Identification of Salmonella enterica biovars Gallinarum and Pullorum and their antibiotic resistance pattern in integrated crop-livestock farms and poultry meats
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Due to consumer demand, many conventional poultry farms are now growing poultry without antibiotics or synthetic chemicals. In addition to this, pasture/organic poultry farms have increased significantly in the USA, and they are also antibiotic- and chemical-free. According to recent reports, both antibiotic-free conventional and pasture poultry farmers are facing the re-emergence of bacterial diseases. Bacterial diseases cause higher mortality rates in birds and lead to non-profitable poultry farming. This study investigated the prevalence of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum ( S . Gallinarum), the causative agent of fowl typhoid, and Salmonella enterica subsp. enterica serovar Gallinarum biovars Pullorum ( S . Pullorum), the causative agent of pullorum disease, within integrated crop-livestock/pasture farm environments and their processed products. Specifically, the study focused on both the pre-harvest period, which includes the conditions and practices on the farm before the crops and livestock are harvested, and the post-harvest period, which encompasses the handling, processing, and storage of the products after harvest. A total of 1286 samples were collected from six farms and adjacent 13 markets to determine the prevalence of S . Gallinarum and S . Pullorum by using both microbiological culture and molecular techniques, specifically PCR. Antimicrobial susceptibility testing was performed using the agar dilution method for the recommended antibiotics as described in the Clinical Laboratory Standards Institute (CLSI). S . Pullorum was detected in 11 samples (2.7%), while S . Gallinarum was found in six samples (1.5%) out of a total of 403 samples at the pre-harvest level. At the post-harvest level, only S . Gallinarum was identified in 14 meat samples out of 883(1.6%) recovered from samples collected from retail markets. Antibiogram showed S . Gallinarum and S . Pullorum to be highly resistant to cephradine, trimethoprim-sulfamethoxazole, amoxicillin, streptomycin, and ampicillin. This data demonstrates that both S . Pullorum and S . Gallinarum are commonly present in farm poultry environments as well as the products sold in the markets, which warrants implementation of regular surveillance and monitoring programmes, as well as potentially requiring future control strategies to reduce S . Pullorum and S . Gallinarum transmission.
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Thank you for responding to all reviewer comments.
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Thank you for providing the gel image, but please could this be incorporated into the manuscript. A supplementary figure will suffice.
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Whilst I appreciate that many PCR reactions and gels have been performed, reporting appropriate controls is essential for robust science. I am not able to accept this manuscript without controls being provided. Additionally, water is not a robust negative for diagnostic-type PCR. The primers need to be shown to be specific for the species you are looking for versus highly related species.
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Thank you for addressing the majority of points raised by the reviewers, the manuscript is much improved as a result. I have some outstanding concerns regarding points raised by Reviewer 2: 1. Reviewer 2 raised the point that BPW is more appropriate than enriched LB. Please address the limitations of your chosen medium in the Discussion. 2. The lack of a negative control is not best practice. Please consider repeating the PCR with an appropriate negative control in order to provide robustness to your analysis. 3. I am not clear on which strains have already been described in the literature and which are new to this study. Please could that point be addressed more clearly. Please ensure that how you have modified the manuscript is detailed in the response document specifically.
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The work presented is of high importance and the manuscript well written. The reviewers raise some points of consideration that need to be addressed. Please pay particular attention to the point raised by Reviewer 2 regarding existing published data.
