DETECTION AND ANTIBIOTIC RESISTANCE OF SALMONELLA ISOLATES FROM SELECTED POULTRY FARMS IN DAR ES SALAAM, TANZANIA.
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Introduction Salmonella, one of the most frequent foodborne bacteria, is linked to 155,000 annual mortality and 93.8 million foodborne illnesses. Rampant use of antibiotic agents to combat Salmonellosis in poultry has contributed to the emergence of resistance against commonly used antibiotics. Methodology A cross-section study was conducted between January and June 2023. Purposive sampling was used to select farms, poultry faecal droppings were collected to determine the occurrence of multidrug-resistant (MDR) Salmonella spp. Samples were examined for the presence of Salmonella by standard microbiological techniques. Conventional biochemical tests and molecular methods such as PCR and partial DNA sequencing were used for the identification of Salmonella isolates. Antimicrobial susceptibility testing was performed to identify Salmonella isolates resistant to seven commonly used classes of antibiotics. Results The overall Salmonella spp isolated from faecal droppings from the selected farms was 6.04% (n=48). Of the PCR-confirmed isolates, 64.3% (n=18) were resistant to more than two classes of antibiotics and hence considered MDR. The highest resistance was observed with ampicillin (92.9%), followed by tetracycline (69%), ciprofloxacin (42.9%), sulfonamide (42.9%), gentamicin (35.7%), and azithromycin (28.9%). All the isolates were susceptible to chloramphenicol (100%). Twenty-eight (28) isolates were sequenced and only sixteen (16) sequences met the criteria for phylogenetic analysis. All the sixteen (16) Sequences clustered together with Salmonella enterica sub spp. enterica with accession number EU348369 which was isolated from chickens, pigs, and foods. Conclusion The observed high level of antibiotic resistance found in this study could be attributed to the unwarranted use of antibiotics. To limit the use of antibiotics in poultry farming at the farm level, immediate action is needed including advocating for prudent use of antibiotics in poultry production systems by strengthening extension services to poultry farmers and use of farm biosecurity.
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Comments to Author
The authors of "Detection and antibiotic resistance of Salmonella isolates from selected poultry farms in Dar es Salaam, Tanzania have written a manuscript that is of interest to researchers in the field of AMR among foodborne bacterial pathogens. But unfortunately the description of their results and discussion requires vast improvement. It is impossible to follow their narrative regarding number of farms visited, number of samples taken, number of Salmonella positive isolates, whether they have AMR genes or were MDR etc. A overview figure and/or an overall supplementary table with all results per farm/isolate would be much appreciated. In its current form I cannot accept the manuscript for publication. Below you will find some specific comments. According to the study design in section 2.2 the …
Comments to Author
The authors of "Detection and antibiotic resistance of Salmonella isolates from selected poultry farms in Dar es Salaam, Tanzania have written a manuscript that is of interest to researchers in the field of AMR among foodborne bacterial pathogens. But unfortunately the description of their results and discussion requires vast improvement. It is impossible to follow their narrative regarding number of farms visited, number of samples taken, number of Salmonella positive isolates, whether they have AMR genes or were MDR etc. A overview figure and/or an overall supplementary table with all results per farm/isolate would be much appreciated. In its current form I cannot accept the manuscript for publication. Below you will find some specific comments. According to the study design in section 2.2 the authors took multiple samples from a total 224 farms. Surprisingly, that is the last time the authors mention these multiple samples, consequently it is unclear whether the obtained Salmonella positive samples all came from different farms. What was the overall prevalence among the farms visited. The farms were visited between January and June 2023, was there a trend in Salmonella positive farm and date of sampling? In addition, regarding the 224 farms visited, adding the number of farms in lines 246-247 results in a total of 221. Please explain this difference. Why was the concentration and purity of the genomic DNA were determined while according to the M&M section, this was not taken into account when performing the invA PCR. The percentage and number of participants of the Ilala district in lines 246-247 do not match with those in Table 2. Please rephrase the sentences in