Influence of the Salmonella Infantis pESI plasmid on disinfectant efficacy when in biofilm
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Salmonella (S.) Infantis strains harbouring multi-drug resistance (MDR) to high-priority critically important antimicrobials have been isolated globally, giving cause for concern. The serovar is highly persistent throughout the poultry industry and is the fourth most reported serovar linked to zoonotic disease. The serovars resistance is attributed to the presence of a megaplasmid termed pESI. Biocides are an important tool to reduce the need to use antimicrobials, including antibiotics. Bacteria can produce a protective exopolysaccharide matrix called biofilm, and biofilm presence can reduce the efficacy of disinfectants. Here, we aimed to assess the role of pESI on disinfectant efficacy and biofilm formation, and as a contributing factor in the serovars persistence. We analysed S. Infantis strains isolated within the United Kingdom (UK) and Austria, using in vitro planktonic and biofilm disinfectant efficacy assays. Commercially available, commonly used Peroxymonosulphate-, Chlorocresol-and Aldehyde-Quaternary Ammonium Compound-based UK poultry disinfectants were assessed. Biofilm formation was evaluated after 72- and 120-hours incubation, and on a variety of surfaces. Comparative genomic analysis was performed between the UK and Austrian isolates, as well as further globally isolated strains. We identified variation in the presence/absence of antimicrobial resistance genes in both the whole genome and plasmid sequences, within and between the UK and Austrian strains. Variation in biofilm formation was observed between strains, and with greater biofilm formation on non-porous surfaces. Despite this, we could not demonstrate an influence of the pESI plasmid on biofilm formation. Additionally, the presence or absence of the plasmid, or variation observed within the plasmid, did not seem to influence planktonic nor biofilm disinfectant tolerance. Further investigation should be undertaken to identify the influence of pESI on the persistence of S. Infantis and to ensure effective prevention and control of the spread of the serovar and the plasmid.
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Dear Dr Davies, Thank you for submitting your work to Access Microbiology, which I will be happy to accept after minor revisions. Three specific points must be addressed in your revised manuscript: 1. BioProject accession PRJNA1403979 could not be retrieved from NCBI/ENA. Please ensure that this accession and associated biosample/SRA accessions are linked to publicly available data/metadata before resubmission. 2. Reviewer 2 has raised concerns about sample stratification by AMR gene carriage and the appropriateness of comparisons given the phylogenetic relationships between isolates. Please acknowledge these as explicit limitations of the study. With only ten isolates, meaningful stratification with phylogenetic controls was not feasible; a more extensive study would be required to address this. 3. Reviewer 2 has also requested …
Dear Dr Davies, Thank you for submitting your work to Access Microbiology, which I will be happy to accept after minor revisions. Three specific points must be addressed in your revised manuscript: 1. BioProject accession PRJNA1403979 could not be retrieved from NCBI/ENA. Please ensure that this accession and associated biosample/SRA accessions are linked to publicly available data/metadata before resubmission. 2. Reviewer 2 has raised concerns about sample stratification by AMR gene carriage and the appropriateness of comparisons given the phylogenetic relationships between isolates. Please acknowledge these as explicit limitations of the study. With only ten isolates, meaningful stratification with phylogenetic controls was not feasible; a more extensive study would be required to address this. 3. Reviewer 2 has also requested additional metrics to allow the reader to judge the completeness of plasmid reconstruction from short reads. Please include assembly QC metrics (e.g. number of contigs, N50) and confirm whether complete circular plasmid assemblies were recovered for each isolate. Where they were not, please acknowledge this as a limitation of the genomic comparisons made. The legend to Figure 2 already notes an incomplete assembly for at least one isolate. The full reviewer reports are appended below. The reviewers raise a number of additional points including typographic corrections, statistical reporting clarifications, and methodological queries, which I ask you to address as appropriate in your revision. With my best regards, Paul
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Comments to Author
Davies et al investigated the effects of disinfectants against eight strains of S. Infantis with pESI. Although the objective of the study is relevant, the approach as several flaws. First, the use of short read sequencing limits the ability to make comparisons about plasmids. The pESI-like plasmids range from 200 - 300 Kb, which is nearly impossible to assemble to complete circular plasmid using short read technology. Therefore, the plasmids become truncated and the assembly is error prone and accurate plasmid comparisons cannot be achieved. Also, the authors should provide all the WGS QC metrics (Number of reads, N50 and Number of contigs). Also, the authors should confirm if they were able to achieve a complete and circular assembled pESI-like plasmid. Without a complete plasmid, genomic comparisons made in the …
Comments to Author
Davies et al investigated the effects of disinfectants against eight strains of S. Infantis with pESI. Although the objective of the study is relevant, the approach as several flaws. First, the use of short read sequencing limits the ability to make comparisons about plasmids. The pESI-like plasmids range from 200 - 300 Kb, which is nearly impossible to assemble to complete circular plasmid using short read technology. Therefore, the plasmids become truncated and the assembly is error prone and accurate plasmid comparisons cannot be achieved. Also, the authors should provide all the WGS QC metrics (Number of reads, N50 and Number of contigs). Also, the authors should confirm if they were able to achieve a complete and circular assembled pESI-like plasmid. Without a complete plasmid, genomic comparisons made in the study are likely misleading and not good for the literature. Furthermore, the grouping of the isolate based on AMR phenotype only is also inappropriate. It is possible that the short read assembly missed some AMR genes, likewise the phenotype may also not match genotype. Therefore, a better approach was to classify or group isolates by core genome phylogenetic analysis. From Figure 3, four clades/groups can be seen and the comparison of results should be made this way. For instance, UK10, UK14 and UK15 belonged to the same clade and carried dfrA genes, while the clade assigned to MRS isolates did not carry dfrA genes. Also, UK15 tended to have better biofilm capacity than UK10 and UK14, even though they were grouped together. The main AMR gene that differentiates this 3 strains is the carriage of BlaCTX by