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Comments to Author
The paper details sampling of environmental and feed samples from poultry enterprises on combined livestock and crop poultry farms in Maryland (USA), with culture for Salmonella plus phenotypic antimicrobial resistance (AMR) testing of biovars Gallinarum (SG) and Pullorum (SP). Resistance testing of some SG and SP isolates from an earlier survey of retail poultry meats is included. Findings include regular detection of the avian-adapted salmonella biovars SG and SP, and frequent antimicrobial resistance among these isolates. GENERAL COMMENTS The fundamental problem with this paper is that it seeks to report, within the M&M and results sections, a mix of new data (Environmental sampling and AMR) and data that have already been published in a peer-reviewed paper in 2016. Therefore the manuscript needs …
Comments to Author
The paper details sampling of environmental and feed samples from poultry enterprises on combined livestock and crop poultry farms in Maryland (USA), with culture for Salmonella plus phenotypic antimicrobial resistance (AMR) testing of biovars Gallinarum (SG) and Pullorum (SP). Resistance testing of some SG and SP isolates from an earlier survey of retail poultry meats is included. Findings include regular detection of the avian-adapted salmonella biovars SG and SP, and frequent antimicrobial resistance among these isolates. GENERAL COMMENTS The fundamental problem with this paper is that it seeks to report, within the M&M and results sections, a mix of new data (Environmental sampling and AMR) and data that have already been published in a peer-reviewed paper in 2016. Therefore the manuscript needs to be extensively revised (including Title, Abstract, figure, tables and all other sections) to place consideration of the already-published data in its proper place (Introduction and Discussion), and to make clear what are the new data. The discussion is not succinct or well-structured, but the above issues with the paper preclude detailed comments or advice on this. In addition, there is a problem throughout with assertions (often broad) that are either not referenced, or not supported by their cited references. I have detailed those which have caught my attention, but there may be others. Generally, I would recommend using 'antimicrobials' or 'antimicrobial drugs' rather than 'antibiotics', as not all drugs described are antibiotics. SPECIFIC COMMENTS Line 61: 'underdeveloped' is a subjective, arbitrary notion in this context, and elsewhere you refer to 'developing' countries/nations, which I suggest you also use here, for consistency. Line 78: You provide no evidence/reference for the control of S. Gallinarum (SG) and S. Pullorum (SP) being a consequence of growth promoter use. In fact the literature indicates that test and slaughter policies, housing, hygiene and biosecurity practices have been the key elements in control. Lines 82-3: This sentence is ambiguous/poorly constructed, and the reference you cite is a review that in turn cites only references specific to the situation in Ethiopia on this matter. You need to better express exactly what you are intending to say, and reference it appropriately. Line 84: ' Highly resistant' implies knowledge of MIC values, which are not reported in the referenced source. 'Frequently resistant' is a better phrase. Lines 86-7: percentage values are the key information in this list, but the numerator values are not useful - advise remove. Lines 89-91: You just repeat an opinion here from the cited reference - an opinion, moreover, that is not actually supported by the data in the reference. If you can't support this assertion by an authoritative source, advise delete it. line 95: You are specifically targeting a particular type of farm, and you should say so here. Line 105-6: how were the study farms recruited / selected? Line 107: It's unclear what you mean by 'actively' here - do they or do they not use any antimicrobials? And what do you mean by 'synthetic chemicals?' - that's an enormously broad category. Lines 109-111: Are the farmers markets close to the sampled farms, an/or likely to have received birds from them? Lines 113-122: All of this belongs in Discussion, not M&M Lines 130-131: A brief description of the environment sampling process is needed - how were samples taken, handled, transported, etc.? Also, how were retail birds sampled and transported to the lab? Line 134: 1 of what PBS? 1mL? Seems like a small volume. Line 136: Incubation in a non-selective medium such as LB broth is pre-enrichment, not enrichment. A rich, non-buffered medium such as LB plus sheep blood is not ideal for pre-enrichment of environmental samples. Buffered peptone water is the accepted standard for pre-enrichment of environmental Salmonella - why did you not use this? Line 139: 'properly rinsed' - How? With what? Lines 153-4: What negative controls were used? Ideally you would have used E.coli DNA or another related bacterial genus to demonstrate specificity. Table 4: Formatting of left hand side entries is confusing - advise have antimicrobial class entries at the top of each corresponding set of antimicrobial drugs, not in the middle. The breakpoint values unit (I assume mg/L) needs to be added to the table. Lines 190-210: None of this is 'results' - it belongs in Discussion Lines 212, 237, 282: In each case you present P-values of
