains were as follows: MRS broth, M17 broth or BM broth which have much more complex composition than BHI agar. Moreover, liquid medium provides limited access of cells to oxygen and since the majority of tested isolates were facultative anaerobes, that could beneficially impact their growth. On solid media, cells are more exposed to that gas, even in the incubator with CO2 flow. Another possibility is that after depositing cells in microbank tubes, cells moved to the non-countable but viable state.
paragraph 2.2: Did the authors consider genes coding for efflux pumps, expecially for the multiresistant strains?
Among tested genes, the following encode efflux pumps: emrB (erythromycin), tetK (tetracycline), marA (rifampicin). Other genes were selected based on the high frequency on plasmids that was found in the CARD database as described in Materials and Methods.
paragraph 2.3: it could be interesting to introduce information about the plasmids present in the strains. Are they contained in all of the analyzed strains? Are there information about the number or the dimension of the different plasmids? Did the used protocols able to even isolate big plasmids?
Response: Such information is not available because at that stage of the project we did not sequence whole plasmids. The protocol that was used in the study enabled isolation of small and large plasmids as well.
paragraph 2.4. Gene expression analysis
Did the author consider that the genes that were not expressed in the tested conditions could be expressed in other environmental conditions?
Response: Yes, however, at the time when the experiments were carried out we didn’t have a chance to create and validate the protocol that would enable investigating such phenomenon under the conditions which are close to the environment similar to the human breast milk. In another project carried out at CDC Poland we were trying to produce the substance that would be similar to human breast milk but produced in cell cultures but our attempts to achieve that, failed.
line 137: why the authors decided to analyze only the strains with 2 or more antibiotic resistance genes? if the aim of the study is to know if the strains are safe for their use as probiotics, even the presence and the expression of a single gene coding for 1 antibiotic resistance is important
Response: In the current paper we focused only on strains carrying at least two antibiotic resistance genes on plasmids because those could be potentially more harmful to people. Due to the fact that the study was carried out in the private sector, not the academia, we were time limited and didn’t have a chance to carry out additional experiments or analyses.
line 172: why the authors decided to exclude the Lactobacillus strains, that are commonly used as probiotics, from their analysis?
Response: Lactobacillus strains were also considered in the study but since those will be the subject of patents, we couldn’t submit their sequences to the GenBank or results obtained for them. In the industrial environment we need to be cautious in regards of the data that we publish so we wouldn’t breach confidentiality agreements with our employers. We also added the following sentence in lines 183-184 of the revised manuscript: We did not include the results obtained for those isolates because their application could be patented in the future after further studies.
line 221 the sentence is probably incomplete
Response: Word ‘among’ in line 237 was deleted from the sentence.
Our paper was proofread by the English native speaker.
Reviewer 2 Report
The manuscript "Enterococcus Faecium Isolates Present in Human Breast Milk might be Carriers of Multi-antibiotic Resistance Genes" is a original study of very high quality.
After careful analysis I really do not see mistakes or other things to improve. The only question I consider is about methodology and results. The authors wrote, that there were 20 isolates from each donor. Do you checked the differences between these single probes from the same donor and are there any differences or variability suggesting that we have to collect multiple probes as better method compared to single probes? Could you explain and comment this.
Author Response
Response: Thank you very much for a very insightful comment.
In the result section we added the following paragraph in lines 144-147: We also noted that if the analysed isolates originated from a common donor, they carried the same AMR genes: CDCP521, 531 and 533 carried ErmB1, CDCP750 and 753 carried dfrA14 gene; CDCP 787, 791 and 795 carried dfrA14 gene; CDCP1446 and 1449 carried dfrA14 gene as well.
In the discussion we added the following sentence in lines 321-326: We also noticed that if isolates originated from the same donor carried the same AMR genes on plasmids. This suggests that either those isolates acquired those genes in the same environment and then they were transferred to human body, the transfer of those genes between bacterial cells in human body is very easy and happens sponta-neously or that human microbiome acquired the resistance in contact with the antibi-otic that was taken incorrectly (the treatment interrupted before it was complete).