lines 248-253, because it is not possible to understand what is meant. What do the authors mean with "being business owners" in line 252. What kind of businesses? Farming? Please rephrase because only in the discussion this becomes more clear Please rephrase the sentences in lines 261-264, because they are not clear. It is impossible to deduce why the authors end up with 48 confirmed Salmonella isolates based on Table 3. The positive and negative controls in the legend of figure 2 are mixed-up. It looks like the authors are rather new to the field of Salmonella characterization. In Figure 3, they call all six isolates retrieved from GenBank Salmonella enterica subsp. enterica, while actually the closest match (OL581592, see https://www.ncbi.nlm.nih.gov/) is a Salmonella enterica subsp. enterica, serovar Kentucky with can also be called S. Kentucky. And for example, OL581595 is a S. Newport. Please address serovars of the other isolates from the public database if you leave the figure as it is. But, a quick blastn search revealed that the invA fragments of the 16 isolates have indeed identical sequences, but their closest matches are S. Agona isolates and not the others included in Figures 3. Please reanalyse and use the proper outcome in the manuscript. Consequently the section in lines 414-437 has to be rewritten accordingly, since in my view OL581592 is not the closest relative. Moreover I was surprised about the 100% matches of the invA PCR fragments. Unfortunately the authors do not indicate from which district these 16 tested isolates came from. Since, a reader cannot determine which isolates are MDR based on Table 3, therefore the text in lines 312-314 are enough and Table 4 can be skipped. I would not call "the use of antimicrobial drugs in animals raised for food has grown to be a significant public health concern" recently when citing papers from 2007 and 2010. Please remove the word pathogens in line 343, since Salmonella itself is a human pathogen. Section 4.2 Isolation and identification of Salmonella spp. using biochemical tests (lines 383-396) is not a real discussion rather an elaborate results description. Please rephrase Lines 409-411; Salmonella is the zoonotic organisms and animals are asymptomatic carriers. Please rephrase. Section "Molecular characterization of Salmonella isolates" describes the confirmation of Salmonella isolates by PCR for 93.3% of the tested isolates. But what about the remaining 19 Salmonella isolates, confirmed by biochemical tests, but which could not be analysed by invA PCR. Please speculate on these as well. The discussion in sections 4.3 and 4.4 is very elaborate. Please shorten. Minor comments Line 133; and level of education Line 146; BPW 1mL Line 147; please change the zero in between 42 and C for a real degree sign Line 157; triple sugar iron test Line 214; because they are Line 214; clinically important Line 219; please explain MHA Lines 259-260; in BPW, RVS, and on XLD agar. Line 261; agar). The Line 261; selective media (XLD) Table 3; please put the total of frequency and percentage of TSI also in bold. And write 0.5 instead of .5 in the percentage column for indole and motility Line 270; invA in italic Line 276; invA in italic twice Line 284; invA in italic Line 289; Fig.3 Lines 298-299; Seven different classes of antibiotics were used Line 302; chloramphenicol (100%) Table 4 shows the Multidrug Resistance patterns of Salmonella isolates and not in poultry faecal droppings. Please rephrase Line 318; Figure 4 shows Line 320; Chloramphenicol resistance is shown and not its effectiveness. And the isolates are resistant to ampicillin. Please rephrase Lines 327-328; Figure 5 Line 332; 4.0 Discussion Lines 402-404; invA does not identify Salmonellosis, the disease, but Salmonella, the bacterium. Please rephrase Line 415; invA
Please rate the manuscript for methodological rigour
Poor
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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Thank you to both reviewers for their comprehensive reports on this manuscript. Together with my own assessment I have gone with a major revisions decision as the manuscript requires a significant rewrite to improve flow and legibility. Please revise in line with reviewers comments below, paying particular attention to consistency of reported numbers throughout the manuscript. Reviewer two has suggested you generate an overview figure to demonstrate the sampling process (number of samples per farm, positivity rate etc)- this is a good suggestion which will improve the readability of the manuscript. In addition to the reviewer comments please also address the following points; Line 138- you have referred to methodology from another publication but state "with modifications"- please detail these so that your methods are reproducible.