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The article describes the prevalence of Salmonella, specifically S. Gallinarum and Pullorum in the poultry environment and in post-harvest chicken meat (processed products). The subject is extremely important, since the presence of Salmonella can harm the poultry environment, as well as impacting on the health of poultry and the economic sector. In addition, the study monitors antimicrobial resistance. This type of monitoring is of great relevance since the indiscriminate use of antibiotics, as well as environments contaminated with antibiotics, can spread these pathogens to different environments and cause outbreaks. Strengths of the article: The article brings up a topic of extreme importance in the poultry farming scenario, addressing epidemiological data, such as the prevalence of Salmonella …
Comments to Author
The article describes the prevalence of Salmonella, specifically S. Gallinarum and Pullorum in the poultry environment and in post-harvest chicken meat (processed products). The subject is extremely important, since the presence of Salmonella can harm the poultry environment, as well as impacting on the health of poultry and the economic sector. In addition, the study monitors antimicrobial resistance. This type of monitoring is of great relevance since the indiscriminate use of antibiotics, as well as environments contaminated with antibiotics, can spread these pathogens to different environments and cause outbreaks. Strengths of the article: The article brings up a topic of extreme importance in the poultry farming scenario, addressing epidemiological data, such as the prevalence of Salmonella biovars in the pre- and post-harvest period that impact the health of birds, increasing mortality rates, impacting directly in the economic sector and food security. Surveillance of antimicrobial resistance was also addressed, and it was demonstrated that isolates are resistant to several antibiotics, bringing an alert to the need to implement measures aimed at controlling the indiscriminate use of antibiotics, in addition to improving surveillance of potential resistant isolates that may be disseminated and can be found in chicken meat, which is widely consumed by the world population. Abstract In general, the abstract summarizes all the content of the article and is well written. Here are some considerations regarding the abstract: Line 26-30: The aim of the study is described. I think the author should make it clear within this paragraph what the pre-harvest and post-harvest periods are, considering that these terms are used to a considerable degree in the writing of the article. Line 32: The author describes ''using both microbiological culture and molecular techniques''. The way described is superficial, it is necessary to specify which molecular techniques were used, in this case PCR. Line 33: the antimicrobial susceptibility test was carried out, in the study you used the agar dilution method, so I wouldn't use the term "antibiogram", I suggest you use the term "agar dilution method" (MIC), and describe that it is a quantitative susceptibility test. Line 34: The author describes '' for relevant antibiotics''. Change the use of the term ''relevant''. I suggest describing it as follows: Antimicrobial susceptibility testing was performed using the agar dilution method for the recommended antibiotics as described in the Clinical Laboratory Standards Institute (CLSI). Introduction The introduction is well written, with up-to-date references, and addresses all the problems of the topic itself. Suggestion: Line 70-75: This paragraph describes Salmonella enterica (SE). There is a lack of references to support the literature Line 82: Replace the term ''number'' with another term, for example: increase or the profile of resistant isolates in SE Line 83-84: The term "have been discovered" is not appropriate for the context of the sentence, Describe: the isolates of S. Gallinarum showed high levels of resistance to oxytetracycline, doxycicline, amoxicillin... Line 90: the term "travel" doesn't fit well within the context of the sentence. Replace with: spread, for example. Methods Line 133: The author quotes '' with some modification''. Briefly describe the modifications made to the protocol for this study Line 152: the ''aceK'' gene was used to identify SE species. Briefly describe the criteria for using this gene to identify Salmonella and its species. Line 155: ratA gene was used to differentiate S. Gallinarum from S. Pullorum. Briefly explain why this gene was used as