Staphylococcus aureus from Minas Artisanal Cheeses: Biocide Tolerance, Antibiotic Resistance and Enterotoxin Genes

Round 1

Reviewer 1 Report

The manuscript by Bernadette Dora Gombossy de Melo Franco et al. reports characteristic of Staphylococcus aureus from Minas artisanal cheeses, their biocide tolerance, antibiotic resistance and enterotoxin genes. The study brings new information on the level of basic research, regarding the relationship between resistance to biocides, antibiotic resistance and presence of enterotoxin genes in S. aureus strains as frequent contaminants in cheese manufacture. The topic is of interest in consideration of the improving the hygiene and manufacturing practesses to safeguard consumers´health. Though, some explanation about materials and methods, results and discussion are needed. 

Below are some points: 

Materials and methods

L85-92,106,116,118: data regarding peptone water, Petrifilm, BHI, Baird-Parker agar, GenElute kit, etc. could be presented, please, collect these data, names of incorporation, city, etc.

L101: TSB – Please, explain the abbreviation at first time 

Results

L182-199: The antibiotic resistance and the presence of efflux pump genes in biocide and antibiotic resistant isolates should be presented in Table 

Discussion

L246: Salmonella – please precise the species or use Salmonella sp. 

L277-280 and 283-284 – The same sentence was written twice, please, correct/rewrite it and explain the main reasons resp. factors of resistance genes´ transport between human-animals-food (One health concept). The staphylococcal strains were isolated from Artisanal cheese – the Authors can be focused on the presence of staphylococci in this type of cheese, the ways how this cheese can be contaminated or if these bacteria can also positively or negatively influence microbiome and/or the microbial quality of Artisanal cheese. However, the Authors should be also added, which biocides are still active against these bacteria and can be used in the cheese production, and/or which another ways (natural) can be also preferred during the manufacturer process.  

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The reviewed article Staphylococcus aureus from Minas Artisanal Cheeses: Biocide Tolerance, Antibiotic Resistance and Enterotoxin Genes is good in terms of relevance, scientific novelty, theoretical and practical significance, reliability of the results obtained, as well as in terms of the volume and level of research conducted.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Report

The present review title: Staphylococcus aureus from Minas Artisanal Cheeses: Biocide Tolerance, Antibiotic Resistance and Enterotoxin Genes” evaluated the tolerance of S. aureus isolated from Minas artisanal cheeses to the biocides benzalkonium chloride, hexadecylpyridinium chloride, cetrimide, triclosan, hexachlorophene and chlorhexidine, and the simultaneous occurrence of genes coding for resistance to antibiotics (mecA, aacA-aphD and tetK), efflux pumps [qacA/B and smr (qacC/D)] and enterotoxins (sea, seb, sec, sed, see,seg, seh, sei and sej). The topic of this research is very interesting and important from the medical point of view particularly in the era of searching new approaches for combating multidrug resistant (MDR)-pathogens. However, I have certain important comments that should be considered before accepting this work for publications, these include:

 

Major Comments:

                                                                                                                

1. The author should explain rational of testing for this particular efflux pumps [qacA/B and smr (qacC/D)].
2. The function and the coded proteins of the nine tested enterotoxins genes (enterotoxin genes sea, seb, sec, sed, see, seg, seh, sei and sej ) should be written in the methods and in the footnote of Table 2. In addition the author should point out the respective types in in the introduction as well as the respective differences in their actions.
3. In the result rection, Line 163, the author mentioned that” Isolates from the same cheese sample presenting 163 identical results were considered as one isolate”. This is not satisfactory to confirm clonal relationship based only on the phenomenon. Therefore, genetic relatedness using ERIC-PCR or RAPD PCR analysis should be done to confirm relationship. Since one cheese sample could have two different aureus isolates exhibiting similar phenotypes but different genotypes. This is of particular importance in this study to confirm origin of S. aueus infection whether from food handlers during cheese production or from skin of animals during milk collection or from infected animals (mastitis, animal infection,….)
4. The author should emphasize the origin or source of the collected 163 isolates collected from cheese samples by comparing the obtained results to those recovered from nasal swabs of food handlers or from skin of animals to explore the potentiality and significance of this study. Otherwise, the obtaining results do not make sense from the medical point of view. Since and based on the obtained results we could not confirm the source and etiology of Staphylococcal infection whether it was human- or animal-borne.
5. The discussion should be modified to point out and discuss the obtained findings in the light of recent literature and should further elaborated on the source and etiology of infection based on the results obtained from ERIC-PCR and/or RAPD-PCR analysis of Staphylococcal isolates recovered from both cheese and human subjects.
6. In the conclusion the author mentioned” Biofilm 291 formation by biocide tolerant strains is a threat to disinfection, and presence of antibiotic resistance and enterotoxins 292 genes may be an additional health concern for consumers of artisanal cheeses: however, the author did not examine the biofilm production. So, I advised revising of the conclusion to only point out the exact conclusion based on the results obtained from this study.