Thank you to both reviewers for their comprehensive reports on this manuscript. Together with my own assessment I have gone with a major revisions decision as the manuscript requires a significant rewrite to improve flow and legibility. Please revise in line with reviewers comments below, paying particular attention to consistency of reported numbers throughout the manuscript. Reviewer two has suggested you generate an overview figure to demonstrate the sampling process (number of samples per farm, positivity rate etc)- this is a good suggestion which will improve the readability of the manuscript. In addition to the reviewer comments please also address the following points; Line 138- you have referred to methodology from another publication but state "with modifications"- please detail these so that your methods are reproducible. PCR methodology- please detail what your +ve and -ve controls are. Please change the colours on your bar charts to be solid blocks rather than hashed lines. Figure 4 is difficult to understand. The figure legend needs more detail to describe I,R,S and what "sum of cro" etc mean. My interpretation of this figure is that there is lower AMR in the Temeke district- but maybe there were just fewer isolates overall. This graph would be more informative if it gave resistance patterns as a % of strains collected rather than absolute numbers. Figure 5- the Y axis maximum should be 100% not 120% Please include a tracked changes document when you resubmit so that changes made are easy to see.
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Comments to Author
Mwambene and colleagues detail a multidisciplinary study that investigates the demographics of poultry farmers in Tanzania, as well as the resistance frequencies and genetic relatedness of 28 Salmonella spp. identified within their farms. I enjoyed reading the study, and in general it has been conducted to a good standard, though it is restricted to detailed characterisation of 28 isolates due to cost. There are some sections which could benefit with modification and inclusion of further clarify details as outlined my comments below. I hope these are helpful to the authors in improving the manuscript. Major comments: - Please provide ethics approval information - Line 194: Please detail the type of sequencing that was carried out (Sanger, Nanopore, Illumina, etc.) and the method used for …
Comments to Author
Mwambene and colleagues detail a multidisciplinary study that investigates the demographics of poultry farmers in Tanzania, as well as the resistance frequencies and genetic relatedness of 28 Salmonella spp. identified within their farms. I enjoyed reading the study, and in general it has been conducted to a good standard, though it is restricted to detailed characterisation of 28 isolates due to cost. There are some sections which could benefit with modification and inclusion of further clarify details as outlined my comments below. I hope these are helpful to the authors in improving the manuscript. Major comments: - Please provide ethics approval information - Line 194: Please detail the type of sequencing that was carried out (Sanger, Nanopore, Illumina, etc.) and the method used for phylogenetic inference (ML, NJ, etc.). Please also include a supplementary table of sequences used in phylogenetic analysis that includes accession numbers and any key metadata. Please also explain how the publicly available sequences were selected for analysis. - Were the authors able to identify the serovar of any of the S. enterica isolates? If so, it would be helpful to detail this information in the manuscript. - Please consider adding some text detailing the limitations of the study to the discussion. Minor comments: - Line 38: Please consider correcting "strain" to "strains" - Line 62: There is some repetition between lines 62-64 that could be reduced - Line 66: There is no need to italicise "subsp." - Line 83: Please define how MDR is being used here - how many drug classes or which drugs? Please also consider rephrasing "Multidrug resistance" to "Multidrug resistant" - Line 83: Please consider rephrasing "resistance" to "resistant" - Line 152: Please provide a citation for ISO/TS 6579-2:2012(E) (here and elsewhere). - Line 172: Please detail any quality criteria used for DNA purity/concentration. - Line 182: Please provide further details of the PCR premixed mastermix used (e.g. name and supplier). - Line 249: It's unclear from the text at present which age group is being discussed, I think 45-54 is intended but please consider rephrasing this to improve clarity - Line 271: Please consider removing the word "asked" from this statement - Line 276: Please consider rephrasing "results is" to "results are" - Figure 2: 28 samples are shown in the gel (excluding controls) but 29 are mentioned in text at line 272. Please clarify which number is correct here and elsewhere. - Line 284: It is unclear how many sequences in total were generated from this section and the methods section. Were 16 sent or were more sent and some failed quality control etc.? Please clarify this in text. - Line 298: Please clarify if AST testing was done on the same isolates shown in the gel in Fig 2. - Line 332: Please correct the spelling of "Discussion". - Line 375: Please define KAP. - Section 4.2 of the discussion largely restates the results and could be omitted. - Line 420/Figure 3: Please clarify which samples are from pigs and which are from poultry. Please also consider rephrasing the language here as the analysis is based on a single gene rather than whole genome sequence (and it is therefore difficult to interpret the data in terms of potential transmission/strain circulation patterns). - Line 425: As per my comments above for line 420, please consider indicating the Indonesian strain in figure 3 and refining the language in relation to transmission. - Please consider mentioning what is known about the demographics of famers in Tanzania the region in the introduction to align with the results and the discussion.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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?
No: No ethics approval information provided.
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