Detecting mecA in Faecal Samples: A Tool for Assessing Carriage of Meticillin-Resistant Staphylococci in Pets and Owners in the Microbiological ‘Fast Age’?

Round 1

Reviewer 1 Report

This is an interesting paper with a novel approach for the detection of MR staph from animals and people. The paper is very well and clearly written for which the authors should be commended. While the results are obviously inconclusive about the utility of the technique- I believe that publication could be helpful to (1) describe the methodology used and (2) as a comparison for future work done in this space. Use of the term "proof of concept" in the intro or discussion may add to contextualizing the preliminary nature of this data. The discussion does a very nice job at discussing limitations of the work and alternative explanations of results.

I have two major concerns:

One is the culture based method for the detection of MRS.  The 6 mg/L of oxacillin concentration seems high particularly when the S. pseudintermedius CLSI breakpoint is 0.5. Is there a chance a significant number of MRSP were missed using this concentration?  Could a reference for this methodology be provided or the rationale explained. I also believe this could be an important limitation to include in the discussion.

Second, there are no reporting of the results of the species identification (both phenotypic and nuc pcr results). I think a table of the 11 MRCoPS isolates would be of tremendous help in understanding the results.

Specific line edits and comments can be found below:

Line 54- Could the "why" this is beneficial be expanded upon here?  Additional background on potnetial utility of this tool in both research and clinical settings would be helpful.

Line 88- what volume of enrichment broth was plated?

Line 96- were slide coagulase negative isolates excluded from future analysis? S. pseudintermedius is traditionally thought of as slide coagulase negative.  Or were classic hemolytic pattern of S. pseud included regardless of slide coag results? Why not perform nuc on all isolates?  Were results consistent?

Line 98: please confirm by conventional or real-time PCR?

Line 150: The decription of the culture results is lacking. Specific results on the isolates identified should be included (see above).

Line 228:  I would remove the word "passive" here- the technique is for the specimen type and not the nature of the specimen collection.

Author Response

Reviewer 1

This is an interesting paper with a novel approach for the detection of MR staph from animals and people. The paper is very well and clearly written for which the authors should be commended. While the results are obviously inconclusive about the utility of the technique- I believe that publication could be helpful to (1) describe the methodology used and (2) as a comparison for future work done in this space. Use of the term "proof of concept" in the intro or discussion may add to contextualizing the preliminary nature of this data. The discussion does a very nice job at discussing limitations of the work and alternative explanations of results.

Thank you very much to the reviewer. We are happy that the paper was enjoyed, and we hope that with the benefit of this review we have been able to make satisfactory changes to the manuscript which further aid its interest and utility for the scientific community. As suggested here, we have added ‘proof of concept’ to the introduction to highlight the utility of this research.

(Line 66-68) ‘This study used quantitative PCR (qPCR) to identify mecA in faecal samples from a population of dogs and their owners as a proof-of-concept study to investigate possible alternative to traditional detection methods from nasal swabs.’

I have two major concerns:

One is the culture based method for the detection of MRS.  The 6 mg/L of oxacillin concentration seems high particularly when the S. pseudintermedius CLSI breakpoint is 0.5. Is there a chance a significant number of MRSP were missed using this concentration?  Could a reference for this methodology be provided or the rationale explained. I also believe this could be an important limitation to include in the discussion.

The concentration of oxacillin used in the mannitol salt agar has been used for many years in our group and has appeared to be very reliable to detect methicillin-resistant staphylococci from carriage sites, following broth enrichment, without also allowing ‘breakthrough’ growth of non-mecA staphylococci. In fact, the Oxoid Brilliance agar resulted in the growth of numerous staphylococcal species that were in fact mecA-negative and oxacillin-susceptible in this case. The discussion has been extended (lines 205-212) to include these comments on concentration of oxacillin within the agar, and the need for confirmation of mecA even when using screening agars. The use of 6 mg/L oxacillin supplementation has been referenced both in the methods (line 99, new references 20 and 21) and the discussion (line 208).

‘It may also be due to the presence of meticillin-resistant coagulase-negative staphylococci (MRCoNS) in these samples, which may have lower oxacillin breakpoints and therefore not have been detected on the 6mg/L oxacillin-impregnated mannitol salt. However, this concentration of plate has been shown reliable for previously detecting MRCoPS [20,21]. In fact, the lower concentrations of beta-lactam antimicrobial in the Oxoid Brilliance agar resulted in a number of non-mecA oxacillin-susceptible staphylococcal isolates being recovered (data not shown), which reinforces the im-portance of confirmatory testing of isolates through mecA PCR when using screening agars.’

Second, there are no reporting of the results of the species identification (both phenotypic and nuc pcr results). I think a table of the 11 MRCoPS isolates would be of tremendous help in understanding the results.