Minor comments:

1. Staphylococcus aureus should be italized in the whole manuscript.
2. Abbreviation should me written in full sentence in the first mention and the abbreviation should be used in the whole manuscript thereafter (Example: in the introduction, line 34, SEA e SED; line 35, SEB, SEC, SEE, SEG, SHE and SEI); (Line 88, BHI; Line 101, TSB; MIC line 107) and also the author should avoid repetition of abbreviation (line 46 and Line 48, Line 66, there is a repetition of the quaternary ammonium compounds (QACs)
3. Insert Source (city and counter of any used kits or apparatus (Example GenElute™ kit line 92)
4. Line 92, the 16S rDNA should be corrected to 16S ribosomal RNA.
5. Line 95 “A total of 159 Staphylococcus aureus isolates were obtained” should be transferred from Methods section to the Result section.
6. Line 107 the citation of Marquez et al., 2017 should be cited as numbers in square brackets ) [17].
7. Line 118 was below 1.7 and 2.0. should be corrected to was in the range 1.7 to 2.0.
8. The author should check the concentration of the primers used in PCR since they mention 1µl (400nM) and normally primer concentration is usually in the range of 25-100 picomole).
9. L122, 100pb marker should be corrected to 100 bp marker
10. In in the footnote of table 1, The author should include explain the abbreviations and the role of each gene being tested (mecA, a gene coded for……..etc). aacA-aphD,….etc
11. The aacA-aphD primers, was it sued to amplify the acetyltrasferase gene (aacA) or aminoglycoside phosphotransferase (aphD) or for certain family transposase (the author should clarify this issue).
12. Taq should be italized (Taq)
13. L141 “The amplification products and a 100pb marker (Promega, Spain) were submitted to 2% agarose gel 141 electrophoresis” should be corrected to “The amplification products and a 100 bp marker (Promega, Spain) were analyzed to 2% agarose gel 141 electrophoresis.

 

 

 

Comments for author File: Comments.docx

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript presents a quite well written research however, it needs improvement. The repetition of the same words is very common in the whole manuscript so I recommend to carefully read the manuscript and rewrite some sentences or use synonyms.

Major comments

The description of  the DNA extraction and estimation of the quality of the extracted DNA is the same in subsections 2.3 Determination of antibiotic resistance, 2.4 Screening for efflux pump genes and 2.5 Investigation of staphylococcal enterotoxin genes. Therefore I suggest to add one subsection to the Material and Methods and describe this procedure once instead of repeting the same phrases in the three mentioned subsections.

The information about the production of staphylococcal enterotoxins by enterotoxigenic strains of S. aureus seems to be missing in the text. It may lead to uncorrect conclusions that all isolates which have SE genes will produce the toxins. I suggest to complement the information about the expression of classical SE genes in the introduction and the discussion section.

The procedure published in 2003 was used to determine the MRSA strains. However, it only allows to detect mecA gene. Nowdays, it is known that a homologue to mecA gene - mecC is also responsible for methicillin resistance. Since the presence of mecC was not analyzed please explain why or add the reults of mecC analysis.

Minor comments:

Line 12-13 rewrite this sentence and avoid the repetition of "and"

Line 15 - Add colon after "biocides"

Line 16 - change "genes coding for resistance to antibiotics" to "genes encoding antibiotic resistance"

Line 22 - change "safeguard" to "ensure"

Line 34 - add "and" between instead of "e"

Line 35 correct SHE into SEH

Line 58-60 rewrite this sentence and avoid the repetition of "and"

Line 98 - Add colon after "biocides"

line 122 and in the whole manuscript - correct "pb" into "bp"

line 157 - correct she into seh

line 168-169 - rewrite this sentence to avoid repetition of  "were tolerant". The word "tolerant is used 7 times in lines 168-171, please use synonyms

line 177 - rewrite the phrase "positivity for antibiotic resistance genes" for example into "the presence of antibiotic resistance genes"

line 180 - add comma before "simultaneously"

line 187 - delete "s"

line 188 - write "9" as "nine"

line 190 - delete "gene" as it is written twice

line 191 - change "gene" into "markers" to avoid words repetition

line 199 - change second "isolates" into "S. aureus"

line 202 - Is 139 it the correct number? or should it be 136?

Table 3 - sec gene the prevalence is 5 (3.87%), so it is 3.9%

Line 210-211 the title of the subsection is too long, I suggest to rewrite it for example: "Tolerance to biocides and presence of antibiotic resistance and SE genes"

Line 212 - delete "occurence of"

line 238 - change "corroborating" into "confirming"

line 264 - change "anyway" into "however"

Line 109 - Add the reference Marquez et al., 2017 as [17] at the end of cited phrase - in line 109 and delete it from line 107

Lines 56, 74, 107, 115, 143, 153, 156, 194, 257 -  too much space between the words

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Tha Authors answered and corrected all questions/comments.

 

Reviewer 3 Report

the authors have responded to all the addressed comments