UK15, which may contribute to better biofilm formation. In a nutshell, the authors should revise the manuscript by making only qualitative conclusions using short read assembled data i.e., limit their conclusions the presence and absence of an AMR gene only. Also, comparison between isolates can only be meaningful if they are grouped by the core genome phylogenetic tree. Minor comments: 1) Overall, the manuscript needs better sentence construction, clarity and grammar improvements. (2) Not clear how many isolates in total were selected. In some parts the authors mention ten groups and later say one isolate from four groups were selected (lines 110 - 128). Also, the number of isolates are very limited for them to assign them country names like Austrian or Uk isolates. It is inappropriate and unfair to suggest a country has particular types of pESI based on 2 - 3 isolates. (3) Line 140: Do you mean the sanitizers that are commonly used in the UK or sanitizer commonly made in the UK? It is not clear. (4) Line 148 - 153: Why store the isolates in different media? Do you think that will introduce another uncontrollable variable ? What is COS agar; spell out COS on first use. (5) Line 129 - 138: The SNIPPY results should have been used for isolate selection and grouping. Also, you have limited number of isolates, therefore it is inappropriate to term them Austrian and UK isolates. they are just isolates that were recovered from random samples in Austria and UK. (6) Line 156 and throughout : After recovery of what and from what? (7) Line 161 : what is excess broth (8) Line 166 -167 and throughout: Not clear how biological replicates versus technical replicates were analyzed. Biological replicates are more important. The error bars of the biological replicates should be used in the figures and not technical replicates. (9) Line 176: What is DEFRA? (10) Line 188: 10uL of the MIC etc does not make sense. How do you pipette 10 ul of the MIC? Also why are neutralizers used, please add a justification. (11) Line 200 - 227: the method is not clear to be reproduced. And what is the Biological replicates (N) for the coupons (12) Line 201: "Instructions"
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
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?
Not at all
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 manuscript presented here is largely well-explained and its materials and methods section enables others to repeat the experiments (although a few issues are commented on below). Many of the methods' shortfalls are appropriately discussed by the authors throughout the manuscript. The authors find that the adequate comparison of disinfectant efficacies on their chosen materials is potentially made more difficult by the combination of methodology (glass beads) and porous surface. The results are appropriately presented through the lens of the underlying challenges in testing disinfectants on biofilms (esp. variability in growth leading to variability in log reduction). These are frequent challenges for this style of experiment. Overall, the manuscript will make a valuable contribution to how native plasmids impact …
Comments to Author
The manuscript presented here is largely well-explained and its materials and methods section enables others to repeat the experiments (although a few issues are commented on below). Many of the methods' shortfalls are appropriately discussed by the authors throughout the manuscript. The authors find that the adequate comparison of disinfectant efficacies on their chosen materials is potentially made more difficult by the combination of methodology (glass beads) and porous surface. The results are appropriately presented through the lens of the underlying challenges in testing disinfectants on biofilms (esp. variability in growth leading to variability in log reduction). These are frequent challenges for this style of experiment. Overall, the manuscript will make a valuable contribution to how native plasmids impact resilience of bacteria to environmental stressors. However, consideration of the following comments could improve the manuscript: Line 97: Typographic error, should presumably read "as opposed to". Line 98: Typographic error, space before the full stop. Lines 155-171: * The cell density of the inoculum for the CV plates was not normalised. Did all isolates grow to similar cell densities in the overnight cultures? * Was the 'edge effect' accounted for in the plate layout? Lines 166, 171: It is not entirely clear what is meant by biological replicates in the context of blank medium wells. Line 201: Typographic error, "instructins" Lines 229-230: Citation 55 appears to be wrong. R should be cited using the R core team citation, available in R via the citation() function. R Studio has an equivalent citation that should also be included. Lines 231-232: The p-adjust method used with the Dunn post-hoc test should be specified. Lines 243-244: Was normality of the differences between pairs assessed for the paired t-tests? Lines 302-303: Is the semicolon on line 302 (between MRS-23-01610 and APHA_UK16) intended? The implied significant difference between APHA_UK16 and _15, but not between _UK10 and _UK15/16, seems notable given figure 4. Figure 4, Lines 301-306: A graphical representation of significant differences between strains would improve readability. Figure 4: The A595 generated by the blank medium seems very high. It also appears to shift two-fold between the time points (as per the low/medium/high threshold values), which is unusual for LB medium. The blanks would be expected to be close to 0 for either time point. The authors might clarify if this is an effect of the length of incubation, or variability between 96 well plates. Line 329, Figure 9: The variability between inoculum densities is very high. Do the less dense inocula correspond to the strains with lower biofilm formation in Fig. 9? Lines 345-346, 355-356, 371-372, Figures 8, 10, and 11: The authors highlight the number of strains that pass the common 4-log threshold for successful disinfection for the different materials. However, several of the mentioned strains (especially in the concrete samples) only barely clear this threshold on average. For samples where only some of the biological repeats showed greater than 4-log reduction, statistical analysis (e.g. one-sample t-test where assumptions are met) would clarify the situation. Lines 462-470: Although the general point of the authors regarding required concentrations for a successful (4-log) disinfection against S. Infantis biofilms is likely true, the data presented in this manuscript only support a reliable 4-log reduction for certain strains, largely on PVC. This follows on from the previous comment on Lines 345-346, 355-356, 371-372, Figures 8, 10, and 11. Discussion of the impact in practice of the variable disinfection efficacy observed here, as well as the implications for persistence (lines 472-473) would improve the manuscript.
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
Please rate the manuscript for methodological rigour
Very good
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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