Reviewer 3 Report
The work is important from a public health point of view because it addresses an important global problem, which is the antimicrobial resistance of Enterococcus faecium isolates. However, the work needs improvement before it can be accepted for publication. There is a lot of talk about probiotic isolates, but there is nothing in the work that proves it. The tables are long and confusing. I suggest improving. More suggestions are below:
Line 11. Why talk about probiotic strains if that was not the purpose of the study? I suggest removing the text that mentions this.
Line 73. Probiotic isolates was not the purpose of the study.
Line 91. “The majority of tested isolates were resistant to erythromycin 91 (96%), followed by trimethoprim (67%), tetracycline (57%) and gentamicin (55%) (Table 2).
Table 2 does not show the percentage of Resistance of the isolates, I suggest adding to the Table or rewriting.
Line 93. There were three antibiotics that demonstrated particularly high efficacy and isolates 93 were susceptible to them: linezolid (98%), then ampicillin (94%) and chloramphenicol 94 (92%).
It is not clear how the isolates were classified into sensitive and resistant, since the methodology only describes the methodology for obtaining the MIC. Even using MIC it is possible to classify the isolates into sensitive and resistant. See Clinical and Laboratory Standards Institute (CLSI, 2017).
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100 (ISBN 1-56238-804-5 [Print]; ISBN 1-56238-805-3 [Electronic]). Clinical and Laboratory Standards Barbara L. Zimmer, PhD. Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2017.
Line 100. Cannot see this in the table.
Line 118. As most isolates do not have resistance genes, I suggest joining similar isolates and leaving them in one line. Improved Table configuration.
Line 123. Why are the numbers of isolates in Tables 3 and 4 different from those in Table 2? Are they different isolates?
Line 149. There is no call from Table 5.
Row 186. Very confusing to discuss susceptibility and resistance of isolates, these data are not clear in Table 2.
Line 239. It is repeated throughout the discussion for each antibiotic that has not been addressed the antibiotics and patient diseases, I suggest putting it together in a single paragraph.
Line 463: Conclusion: the conclusion needs to respond to the objective of the work.
Line 506: References
50% of references are more than 10 years old. I suggest upgrading to the last 5 years.
Author Response
Line 11. Why talk about probiotic strains if that was not the purpose of the study? I suggest removing the text that mentions this.
Response: The main goal of the funded research project carried out by us was to find candidates for probiotics in human breast milk We discovered antibiotic resistance while carrying out the analysis and decided to investigate that phenomenon further. Therefore, to comply with the requirements of the funding body, we insist on keeping the information regarding the search for probiotics. We also added the following sentence in lines 64-67: Originally, the project was focused on searching putative probiotics in human milk microbiota but since antibiotic resistance was shown to be very common among tested isolates, we decided to take a closer look into that phenomenon.
Line 73. Probiotic isolates was not the purpose of the study.
Response: The main goal of the funded research project carried out by us was to find candidates for probiotics in human breast milk We discovered antibiotic resistance while carrying out the analysis and decided to investigate that phenomenon further. Therefore, to comply with the requirements of the funding body, we insist on keeping the information regarding the search for probiotics. We also added the following sentence in lines 62-64: Originally, the project was focused on searching putative probiotics in human milk microbiota but since antibiotic resistance was shown to be very common among tested isolates, we decided to take a closer look into that phenomenon.
Line 91. “The majority of tested isolates were resistant to erythromycin 91 (96%), followed by trimethoprim (67%), tetracycline (57%) and gentamicin (55%) (Table 2).
Table 2 does not show the percentage of Resistance of the isolates, I suggest adding to the Table or rewriting.
Response: The verse with the percentage of resistant strains was added in the bottom of Table 2.
Line 93. There were three antibiotics that demonstrated particularly high efficacy and isolates 93 were susceptible to them: linezolid (98%), then ampicillin (94%) and chloramphenicol 94 (92%).
It is not clear how the isolates were classified into sensitive and resistant, since the methodology only describes the methodology for obtaining the MIC. Even using MIC it is possible to classify the isolates into sensitive and resistant. See Clinical and Laboratory Standards Institute (CLSI, 2017).
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100 (ISBN 1-56238-804-5 [Print]; ISBN 1-56238-805-3 [Electronic]). Clinical and Laboratory Standards Barbara L. Zimmer, PhD. Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2017.