a species differentiator. The reader understands that the gene has an characteristic that allows the differentiation of S. Gallinarum and S. Pullorum, however, in the way described, it is not clear. Please provide this information clearly and objectively. Table 3: A table describing the primers that were used for the PCR. The primers ''SalOriC-F and SalOriC-R'' are not in the text. In the previous text, describe the table, objectively, why this region is used for research. Line 169: it is mentioned that three different concentrations of antibiotic were used in preparing the plates, but it does not mention which concentrations. Describe in this paragraph. Line 177-179: The author describes that isolates can be treated with doses higher than the usual dose of antibiotic. Clearly explain the criteria used to classify strains with an intermediate profile (I) as sensitive (S). Were they based on any studies? If yes, add reference. Table 4: Include reference to the breakpoints used. Ex: CLSI, as mentioned in the text. Add according to the format of the magazine to be published Results Line 203-206: Paragraph is out of context within the results. Within discussion it would be more appropriate. Line 210-211: The author describes that table 5 presents the prevalence of SE serovars in the samples. However, it does not correspond to what is actually described in table 5. Table 5 shows the sources (samples) at the pre-harvest level and the number of SE positivity, at no point are the serovars described. Review text and table. Line 214: cites the highest percentage (31.6%, 18/57). The total number of samples is 60, why did they use 57? Reporting the results more clearly, as described, is confusing. Furthermore, the highest percentage, according to table 5, was in poultry feces (n=21). Review text, table. If the information is correct, explain the results more clearly Line 215-224: clarify numbers and percentages. As described, it is not clear to the reader. Line 233: this describes "meticulous sampling". I would recommend changing the use of this term. Use a more scientific term. Line 234-248: Are the results (numbers and percentages) correct? If yes, justify, because the way it is described is not clear to the reader. Line 249-250: Figure 1A is not referenced in the text, making it difficult for the reader to associate the text with the figure. Line 261: Explain why there are 20 S. Gallinarum and 11 S. Pullorum. The way it is described, it is not possible to understand exactly the number of isolates of each serovar. Line 260 - Sensitivity testing was carried out using agar dilution, so this paragraph should describe the minimum inhibitory concentrations (MIC) of each antibiotic, since this is a quantitative sensitivity methodology. Describe at least for the antibiotics that showed resistance, since it is extremely important to know the MIC value both in treatment and in relation to resistance surveillance. Line 263-264: Enter the percentages (100%) of sensitivity of each antibiotic mentioned. Line 269: In the sentence: '' All S. Pullorum isolates were found susceptible, the term ''found'' does not seem appropriate, I suggest changing it to '' All S. Pullorum isolates were susceptible to gentamicin, ciprofloxacin.... Line 269-273: I suggest reorganizing the results by first describing the highest percentage of resistance, followed by lower percentage profiles, and finally describing the sensitivity of the bacterial strains. Discussion Overall, the discussion is objective and the author has not failed to cover all the important points of the study. I believe that all the subjects that should have been discussed were covered with excellence, such as the prevalence of serovars, explanations of possible contamination, as well as the antimicrobial resistance section. There will be a few comments on this topic: Line 287: The author describes the highest prevalence of SE was in soil samples, however, in table 5 it appears that the prevalence was higher in feces. Is this correct? If yes, ok, if no, rewrite. paragraph from line 356-359, is well written, where reflection is made on the importance of the study is not only focused on the health and welfare of poultry, but on human issues, such as consumer confidence, economic viability, in addition to the safety and quality of products, seeking to carry out efficient surveillance of chicken meat. Line 362: The author describes ''In certain nations''. Cite studies from countries where poultry diseases are still treated with antibiotics Line 368- I suggest changing the use of the expression ''travel'' as has been mentioned in this document previously. Line 373: The author mentions that the isolates have high resistance to cephaldine. Does this antibiotic have any importance in the poultry farming scenario? If yes, describe the reason for the high resistance rate. Line 400-405: The paragraph is not referenced.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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