Occurrence of Methicillin-Resistant Staphylococcus spp. on Brazilian Dairy Farms that Produce Unpasteurized Cheese

Round 1

Reviewer 1 Report

At first read, this paper appears to have interesting results and is well written. However, I feel that the detection of methicillin-resistant coagulase-negative staphylococcal species, which are found relatively readily in the environment, are commonly not multidrug-resistant in the same way as their coagulase-positive counterparts, and are less pathogenic in nature, has been over-emphasised in a parallel to MRSA.

The introduction discusses MRSA almost exclusively, and the discussion further draws parallels to the risk of MRSA in food production despite the finding of no MRSA isolates in this study. I think that the paper has scientific merit, but the introduction and discussion should reflect the lack of MRSA and not attempt to hide that with reference to other literature. Further in depth comments are added below. 

Introduction

Line 25-30: This should discuss in more depth the difference between MRSA and MR-Coagulase-negative spp. Throughout 'MRS' and 'MRSA' appear to be used interchangeably however they are very specific terms and not the same. The single line of 'MRS strains in veterinary medicine' should be expanded to discuss the difference between MRSA in bovine/companion animal medicine and vet-specific MRSP and MRSS isolates. Some discussion of the much higher prevalence of MR-coagulase negative staphylococci (including in the environment) would be of benefit here, and then in the discussion, to aid interpretation of the study's results.

Line 33-35 - This sentence is not clear as to how farm workers are infected and then turned into sources of contagion (is this a reference to MRSA carriage status?)

Line 36 - MRS is used here when it should be MRSA.

Overall the introduction lacks information on why MRSA is a risk in the food chain (e.g. enterotoxins) and the role of other staphylococcal species in food-borne infections

Materials and Methods

Line 49 - repetition of unpasteurized

Line 54 - Staphylococcus spp. vary greatly in their appearance; please explain how 'typical' colonies were selected

Line 48-57 - Description of how swabs / faecal samples were processed is missing (e.g. use of enrichment culture)

Line 58-60 - This reiterates lines 544-55. The Aquino reference includes enrichment culture, among other things. Please be clear as to which parts of this method were followed. A longer description of the processing of these samples would be helpful to allow the methods to be replicated.

Line 61-66 and 75-78 - Primers do not need to be listed as the reference is given.

Line 68 - Disk (not plate) diffusion method

Line 73-113 - It is not clear why these two methods for phylogenetic analysis were used? They produce identical (to my eye) dendograms in the results. Some description in the methods / results / discussion clarifying why both methods were used would be helpful. 

Results

Line 120-1 - The three antibiotics quoted are all B lactams, which is therefore unsurprising for mecA containing isolates. 'Highly resistant' is not a term that I am familiar with.

Line 121-3 - This is interpretation and best suited to the discussion.

Line 125 - This should highlight the species of these isolates to be clear that they are not MRSA. All antibiotics tested should be included in this table for clarity of the overall susceptibility to a number of antimicrobial agents in the tested bacteria. 

Line 130-132 - Genes should always be italicised. It is unclear when talking about the 5 isolates specified what is being said: are these S. haemolyticus or S. hominis? 

Figure 1/2 - These phylognetic trees appear identical to my eye (I am not a molecular microbiologist though). Some explanation as to why both are needed, and what they show that is different from each other would be valuable. 

Line 144 - 100% similarity = identical?

Line 144-148 - This is unclear and repeats what can be seen in the figures.

Line 150-1 - Interpretation should be left to the discussion

Discussion

Line 158-163 - The sudden introduction of bovine mastitis needs to be introduced earlier (e.g. occurence of coagulase-negative vs S. aureus mastitis). Is there a reference for intra-mammary antibiotics causing resistance? And is there any clinical support to the statement that farm C 'probably' had all animals infected with mastitis?

Line 165 - Although mecA has its origins in coagulase-negative species, which has then spread to S. aureus etc, I do not think that antimicrobial resistance gene exchange can be described as 'easy'. The emphasis here then goes onto MRSA in milk - but no MRSA was detected in this study. The risk of MR-coagulase-negative spps should instead me discussed. 

Line 170-5 - Again, this focuses on antimicrobial resistance in MRSA (a MDR-pathogen) but MR-coagulase-negatives are often not MDR. Further interpretation of the resistance patterns in these species should be made. 

Line 176-195 - This is making the same point a number of times. Should there be discussion of the environmental nature of coagulase-negatives and the risk posed from this?

Line 199 - This study does not perform enough molecular assessment to determine transmission direction.

Line 207 - What is the risk of MR-coagulase-negative staphylococci in food? Do they produce the same enterotoxins as S. aureus?

Author Response

Please find the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This study deals with an interesting topic in dairy farms. S. aureus causes important economic loss associated with several lesions, such as mastitis, and the associated costs are high in the dairy industry (Graber and Bodmer, 2019). Focuses on the important transmission of MRS from workers to dairy products.

The following changes are suggested:

2017. i) In Introduction. Line 28: A 2007 article is cited. I would add more current citations as Aires de Sousa, M. 2017.

1. ii) In Material and Methods. Line 51: I don’t understand why it cites a review article in the sampling data.

iii) In Material and Methods. Line 72: Don’t use positive control for susceptibility testing of MRS isolates. For example S. aureus strain ATCC 25923.

Author Response

Please find the attachment.

Author Response File: Author Response.docx