This confirmation has been added as a single sentence in line 161-2, as all canine MRCoPS were S. pseudintermedius, and all human as S. aureus, as may have been expected.

‘All MRCoPS isolated from dogs were confirmed as MRSP and all MRCoPS isolated from humans as MRSA.’

Specific line edits and comments can be found below:

Line 54- Could the "why" this is beneficial be expanded upon here?  Additional background on potnetial utility of this tool in both research and clinical settings would be helpful.

As suggested, this has been expanded on in lines 57-63.

‘This would allow veterinary clinics to more easily request pre-admission screening for carriage of these staphylococci, especially in the case of high-risk elective or non-emergency procedures, without delaying for multiple days. The ability for owners to take samples themselves further reduces the potential costs of sampling that are as-sociated with veterinarian-time and would open the study of MRCoPS-carriage to re-searchers who are not closely-linked with suitably-equipped veterinary premises for sampling of large numbers of animals.’

Line 88- what volume of enrichment broth was plated?

100µL was plated, and this has been added to line 97.

Line 96- were slide coagulase negative isolates excluded from future analysis? S. pseudintermedius is traditionally thought of as slide coagulase negative.  Or were classic hemolytic pattern of S. pseud included regardless of slide coag results? Why not perform nuc on all isolates?  Were results consistent?

This is why DNAse production was also included. Slide coagulase positive isolates (likely all the S. aureus, and some of the S. pseudintermedius) did not require DNAse-testing, however if isolates were slide coagulase negative, they were then tested overnight for DNAse production and if positive (as expected for both S. aureus and S. pseudintermedius) they would be included in VP testing for phenotypic speciation, and then nuc confirmation. The results of nuc PCR were all consistent with phenotype expected. This has been made clearer by including ‘…a range of tests, including…’ (line 104) to this sentence, to hope to clarify that it was not a step-by-step list of tests at which point any isolate could be excluded.

Line 98: please confirm by conventional or real-time PCR?

This has been clarified by adding ‘conventional’ to this sentence in line 107.

Line 150: The decription of the culture results is lacking. Specific results on the isolates identified should be included (see above).

This confirmation of species identification has been added as a single sentence in line 161-2, as all canine MRCoPS were S. pseudintermedius, and all human as S. aureus, as may have been expected.

‘All MRCoPS isolated from dogs were confirmed as MRSP and all MRCoPS isolated from humans as MRSA.’

Line 228:  I would remove the word "passive" here- the technique is for the specimen type and not the nature of the specimen collection.

The reviewer is correct, and we have removed the word passive from this sentence (line 245).

Author Response File: Author Response.docx

Reviewer 2 Report

1. There is some confusion regarding how the number of samples tested was arrived at, given that 13 canine and 20 human paired samples were obtained. Taken together with the study design, this needs further clarifications.

2. MRSP as a key word should have been reflected in the abstract as well as in the results and discussion.

Author Response

Thank you to the reviewer for their comments to help us improve the manuscript, we have included their suggestions in the text as detailed below.

1. There is some confusion regarding how the number of samples tested was arrived at, given that 13 canine and 20 human paired samples were obtained. Taken together with the study design, this needs further clarifications.

For further clarification the breakdown of how many samples were recovered from each of the dogs/humans has been further clarified as suggested in lines 78-80.

‘For this study, 35 canine and 51 human faecal samples and their corresponding nasal swab results were included from 13 dogs and 20 humans (one sample n=1 dog, n=2 humans; two samples n=5 dogs, n=8 humans; three sample n=4 dogs, n=7 humans; four samples, n=3 dogs, n=3 humans).’

2. MRSP as a key word should have been reflected in the abstract as well as in the results and discussion.

To ensure that MRSP is included where appropriate, this has been briefly added to the MRCoPS-carriage PCR confirmation sentence in the abstract (line 22-3).

‘Nasal MRCoPS-carriage (either MR-Staphylococcus aureus or MR-Staphylococcus pseudintermedius was confirmed by identification of species (nuc) and meticillin-resistance (mecA) (PCR).’

Following the other reviewer’s comments, the confirmation of some isolates as MRSP now features within the results (line 161-2).

‘All MRCoPS isolated from dogs were confirmed as MRSP and all MRCoPS isolated from humans as MRSA.’

We have also included MRSP explicitly as one of the MRCoPS considered, when discussing the need for sampling multiple body sites for conventional culture-based techniques as this has been demonstrated in both MRSA in humans and MRSP in dogs (line 203-4).

‘This may be due to the need for sampling of multiple body sites to identify all potential MRCoPS-carriers, as has been shown for both MRSA and MRSP [27,28].’

Author Response File: Author Response.docx