Response: We provided the explanation to that in the introduction section, lines 38-40: In order to distinguish resistant from susceptible strains, the cut-off values of antimi-crobial concentration were established. Strains which growth is inhibited at minimum inhibitory concentration (MIC) or below it are considered susceptible.
Line 100. Cannot see this in the table.
Response: The verse with the percentage of resistant strains was added in the bottom of Table 2.
Line 118. As most isolates do not have resistance genes, I suggest joining similar isolates and leaving them in one line. Improved Table configuration.
Response: Changes in Table 3 and 4 were made
Line 123. Why are the numbers of isolates in Tables 3 and 4 different from those in Table 2? Are they different isolates?
Response: Those numbers are the same – we double checked that. Due to the editing of tables last digits of the numbers were moved to the next verse which could have caused some confusion.
Line 149. There is no call from Table 5.
Response: Table 5 was mentioned in lines 155-156.
Row 186. Very confusing to discuss susceptibility and resistance of isolates, these data are not clear in Table 2.
Response: We added letter R next to numbers if the isolate is resistant to particular antibiotic and letter S if the isolate is susceptible to a particular antibiotic. We did that only in the case of antibiotics for which MIC values were specified in the EFSA guideline.
Line 239. It is repeated throughout the discussion for each antibiotic that has not been addressed the antibiotics and patient diseases, I suggest putting it together in a single paragraph.
We removed that sentence from all paragraphs when it was mentioned and inserted a single paragraph in lines 232-234.
Line 463: Conclusion: the conclusion needs to respond to the objective of the work.
Response: We made changes to the conclusion section in lines 497-507.
Line 506: References
50% of references are more than 10 years old. I suggest upgrading to the last 5 years.
Response: We selected our references based on the relevance to our findings. We believe that using references that report some findings for the first time should be used so we cited those papers in our manuscripts.
Our paper was proofread by the English native speaker.
Round 2
Reviewer 1 Report
The paper has been in part revised. In particular:
Lines 23-24: remove the last sentence in the abstract (“Moreover, we found one isolate that is a putative probiotic 23 (CDCP539) but it requires further investigation”). This is not the aim of the work and data reporting the probiotic features of the strain are missing
Table 2: the table is not correctly formatted. Probably the font size should be reduced.
Table 3 is confusing and is not easy to read. Probably it is better to report only the strains carrying the antibiotic resistance genes. In other words, the data summarized at lines 133-141 can be summarized in a unique table that substitutes tables 3 and 4.
One possibility for the new table could be to indicate the genes in vertical orientation and the strains containing 1 or more of them in horizontal orientation.
Lines 142-145: how do you know that the strains isolated from the same donor and showing the same antimicrobial resistances, are not the same strain? Why do you suppose that are different strains?
Author Response
Lines 23-24: remove the last sentence in the abstract (“Moreover, we found one isolate that is a putative probiotic 23 (CDCP539) but it requires further investigation”). This is not the aim of the work and data reporting the probiotic features of the strain are missing
Response: The sentence was removed from the manuscript.
Table 2: the table is not correctly formatted. Probably the font size should be reduced.
Response: Font size was changed from 9 to 10 and orientation of the page was changed to landscape to make the table easier to read.
Table 3 is confusing and is not easy to read. Probably it is better to report only the strains carrying the antibiotic resistance genes. In other words, the data summarized at lines 133-141 can be summarized in a unique table that substitutes tables 3 and 4.
One possibility for the new table could be to indicate the genes in vertical orientation and the strains containing 1 or more of them in horizontal orientation.
Response: We merged both tables and left only records for those isolates for which AMR genes were detected
Lines 142-145: how do you know that the strains isolated from the same donor and showing the same antimicrobial resistances, are not the same strain? Why do you suppose that are different strains?
Response: We added the following explanation in lines 425-427: Additionally, we carried out the alignment of all collected sequences to exclude the possibility that obtained isolates could be the same strains. For that purpose we used phylogeny.fr platform.
The paper was proof read by an English native speaker.
Reviewer 3 Report
Dear authors
After the corrections have been made
Author Response
Response: The reviewer had no comments.
